JