WO2023106261A1 - 鉄蓄積性神経変性疾患の治療のための組換えアデノ随伴ウイルスベクター - Google Patents

鉄蓄積性神経変性疾患の治療のための組換えアデノ随伴ウイルスベクター Download PDF

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WO2023106261A1
WO2023106261A1 PCT/JP2022/044719 JP2022044719W WO2023106261A1 WO 2023106261 A1 WO2023106261 A1 WO 2023106261A1 JP 2022044719 W JP2022044719 W JP 2022044719W WO 2023106261 A1 WO2023106261 A1 WO 2023106261A1
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amino acid
promoter
acid sequence
iron
sequence
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一洋 村松
貴和子 月田
慎一 村松
崇倫 山形
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Jichi Medical University
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Definitions

  • the present invention relates to a recombinant adeno-associated virus (rAAV) vector for treating abnormal iron metabolism in the central nervous system, a pharmaceutical composition containing the same, and the like.
  • rAAV adeno-associated virus
  • Neurodegenerative diseases associated with iron deposition in the brain are progressive diseases that show evidence of iron deposition mainly in the basal ganglia and cause intellectual disability and motor dysfunction from infancy or adulthood. In addition, the disease is characterized by dystonia, Parkinson-like symptoms, and intellectual decline.
  • Non-Patent Documents 1 to 4 A type of neurodegeneration with brain iron accumulation (NBIA) that involves non-progressive intellectual disability from childhood and rapidly progressing dystonia, parkinsonian symptoms, and other movements in adulthood.
  • SEDA/BPAN static encephalopathy of childhood with neurodegeneration in adulthood/Beta-propeller protein-associated neurodegeneration
  • SENDA SENDA/BPAN
  • the inventors of the present application further used SENDA/BPAN patient-derived cells to analyze in detail the expression of various molecules involved in iron metabolism.
  • the expression level of NCOA4, which is essential for iron metabolism, is greatly decreased, and the decrease in the expression level of NCOA4 is a factor in the deposition of iron in cells of the central nervous system. Found it.
  • NCOA4 is reduced or lost, intracellular ferritin degradation is suppressed, which is a factor in intracellular iron accumulation in this disease.
  • the WDR45 gene was actually expressed in patient-derived cells using an AAV vector, thereby restoring the intracellular NCOA4 expression level and, as a result, correcting ferritin accumulation, thereby completing the present invention.
  • the present application provides, for example, the adeno-associated virus vectors described below, pharmaceutical compositions comprising the same, and the like.
  • An adeno-associated viral vector comprising a polynucleotide encoding an amino acid sequence having about 90% or more identity to an amino acid sequence, preferably said amino acid sequence forming a viral vector similar to the wild-type sequence.
  • [3] The adeno-associated virus vector of [1] above, which is used for the treatment of iron-accumulating neurodegenerative diseases.
  • [4] The adeno-associated virus vector of [1] above, which is used for the treatment of SENDA/BPAN (WDR45 disorder).
  • [5] The recombinant adeno-associated virus vector of [1] above, which contains a wild-type AAV1, AAV2, AAV9, or AAVrh10 capsid protein.
  • Adeno-associated virus recombinant vector Adeno-associated virus recombinant vector.
  • ITR inverted terminal repeat
  • the polynucleotide contains a synapsin I promoter sequence, a myelin basic protein promoter sequence, a neuron-specific enolase promoter sequence, a calcium/calmodulin-dependent protein kinase II (CMKII) promoter sequence, a tubulin ⁇ I promoter sequence, and a platelet-derived growth sequence.
  • ITR inverted terminal repeat
  • GFAP glial fibrillary acidic protein
  • L7 promoter sequence (cerebellar Purkinje cell-specific promoter), glial fibrillary acidic protein (hGfa2) promoter sequence, glutamate receptor delta 2 promoter (cerebellar Purkinje cell-specific specific promoter) sequence, glutamic acid decarboxylase (GAD65/GAD67) promoter sequence, WDR45 promoter, NCOA4 promoter, cytomegalovirus promoter, and CAG promoter.
  • GFD65/GAD67 glutamic acid decarboxylase
  • a pharmaceutical composition comprising the recombinant adeno-associated virus recombinant vector of any one of [1] to [9] above.
  • the recombinant adeno-associated virus recombinant vector according to any one of [1] to [8] above and the amino acid sequence of any one of SEQ ID NOs: 3 to 6 or any one of SEQ ID NOs: 3 to 6 A pharmaceutical composition comprising a recombinant adeno-associated virus vector comprising a polynucleotide encoding an amino acid sequence having about 90% or more identity with the amino acid sequence.
  • a method for identifying, in vitro or ex vivo, cells that cause an iron storage neurodegenerative disease in an organism comprising: providing sample cells derived from a living organism; measuring the expression level of the amino acid sequence of any one of SEQ ID NOs: 3 to 6 or its coding sequence in the sample cells; Comparing the measured expression level with the expression level of any one of the amino acid sequences of SEQ ID NOS: 3-6 or its coding sequence in said cell in a control cell;
  • the vector according to the present invention can regulate iron metabolism in the central nervous system and provide a useful therapeutic means for iron-accumulating neurodegenerative diseases. Furthermore, by using the method of the present invention, it is possible to diagnose patients at risk of iron-accumulating neurodegenerative diseases or patients with such diseases.
  • FIG. 1 is a schematic diagram of the pathway of iron uptake from outside the cell, intracellular transport, and subsequent excretion in cells with impaired WDR45 and NCOA4 deficiency.
  • Figure 2 shows the expression levels of WDR45, NCOA4, ferritin heavy chain, ferritin light chain, transferrin receptor, divarent metal transporter 1 (DMT1), and ferroportin between cells derived from healthy subjects and cells derived from patients 1 to 4. shows the results of comparison.
  • FIG. 3 shows phenotypic recovery of NCOA4, ferritin heavy chain, ferritin light chain, transferrin receptor, and ferroportin when WDR45 is transfected into patient-derived cells.
  • the present application provides a recombinant adeno-associated virus vector for the treatment of iron-accumulating neurodegenerative diseases by restoring iron metabolism to normal levels and inhibiting iron accumulation in the central nervous system of the body.
  • the iron content in the adult brain is generally about 60 mg.
  • iron is found more in the extrapyramidal system such as the globus pallidum, red nucleus, substantia nigra, and putamen.
  • Iron content in cells of the central nervous system is high in oligodendrocytes, followed by neurons, microglia, and low in astrocytes.
  • iron content in the brain generally increases with aging (Clinical Neurology 2012; 52: 943-946).
  • iron in the brain The functions of iron in the brain are diverse. Iron is also contained in the iron-sulfur centers of complexes I to III, which are major components of the mitochondrial electron transport chain, and in cytochromes, and acts as an electron carrier. In addition, iron, in the form of iron-containing enzymes (or coenzymes), is involved in maintenance of TCA cycle function, formation of myelin sheath, biosynthesis of nucleic acids, biosynthesis and metabolism of various neurotransmitters, and the like.
  • the main kinetics of iron in living cells include (1) uptake from the blood, (2) intracellular metabolism, transport, storage, etc., and (3) extracellular excretion. Various factors are known to act in each kinetic (Fig. 1) (Seikagaku 2018; Vol. 90, No. 3: 272-278).
  • Fig. 1 Seikagaku 2018; Vol. 90, No. 3: 272-278.
  • bivalent iron ions are transported to various sites in the body in the form of holotransferrin or the like in which trivalent iron ions are bound to apotransferrin protein in a free state.
  • Holotransferrin in blood can be taken up into cells via transferrin receptors on the cell membrane.
  • Ferric ions can be taken up via DMT1 on the cell membrane. Transferrin taken up into cells undergoes transferrin degradation via endosomes.
  • Trivalent iron ions released by transferrin degradation are reduced by STEAP3, and ferric ions are released into the cytoplasm.
  • trivalent iron ions are accumulated in ferritin particles intracellularly.
  • Ferritin consists of 24 subunits, which contain two proteins, a heavy chain of approximately 21 kDa and a light chain of approximately 19 kDa.
  • Ferritin heavy chain oxidizes divalent iron to less reactive and less toxic trivalent iron and stores trivalent iron in ferritin light chain (Biasiotto G, et al., Mol Neurobiol.
  • Ferric ions can also be taken up from outside the cell, generated from ferric ions in the process of intracellular endosomal degradation, or released by ferritin degradation by lysosomes. This divalent iron is taken up by mitochondria and plays a role such as redox reaction, or is transported to the endoplasmic reticulum.
  • Iron that is not used in nerve cells is either stored as ferritin iron in the cells or excreted out of the cells via ferroportin. Excreted iron associates with apotransferrin and moves in the blood in the form of holotransferrin.
  • Non-Patent Document 2 Iron-accumulating neurodegenerative diseases It is known that accumulation of iron in the central nervous system, particularly in the brain, causes various diseases. This disease is commonly referred to as neurodegeneration with brain iron accumulation (NBIA). Neurological symptoms common to this disease include progressive dystonia, ataxia, and parkinsonism, and specific symptoms include gait disturbance, dysarthria, and cognitive dysfunction (Non-Patent Document 2). .
  • NBIA can be broadly divided into two categories. One is a disease related to intracellular-to-extracellular iron excretion, iron storage, and the like, and the other is a disease related to lipid metabolism, energy production, autophagy, and the like in nerve cells.
  • the process of autophagy includes a process in which an isolation membrane that appears in the cytoplasm surrounds cytoplasmic components to form vesicles (autophagosomes), the formed autophagosomes fuse with lysosomes, and the cytoplasmic components that have been taken up are separated.
  • a step of decomposing is included.
  • Various membrane components derived from the endoplasmic reticulum, mitochondria, Golgi apparatus, cell membrane, and recycling endosomes are involved in autophagosomal membrane formation.
  • Autophagy-related genes ATG is regulated by the Agt protein, etc. (Seikagaku 2018; Vol. 90, No. 3: 272-278).
  • Autophagy-related neurodegenerative diseases included in NBIA include SENDA.
  • the pathology of SENDA includes non-progressive intellectual disability from childhood, dystonia that progresses rapidly in adulthood, parkinsonian symptoms and dementia.
  • Patients with SENDA have iron deposits in the substantia nigra and globus pallidus and cerebral atrophy.
  • Treatment methods for this SENDA include the use of dopamine preparations and intrathecal baclofen therapy (ITB therapy), but no highly useful treatment method has been established (Non-Patent Document 2).
  • This SENDA patient-derived lymphoblastoid cell line showed markedly decreased expression of WDR45, decreased activity of odophagy, and impaired autophagosome formation.
  • increased expression of Atg9L1 and LC3-II which are markers of autophagy, was also observed (Non-Patent Document 2).
  • WDR45 used in the present invention
  • Human WDR45 (WR repeat-containing protein 45) (also referred to herein as "WIPI4") encoded by this gene is a protein containing multiple isoforms.
  • isoform 1 has 361 amino acids.
  • isoform 2 has 360 amino acid residues (GenBank: NP_001025067.1).
  • WDR45 is one of four human homologues (WIPI1-WIPI4) of the budding yeast autophagy-related gene ATG18.
  • a more detailed function of ATG18 in Saccharomyces cerevisiae is thought to be involved in the process of intracellular autophagosome formation by binding to intracellular phosphatidylinositol-3-phosphate (PI3P) (domain fusion Review 2014; 3, e006, 1-11). Subsequently, it was reported that it is involved in the supply of lipids such as phospholipids from the endoplasmic reticulum together with ATG2 (Osawa T, et al. Nat Struct Mol Biol 26, 281-288 (2019)).
  • WDR45 Specific nucleotide sequences of WDR45 include GenBank Accession No. NM_007075.4.
  • the amino acid sequences of human WDR45 used in the present invention are shown in SEQ ID NO: 1 (isoform 1) and SEQ ID NO: 2 (isoform 2) (excluding the first methionine).
  • the expression of WDR45 can be measured by Western blotting using an anti-WDR45 antibody (Cat# 19194-1-AP) provided by Proteintech, RT-PCR using appropriately designed primers based on the coding sequence, and the like. Detectable by means known in the art.
  • NCOA4 nuclear receptor coactivator 4
  • NCOA4 nuclear receptor coactivator 4
  • ARA70 nuclear receptor coactivator 4
  • This NCOA4 functions as a coactivator of nuclear receptors (androgen receptor, estrogen receptor, glucocorticoid receptor, etc.) (Kollara, A. et al., (2010) J.
  • NCOA4 in intracellular iron transport is as follows. The binding of ferritin to NCOA4 coats the isolation membrane. When the isolation membrane closes, it becomes an autophagosome, and when a lysosome fuses with it, it becomes an autolysosome.
  • NCOA4 When the contents are decomposed by the hydrolase in the lysosome, the trivalent iron contained in ferritin is reduced to divalent iron and released into the cytoplasm. This divalent iron is taken up by intracellular organelles and utilized for various cellular activities. It has been reported that when this NCOA4 is overexpressed in cells, it promotes ferroptosis and causes cell death (Hou, W., et al., AUTOPHAGY 2016, VOL. 12, NO. 8, 1425-1428). This suggests that the optimal expression level of NCOA4 is important for normal intracellular iron metabolism. expected to be possible. NOCA4 protein expression can be directly detected by immunostaining such as Western blot using commercially available antibodies, such as the anti-ARA70 antibody (#Cat no. A302-272A) provided by Bethyl Laboratories, and by RT-PCR. (or quantitative RT-PCR (qRT-PCR)) or the like.
  • sequence identity As the amino acid sequence of the protein expressed by the vector of the present invention, heterologous sequences corresponding to the human sequences of SEQ ID NOS: 1 to 6 above can also be used. Examples of such amino acid sequences include those derived from mammals such as mice, rats, monkeys, dogs, pigs, cows and horses.
  • the vectors of the present invention are the amino acid sequences of SEQ ID NO: 1 or SEQ ID NO: 2, optionally further the amino acid sequences of SEQ ID NOS: 3-6, or human vectors having about 90% or more identity to these sequences. including polynucleotides encoding each corresponding amino acid sequence derived from animals other than . More preferably, the vector of the invention comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2, optionally additionally the amino acid sequences of SEQ ID NOs: 3-6.
  • the protein used in the present invention is also about 85% or more, about 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% of each amino acid sequence of SEQ ID NOS: 1 to 6 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, Proteins having amino acid sequences with greater than 99.9% identity and having the same function as the original protein under physiological conditions are included. Generally, the larger the numerical value, the better.
  • the protein having the above identity when it has the same function as the original protein, it preferably functions to the same extent.
  • functioning to the same extent means, for example, that the specific activity is within the range of about 0.01 to 100, preferably about 0.5 to 20, more preferably about 0.5 to 2 under physiological conditions. , but not limited to.
  • proteins used in the present invention include amino acid sequences in which one or more amino acids are deleted, substituted, inserted and/or added to the amino acid sequences of SEQ ID NOS: 1 to 6 or the amino acid sequences having the above identity. , and have the same function as the original protein under physiological conditions. Two or more of the above amino acid deletions, substitutions, insertions and additions may occur simultaneously.
  • Such proteins include, for example, 1 to 50, 1 to 40, 1 to 39, 1 to 38, 1 to 37, 1 to 36 amino acid sequences of SEQ ID NO: 1 or 2.
  • Examples of mutually substitutable amino acid residues in the protein (polypeptide) of the present invention are shown below.
  • Amino acid residues included in the same group can be substituted for each other.
  • Group A leucine, isoleucine, norleucine, valine, norvaline, alanine, 2-aminobutanoic acid, methionine, o-methylserine, t-butylglycine, t-butylalanine, cyclohexylalanine;
  • Group B aspartic acid, glutamic acid, isoaspartic acid, isoglutamic acid, 2-aminoadipic acid, 2-aminosuberic acid;
  • Group C Asparagine, Glutamine;
  • Group D lysine, arginine, ornithine, 2,4-diaminobutanoic acid, 2,3-diaminopropionic acid;
  • Group E proline, 3-hydroxyproline, 4-hydroxyproline;
  • Proteins with substituted amino acid residues can be produced according to methods known to those skilled in the art, such as conventional genetic engineering techniques. For such genetic manipulation procedures, see, for example, Molecular Cloning 3rd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2001, Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997. can be done.
  • Preferable polynucleotides used in the present invention include, for example, one or more (eg, 1 to 50, 1 to 40, 1 to 40, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9 (1 to several), 1 to 8, 1 to 7, 1 to 6 , 1-5, 1-4, 1-3, 1-2, 1, etc.) nucleotide deletions, substitutions, insertions and/or additions, wherein SEQ ID NOS: 1- 6, or an amino acid sequence in which one or more of the above amino acids are deleted, substituted, inserted and/or added in any of the amino acid sequences of SEQ ID NOS: 1 to 6, and the original Includes polynucleotides that encode proteins that have the same function as proteins.
  • one or more eg, 1 to 50, 1 to 40, 1 to 40, 1 to 50, 1 to 40, 1 to 30, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 9 (1 to several), 1 to 8, 1 to 7, 1 to 6
  • a preferred polynucleotide in the present invention is, for example, a polynucleotide that can hybridize under stringent hybridization conditions to a complementary sequence of a polynucleotide that encodes any of the amino acid sequences of SEQ ID NOs: 1 to 6,
  • Hybridization can be performed according to a known method or a method analogous thereto, for example, the method described in Molecular Cloning (3rd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2001). Alternatively, when using a commercially available library, it can be performed according to the method described in the instructions provided by the manufacturer.
  • stringent conditions may be any of low stringent conditions, medium stringent conditions and high stringent conditions.
  • “Low stringent conditions” are, for example, 5 ⁇ SSC, 5 ⁇ Denhardt's solution, 0.5% SDS, 50% formamide, and 32°C.
  • “moderately stringent conditions” are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide, and 42°C.
  • “Highly stringent conditions” are, for example, 5 x SSC, 5 x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, it can be expected that the higher the temperature, the more efficiently DNAs with higher homology can be obtained. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect the stringency of hybridization, and those skilled in the art can select these factors as appropriate. It is possible to achieve similar stringency in .
  • hybridizable polynucleotides include a polynucleotide sequence encoding the amino acid sequence of any one of SEQ ID NOs: 1 to 6 when calculated by homology search software such as FASTA and BLAST using default parameters; For example, 70% or more, 80% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, Polynucleotides having 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, 99.9% or more identity can be included. Generally, the higher the numerical value of homology, the better.
  • rAAV adeno-associated virus
  • recombinant adeno-associated virus (rAAV) vectors are mainly used as a means for delivering therapeutic genes to nervous system cells.
  • rAAV adeno-associated virus
  • examples of such rAAV include those described in WO 2012/057363, 2008/124724, 2003/093479 and the like.
  • the vector for delivering the gene encoding WDR45 protein and/or the gene encoding NCOA4 as a therapeutic gene to nervous system cells includes a mutant combination described in WO 2012/057363 Recombinant adeno-associated virus vectors, or recombinant adeno-associated virus vectors such as those described in WO 2008/124724 can be used.
  • the rAAV vectors used in the present invention are capable of crossing the blood-brain barrier of a living organism, and therefore can be administered to a patient by administration means that are delivered across the blood-brain barrier to the brain, e.g., by peripheral administration, intrathecal administration, etc.
  • a therapeutic gene of interest can be introduced into nervous system cells such as the brain and spinal cord.
  • the rAAV vector used in the present invention enables highly efficient gene transfer even when directly administered to or near the target site in the brain.
  • the rAAV vectors of the present invention are preferably derived from native adeno-associated viruses type 1 (AAV1), type 2 (AAV2), type 3 (AAV3), type 4 (AAV4), type 5 (AAV5), type 6 (AAV6). ), type 7 (AAV7), type 8 (AAV8), type 9 (AAV9), type AAVrh10, etc., but are not limited thereto.
  • Nucleotide sequences of these adeno-associated virus genomes are known, for example GenBank accession numbers: AF063497.1 (AAV1), AF043303 (AAV2), NC_001729 (AAV3), NC_001829.1 (AAV4), NC_006152.1 (AAV5 ), AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), AY530579 (AAV9), JC105350.1 (AAVrhlO) (WO2013174760). Of these, types 2, 3, 5 and 9 are of human origin.
  • capsid proteins VP1, VP2, VP3, etc.
  • AAV1, AAV2, AAV9 or AAVrh10 capsid proteins
  • the capsid protein contained in the rAAV vector of the present invention is preferably such that at least one tyrosine has another A mutant protein having an amino acid sequence with amino acid substitutions.
  • an amino acid sequence in which tyrosine at position 445 in the amino acid sequence of the wild-type AAV1 capsid protein is substituted with phenylalanine SEQ ID NO: 7
  • the capsid protein contained in the rAAV of the present invention is about 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% of any one of the above amino acid sequences 7 to 9 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, Proteins having amino acid sequences with greater than 99.9% identity and capable of forming capsomeres are included.
  • any one of the amino acid sequences of SEQ ID NOS: 7-9 for example, 1-50, 1-40, 1-35, 1-30, 1-25, 1-20, 1 ⁇ 15, 1 ⁇ 14, 1 ⁇ 13, 1 ⁇ 12, 1 ⁇ 11, 1 ⁇ 10, 1 ⁇ 9 (1 ⁇ several), 1 ⁇ 8, 1 ⁇ 7,
  • An amino acid sequence in which 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 amino acid residue is deleted, substituted, inserted and/or added, and Proteins capable of forming capsomeres are included. Combinations of two or more of these deletions, substitutions, insertions and additions may be included at the same time.
  • the capsid protein used in the present invention has the function of forming capsomeres alone or together with other capsid protein members (eg, VP2, VP3, etc.). Also packaged within the capsomere are polynucleotides, including therapeutic genes of interest for delivery to nervous system cells.
  • the rAAV vector of the present invention can pass through the blood-brain barrier of living organisms including adults and fetuses when administered into the blood stream.
  • nervous system cells as gene transfer targets include at least nerve cells contained in the central nervous system such as the brain and spinal cord, and further include glial cells, microglial cells, astrocytes, oligodendrocytes, Ventricular ependymal cells, cerebrovascular endothelial cells, and the like may also be included.
  • the ratio of neurons among the nervous system cells to be transfected is preferably 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or higher, 96% or higher, 97% or higher, 98% or higher, 99% or higher, 99.1% or higher, 99.2% or higher, 99.3% or higher, 99.4% or higher, 99.5% or higher, 99.6% or higher, 99.7% or higher, 99.8% or higher, 99.9% or higher, or 100%.
  • the Rep protein used for the preparation of the rAAV vector of the present invention has the function of recognizing the ITR sequence and performing genome replication depending on the sequence, and recruiting the wild-type AAV genome (or rAAV genome) into the viral vector.
  • it may have the same number of amino acid sequence identity as above, or the same number of amino acids as above. It may involve deletions, substitutions, insertions and/or additions of residues. Functionally equivalent ranges include the ranges described in the discussion of specific activity above.
  • a known AAV3-derived Rep protein is preferably used.
  • the polynucleotide encoding the Rep protein used in the present invention has the function of recognizing the ITR sequence and carrying out genome replication depending on the sequence, packaging the wild-type AAV genome (or rAAV genome) into a viral vector. As long as it encodes a Rep protein having the same degree of known function such as the function, the function of forming the rAAV vector of the present invention, it may have the same number of identities as above, or the same number as above. Nucleotide deletions, substitutions, insertions and/or additions may be included. Functionally equivalent ranges include the ranges described in the discussion of specific activity above. In the present invention, rep genes derived from AAV2 or AAV3 are preferably used.
  • the capsid protein VP1, etc. (VP1, VP2 and/or VP3), encoded by the internal region of the wild-type AAV genome described above, and the Rep protein, the polynucleotides encoding which are encoded in the AAV helper plasmid It is incorporated into and used.
  • Capsid proteins (VP1, VP2 and/or VP3) and Rep proteins used in the present invention may be incorporated into one, two, three or more plasmids as required.
  • one or more of these capsid proteins and Rep proteins may be included in the AAV genome.
  • the capsid proteins (VP1, VP2 and/or VP3) and Rep proteins are all encoded in one polynucleotide and provided as an AAV helper plasmid.
  • the polynucleotide (ie, polynucleotide) packaged into the rAAV vector of the present invention is an internal region (ie, the rep gene and cap one or both of the genes) by a gene cassette containing a polynucleotide encoding the protein of interest (therapeutic gene) and a promoter sequence for transcribing this polynucleotide, and the like.
  • the 5' and 3' located ITRs are located at the 5' and 3' ends of the AAV genome, respectively.
  • the rAAV genome of the present invention has ITRs located at the 5' and 3' ends of the 5' and 3' ITRs contained in the genome of AAV1, AAV2, AAV3, AAV4, AAV8, AAV9, or AAVrh10. Including ITRs.
  • the ITR portion easily adopts a flip-and-flop structure, the ITRs contained in the rAAV genome of the present invention may have their 5' and 3' orientations reversed. .
  • the length of the polynucleotide to be replaced with the internal region ie, therapeutic gene
  • the total length of the rAAV genome of the present invention is preferably about the same as the wild-type total length of 5 kb, for example, about 2-6 kb, preferably about 4-6 kb.
  • the length of the therapeutic gene to be incorporated into the rAAV genome of the present invention is preferably The length is about 0.01-3.7 kb, preferably about 0.01-2.5 kb, more preferably about 0.01-2 kb, but not limited thereto.
  • polynucleotide (viral genome) packaged into the recombinant adeno-associated virus vector when the polynucleotide (viral genome) packaged into the recombinant adeno-associated virus vector is single-stranded, it may take time (several days) to express the therapeutic protein of interest. be. In this case, it is also possible to design the introduced therapeutic gene to be of the sc (self-complementary) type having self-complementarity and to exhibit the effect in a shorter period of time. Specific details are described, for example, in Foust KD, et al. (Nat Biotechnol. 2009 Jan;27(1):59-65). Polynucleotides packaged in rAAV vectors of the invention may be of the non-sc or sc type.
  • the rAAV vector of the present invention preferably comprises a polynucleotide comprising a central nervous cell-specific promoter sequence and a therapeutic gene operably linked to the promoter sequence (i.e., such are packaged in viral vectors).
  • Promoter sequences used in the present invention are derived from, but are not limited to, nerve cells, glial cells, oligodendrocytes, cerebrovascular endothelial cells, microglia, ventricular epithelial cells, and the like.
  • promoter sequences include synapsin I promoter sequence, glutamic acid decarboxylase (GAD65/GAD67) promoter sequence, Tie2 promoter sequence (cerebral capillary-specific promoter), myelin basic protein promoter sequence, neuron specific enolase promoter sequence, glial fibrillary acidic protein promoter sequence, L7 promoter sequence (cerebellar Purkinje cell-specific promoter), glutamate receptor delta2 promoter (cerebellar Purkinje cell-specific promoter), oligodendrocyte-specific promoter (2' , 3'-Cyclic nucleotide 3'-phosphodiesterase (CNP) promoter) sequences, but are not limited to these.
  • CNP 3'-Cyclic nucleotide 3'-phosphodiesterase
  • WDR45 promoter (NM_001029896.2, NM_007075.4), NCOA4 promoter (NM_001145260.2, NM_001145261.2, NM_001145263.2, NM_001145262.2, NM_005437.4), calcium /calmodulin-dependent Promoter sequences such as sex protein kinase II (CMKII) promoter, tubulin ⁇ I promoter, platelet-derived growth factor ⁇ -chain promoter may also be used.
  • CMKII sex protein kinase II
  • tubulin ⁇ I promoter tubulin ⁇ I promoter
  • platelet-derived growth factor ⁇ -chain promoter may also be used.
  • promoter sequences used in the present invention include strong promoter sequences and enhancer sequences commonly used in the art, such as cytomegalovirus (CMV) promoter, CAG promoter, EF1- ⁇ promoter, SV40 promoter, alone or with other promoter sequences. It may be used in combination with a promoter.
  • CMV cytomegalovirus
  • promoter sequences for use in the present invention include myelin basic protein promoter sequence, oligodendrocyte-specific promoter sequence, synapsin I promoter sequence, myelin basic protein promoter sequence, L7 promoter sequence (cerebellar Purkinje cell-specific promoter). , glutamate receptor delta 2 promoter (cerebellar Purkinje cell-specific promoter), WDR45 promoter, NCOA4 promoter, CMV promoter, CAG promoter and the like.
  • sequences used in the rAAV vectors of the present invention may contain known sequences such as enhancer sequences, untranslated region sequences, Kozak sequences, suitable polyadenylation signal sequences, etc., which assist in transcription of mRNA, translation into proteins, and the like. good.
  • the therapeutic gene of interest integrated into the viral genome packaged in the rAAV vector of the present invention is delivered to nervous system cells with high efficiency.
  • the number of neurons is about 10 times or more, about 20 times or more, about 30 times or more, about 40 times or more, or about 50 times or more compared to the case of using a conventional rAAV vector. It can be transfected into cells. The number of transfected neurons can be determined, for example, by preparing an rAAV vector that packages the rAAV vector genome into which an arbitrary marker gene is integrated, and then administering this rAAV vector to a test animal to increase the number of rAAV vector genomes.
  • a known marker gene can be selected as the marker gene.
  • marker genes include LacZ gene, green fluorescent protein (GFP) gene, luminescent protein gene (firefly luciferase, etc.) and the like.
  • a means for suppressing the expression or function of endogenous de novo mutated WDR45 protein is used.
  • Such means include, for example, antisense molecules, ribozymes, interfering RNAs (iRNAs), microRNAs (miRNAs), CRISPR-Cas9, etc., which target the mutated portion of the mutant WDR45 to disrupt the gene or reduce its expression.
  • Generating vectors or polynucleotides may be used.
  • the length of the antisense nucleic acid is preferably 10 bases or more, 15 bases or more, 20 bases or more, or 100 bases or more. Yes, more preferably 500 bases or more. Generally, the length of the antisense nucleic acid used is shorter than 5 kb, preferably shorter than 2.5 kb.
  • various publicly known documents can be referred to for the design of the desired ribozyme (e.g., FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285, 1988; Nucl. Acids. Res. 17: 7059 , 1989; Nature 323: 349, 1986).
  • RNAi is a phenomenon in which the expression of both the introduced exogenous gene and the target endogenous gene is suppressed when double-stranded RNA having a sequence identical or similar to the target gene sequence is introduced into the cell.
  • the RNA used here includes, for example, a 21 to 25-base-long double-stranded RNA that causes RNA interference, such as dsRNA (double strand RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA), or miRNA. (microRNA).
  • dsRNA double strand RNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA miRNA.
  • dsRNA, siRNA, shRNA or miRNA double-stranded RNA
  • the polynucleotide contained in the vector of the present invention can be interposed with a known internal ribosome entry site (IRES) sequence.
  • ITR internal ribosome entry site
  • the rAAV genome of the invention is non-sc, a wider range of promoter lengths and genes of interest can be selected, and multiple genes of interest can be utilized.
  • the total length of the polynucleotide packaged into the rAAV vector of the invention is preferably no greater than about 5 kb (no greater than about 4.7 kb excluding the ITR region).
  • rAAV vector of the present invention General methods can be used to prepare the rAAV vector of the present invention. and (b) transfecting the cultured cells with a second polynucleotide (comprising the therapeutic gene of interest) packaged within the rAAV vector of the invention.
  • the preparation method of the present invention further includes the step of (c) transfecting the cultured cells with a plasmid encoding an adenovirus-derived factor, referred to as adenovirus (AdV) helper plasmid, or infecting the cultured cells with adenovirus.
  • AdV adenovirus
  • the steps of culturing the transfected cultured cells and collecting the recombinant adeno-associated virus vector from the culture supernatant can also be included. Such methods are already known and are also used in the examples of this specification.
  • the nucleotide encoding the capsid protein of the present invention in the first polynucleotide (a) is preferably operably linked to a known promoter sequence that is operable in cultured cells.
  • a promoter sequence for example, cytomegalovirus (CMV) promoter, EF-1 ⁇ promoter, SV40 promoter and the like can be appropriately used.
  • CMV cytomegalovirus
  • EF-1 ⁇ promoter EF-1 ⁇ promoter
  • SV40 promoter SV40 promoter
  • known enhancer sequences, untranslated region (UTR) sequences, Kozak sequences, poly-A addition signal sequences and the like may be included as appropriate.
  • the second polynucleotide (b) contains a therapeutic gene at a position operable with a promoter sequence such as the nervous system cell-specific promoter described above. Furthermore, known enhancer sequences, Kozak sequences, poly-A addition signal sequences and the like may be included as appropriate.
  • This first polynucleotide may further include a cloning site cleavable by various known restriction enzymes downstream of the neural cell-specific promoter sequence. A multiple cloning site containing multiple restriction enzyme recognition sites is more preferred.
  • a therapeutic gene of interest can be integrated downstream of a nervous system cell-specific promoter according to known genetic engineering procedures by those skilled in the art. For such genetic engineering procedures, see, for example, Molecular Cloning (3rd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2001).
  • a helper virus plasmid eg, adenovirus, herpesvirus or vaccinia
  • the preparation method of the present invention further comprises the step of introducing an adenovirus (AdV) helper plasmid.
  • AdV helpers are preferably derived from the same species of virus as the cultured cells.
  • a helper virus vector derived from human AdV can be used.
  • AdV helper vector a commercially available one (eg, AAV Helper-Free System (Catalogue No. 240071) available from Agilent Technologies) can be used.
  • rAAV vector of the present invention various known methods such as the calcium phosphate method, lipofection method, and electroporation method can be used to transfect cultured cells with one or more of the above plasmids. can. Such methods are described, for example, in Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997.
  • compositions containing the rAAV vectors of the present invention contain genes that are useful in the treatment of iron metabolism diseases (e.g., iron-accumulating neurodegenerative diseases) caused by dysfunction of WDR45 and NCOA4. be able to.
  • iron metabolism diseases e.g., iron-accumulating neurodegenerative diseases
  • the vectors of the present invention containing these genes are administered intravascularly, they can pass through the blood-brain barrier and be integrated into nerve cells such as the brain and spinal cord.
  • rAAV vectors containing such therapeutic genes are included in the pharmaceutical compositions of the invention.
  • the active ingredients of the pharmaceutical composition of the present invention may be formulated singly or in combination, and may also be provided in the form of preparations by blending them with pharmaceutically acceptable carriers or pharmaceutical additives. can.
  • the active ingredient of the present invention can be contained, for example, in an amount of 0.1-99.9% by weight in the formulation.
  • Pharmaceutically acceptable carriers or additives include, for example, excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilizers, isotonic Agents, pH adjusters, stabilizers and the like can be used.
  • excipients such as microcrystalline cellulose, sodium citrate and calcium carbonate, disintegrants such as starch and alginic acid, granule-forming binders such as polyvinylpyrrolidone, lubricants, etc., commonly used in the art. Can be used with excipients.
  • formulations suitable for parenteral administration include injections, intrathecal injections, suppositories, and the like.
  • parenteral administration a solution of the active ingredient of the present invention in either sesame oil or peanut oil, or in aqueous propylene glycol solution can be used.
  • the aqueous solutions should be suitably buffered (preferably pH 8 or greater) if necessary and the liquid diluent first rendered isotonic.
  • Physiological saline for example, can be used as such a liquid diluent.
  • the aqueous solutions prepared are suitable for intravenous injection, while the oily solutions are suitable for intra-articular, intramuscular and subcutaneous injection.
  • the active ingredient when preparing aqueous suspensions and/or elixirs for oral administration, is combined with various sweetening or flavoring agents, colorings or dyes, and if desired, emulsifying and/or suspending agents. can also be used with diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.
  • diluents such as water, ethanol, propylene glycol, glycerin, and combinations thereof.
  • formulations suitable for oral administration include powders, tablets, capsules, fine granules, granules, liquids, syrups, and the like. The aseptic production of all these solutions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • the dosage of the pharmaceutical composition of the present invention is not particularly limited, and an appropriate dosage is selected according to various conditions such as the type of disease, age and symptoms of the patient, administration route, purpose of treatment, and presence or absence of concomitant drugs. It is possible to The dosage of the pharmaceutical composition of the present invention is, for example, 1 to 5000 mg, preferably 10 to 1000 mg per day for an adult (eg, body weight 60 kg), but is not limited thereto. These daily doses may be administered in 2 to 4 divided doses.
  • vg vector genome
  • the rAAV of the present invention can cross the blood-brain barrier of the body (including the immature fetal and neonatal blood-brain barriers, and the established adult blood-brain barrier), the body ( Gene delivery of the rAAV of the present invention to nervous system cells such as the brain and spinal cord is possible by peripheral administration to adults and fetuses or neonates. Furthermore, the rAAV vector used in the present invention can target nerve cells contained in the adult brain, spinal cord, etc. by peripheral administration.
  • Peripheral administration refers to intravenous administration, intraarterial administration, intraperitoneal administration, intracardiac administration, intramuscular administration, umbilical cord intravascular administration (for example, when targeting a fetus), etc. It refers to a route of administration as commonly understood as administration.
  • administration methods other than blood that result in fluid communication with the brain such as intrathecal administration, can also be used for the rAAV vectors of the present invention.
  • the rAAV vectors of the invention can be administered locally to target sites in the brain such as the striatum, hippocampus, and the like.
  • the rAAV of the present invention can be administered into the cerebrospinal fluid by intrathecal administration, into the cisterna magna by insertion of a small diameter catheter into the spinal cavity, or into the blood by peripheral administration.
  • kits for producing the rAAV vectors of the invention can include, for example, (a) a first polynucleotide for expressing capsid protein VP1 or the like, and (b) a second polynucleotide packaged within a rAAV vector.
  • the first polynucleotide comprises a polynucleotide encoding the amino acids of SEQ ID NO:
  • the second polynucleotide may or may not contain a therapeutic gene of interest, but preferably contains various restriction enzyme cleavage sites for incorporating such a therapeutic gene of interest. .
  • kits for producing the rAAV vector of the present invention can further include any of the components described herein (eg, AdV helper, etc.). Kits of the invention can also further include instructions describing a protocol for generating rAAV vectors using the kits of the invention.
  • Measurement of the therapeutic effect of the rAAV of the present invention (a) Measurement of iron metabolism ability in vivo Depletion of iron accumulation can be determined by monitoring over time using, for example, MRI. Brain MRI is the easiest and most sensitive way to evaluate iron accumulation in the brain, and it is also suitable for chronological evaluation. In addition to the measurement results such as the evaluation by brain MRI described above, or as an alternative, it is also possible to judge the therapeutic effect by a more comprehensive evaluation such as the exercise ability of the living body (Barthel Index: MAHONEY FI, BARTHEL DW. FUNCTIONAL EVALUATION: THE BARTHEL INDEX. Md State Med J.
  • the ability of the body to metabolize iron can be estimated by measuring serum ferritin and serum iron, which are general serum biochemical tests, and can be determined by measuring serum ferritin and serum iron over time.
  • serum ferritin and serum iron which are general serum biochemical tests
  • serum ferritin and serum iron can be determined by measuring serum ferritin and serum iron over time.
  • skin fibroblasts or lymphocytic cells can usually be used as the cell type.
  • cells differentiated from pluripotent stem cells can also be used.
  • the cell sample is preferably derived from a patient.
  • cells obtained from a patient may be cultured and proliferated in vitro.
  • Preferred cell types for cell samples include, but are not limited to, cells derived from, for example, oligodendrocytes, neurons, astrocytes, glial cells, lymphoid cells, skin fibroblasts, and the like.
  • Cells such as blast cells can also be used as samples.
  • Methods may be used that include comparing receptor expression levels and/or hepcidin expression levels to control cell expression levels obtained from a healthy individual (such as a healthy subject).
  • the regulation of intracellular iron metabolism means the ability to improve or decrease the iron metabolism ability in cell samples such as cells derived from living organisms or cultured cells with reduced iron metabolism ability. and preferably refers to increasing activity related to iron metabolism.
  • divalent iron is toxic, for example, it produces reactive oxygen species, but when it is in short supply, it has adverse effects on living organisms, such as decreased mitochondrial function, so strict regulation is required.
  • trivalent iron produced by oxidation of divalent iron is stored in ferritin and becomes non-toxic.
  • evaluation items for iron metabolism ability include, for example, measurement of intracellular iron ion levels (ferric iron, trivalent iron) and ferritin levels, and decomposability of stored iron.
  • Intracellularly the amount of iron ions is measured by measuring the total ion amount of ferric iron and trivalent iron, or indirectly by measuring the amount of ferritin.
  • an iron ion measurement kit (Sigma MAK025 https://www.sigmaaldrich.com/JP/ja/product/sigma/mak025), etc. Means known in the art can be used.
  • the amount of trivalent iron in cells is measured by, for example, Berlin Blue staining (or Prussian Blue staining; https://labchem-wako.fujifilm.com/jp/product/detail /W01W0129-2154.html), etc. can be quantified based on the amount of color development.
  • the iron metabolism capacity (or an indicator of iron accumulation) is improved in healthy individuals or in healthy individuals.
  • the quantification by staining of each iron ion is only an approximate index.
  • iron decomposing can be judged from the increase or decrease in the amount of ferritin compared to the control.
  • Known means such as Western blotting and quantitative RT-PCR (qRT-PCR) can be used to measure intracellular ferritin levels.
  • the increase or decrease in the ferritin amount obtained is directly related to the decomposition of stored iron, and it is judged that the decomposition of stored iron has progressed if the amount of ferritin decreases (Fig. 1).
  • the amount of ferritin in this case the amount of ferritin heavy chain, the amount of ferritin light chain, or the total amount of both can be used.
  • the expression level of ferritin heavy chain can be measured based on the action of oxidizing divalent iron to trivalent iron.
  • NCOA1 expression level for example, NCOA1 expression level, ferroportin expression level, DMT1 expression level, transferrin receptor expression level, and hepcidin expression level in sample cells. It is not limited to these.
  • means for measuring the expression levels of NCOA4, ferroportin, DMT1, transferrin receptor, hepcidin, etc. Western blotting using antibodies against each protein, RT-PCR for mRNA of each protein, Northern blotting, etc. are known to those skilled in the art. can be used.
  • changes in expression levels of NCOA4, ferroportin, DMT1, transferrin receptor, and hepcidin are related to decomposing iron storage.
  • the recovery of the target protein expression level in the cells used means that compared to the expression level of normal control cells (such as healthy human-derived cells) , in the range of about 50-200% of the protein level detected, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% % or more and 200% or less, 190% or less, 180% or less, 170% or less, 160% or less, 150% or less, 140% or less, 130% or less, 120% or less, 110% or less, or 100%
  • Methods known in the art can be used as means for detecting protein expression levels, such as immunostaining such as Western blotting, color development by direct staining of protein components such as Coomassie staining, and quantitative RT-PCR.
  • Patients derived from cells identified by the above method can be evaluated for iron accumulation, further confirmed for iron accumulation in the brain using the above-described evaluation means, such as MRI, and evaluated for exercise capacity and the like. can.
  • a fluorescent probe (GFP-LC3-RFP) for autophagy activity measurement can be retrovirally introduced into target cells, and degradation efficiency can be evaluated by the GFP/RFP ratio (Kaizuka, T., et al. al. (2016). Mol Cell 64(4): 835-849.). More specifically, we evaluated the GFP/RFP ratio after incubation for a certain period of time under autophagy-inducing conditions (amino acid starvation conditions) and autophagy-inhibiting conditions (bafilomycin A1 addition). Autophagy activity can be compared.
  • the activity or expression level obtained from the sample of interest is different from that of the control. in the range of about 50-200%, preferably 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more or 95% or more and is 200% or less, 190% or less, 180% or less, 170% or less, 160% or less, 150% or less, 140% or less, 130% or less, 120% or less, 110% or less, or 100% or less point to
  • the present invention also provides methods of screening substances for restoring iron metabolism.
  • This screening method is, for example, a method for obtaining a candidate substance that increases the expression level (protein or polynucleotide) of NCOA4.
  • target cells such as patient-derived cells
  • the steps of contacting a test substance with target cells such as patient-derived cells
  • measuring the expression level of NCOA4 in the target cells in the presence of the test substance and obtaining the expression of NCOA4 comparing the amount with the expression level of NCOA4 in the subject cells in the absence of the test substance, and obtaining a candidate substance that increases the expression level of NCOA4 in the subject cells.
  • It may also include a step of comparing the expression level of NCOA4 in control cells (such as cells derived from healthy subjects).
  • test substance substances that act on the NCOA4 promoter can be used.
  • various substances known in the art can be used, such as organic substances such as low-molecular-weight compounds, peptides, proteins, and lipids.
  • the promoter region sequence of NCOA4 can also be referred to.
  • patient-derived cell samples include, but are not limited to, oligodendrocytes, nerve cells, astrocytes, glial cells, lymphoid cells, and skin fibroblasts.
  • the above method may include a step of comparing the intracellular ferroportin expression level, the ferritin heavy chain expression level, and/or the ferritin light chain expression level with the expression level of a healthy control cell. The expression levels of these substances can be measured by means known to those skilled in the art, such as antibodies against each substance and RT-PCR.
  • Candidate substances obtained as a result of screening in the above method can further include a step of applying them to model cells or model animals of iron-accumulating neurodegenerative diseases to verify their abilities related to iron metabolism. For this verification, the procedure for evaluating the iron storage capacity described above can be used.
  • viral vector As used herein, the terms “viral vector”, “viral particle”, and “viral virion” are used interchangeably unless otherwise specified.
  • the term “nervous system” refers to an organ system composed of nerve tissue.
  • the term “nervous system cells” includes at least nerve cells contained in the central nervous system such as the brain and spinal cord, and further includes glial cells, microglial cells, astrocytes, Oligodendrocytes, ventricular ependymal cells, cerebrovascular endothelial cells and the like may also be included.
  • polynucleotide is used interchangeably with “nucleic acid”, “gene” or “nucleic acid molecule” and is intended to be a polymer of nucleotides.
  • nucleotide sequence is used interchangeably with “nucleic acid sequence” or “base sequence” and is a sequence of deoxyribonucleotides (abbreviated as A, G, C and T) is shown as
  • A, G, C and T deoxyribonucleotides
  • a “polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a fragment thereof” is intended to be a polynucleotide comprising the sequence represented by each deoxynucleotide A, G, C and/or T of SEQ ID NO: 1, or a fragment portion thereof. be done.
  • Each of the "viral genome” and “polynucleotide” according to the present invention can exist in the form of DNA (eg, cDNA or genomic DNA), but in some cases may be in the form of RNA (eg, mRNA).
  • RNA eg, mRNA
  • Each of the viral genome and polynucleotides used herein can be double-stranded or single-stranded DNA.
  • single-stranded DNA or RNA it may be the coding strand (also known as the sense strand) or the non-coding strand (also known as the antisense strand).
  • polypeptide proteins and polypeptide are used interchangeably, and are intended to be polymers of amino acids.
  • the left end is the N-terminus (amino terminus) and the right end is the C-terminus (carboxyl terminus) according to the convention of peptide designation.
  • the partial peptide of the polypeptide of the present invention (which may be abbreviated as the partial peptide of the present invention in this specification) is a partial peptide of the above-described polypeptide of the present invention, preferably the above-described polypeptide of the present invention. It has properties similar to those of peptides.
  • Plasmids means various known genetic elements, such as plasmids, phages, transposons, cosmids, chromosomes, and the like. Plasmids are capable of replication in a particular host and transport gene sequences between cells. As used herein, plasmids contain various known nucleotides (DNA, RNA, PNA and mixtures thereof) and may be single-stranded or double-stranded, preferably double-stranded. . For example, as used herein, unless otherwise specified, the term "rAAV vector plasmid” is intended to include the duplex formed by the rAAV vector genome and its complement. Plasmids used in the present invention may be linear or circular.
  • packaging refers to events including preparation of the single-stranded viral genome, assembly of the coat protein (capsid), and encapsidation of the viral genome.
  • a suitable plasmid vector usually multiple plasmids
  • recombinant viral particles i.e., viral virions, viral vectors
  • Equal amounts of protein were separated by SDS-PAGE on 4-12% gels (Thermo Fisher, WG1403BX10) and transferred to PVDF membranes (Thermo Fisher, IB24001) using iBlot2 (Thermo Fisher). After transfer, the membrane was blocked with a blocking buffer (5% skimmed milk in PBS-T) at room temperature for 1 hour. After washing with PBS-T, the cells were incubated overnight at 4°C with an antibody diluent (1% skimmed milk in PBS-T) containing a primary antibody. After washing, the plate was incubated with an antibody diluent containing HRP-labeled secondary antibody at room temperature for 1 hour. After washing, HRP detection reagents (Takara, T7103A or Thermo Fisher, A38554) were used and detected and analyzed by Amersham Imager (GE Healthcare).
  • AAV.GTX-CMV-WDR45-WPRE vector The vector used in this example (hereinafter also referred to as "AAV.GTX”) is AAV9/3 (AAV9 capsid A viral vector having a tyrosine mutation (Y446 ⁇ F) (SEQ ID NO: 9) and having an ITR sequence derived from AAV3), human cytomegalovirus immediate-early (CMV) promoter, cDNA of WDR45 (NM_007075.4), An expression cassette consisting of the simian virus 40 polyadenylation signal sequence was inserted.
  • the central nervous system in which the vector of the present invention is administered for example, by inserting a thin catheter into the spinal cavity of the subject and allowing the tip to reach the cisterna magna.
  • Subjects are administered by direct route and/or intravenous systemic administration.
  • WDR45, ferritin heavy chain, ferritin light chain, and NCOA4 were detected over time by monitoring the status of iron accumulation by in vivo brain MRI measurement, or in sample cells (lymphoid cells, skin fibroblasts, etc.).
  • transferrin receptor, DMT1, ferroportin and/or LC3 expression levels are measured to verify the effect of the introduction of the vector of the present invention.
  • the vector of the present invention can reduce iron accumulation in the central nervous system by normalizing iron metabolism, it is expected to be applied for the treatment of these diseases.
  • SEQ ID NO: 1 Amino acid sequence of human WDR45 (isoform 1) (NP_009006.2) (excluding the first Met)
  • SEQ ID NO: 2 Amino acid sequence of human WDR45 (isoform 2) (NP_001025067.1) (excluding the first Met)
  • SEQ ID NO: 3 amino acid sequence of human NCOA4 (650aa) (isoform 1) (NM_001145260.2)
  • SEQ ID NO: 4 amino acid sequence of human NCOA4 (630aa) (isoform 2) (NP_001138733.1)
  • SEQ ID NO: 6 amino acid sequence of human NCOA4 (575aa) (AAH12736.1)
  • SEQ ID NO: 7 wild Amino acid sequence in which tyrosine at position 445 in

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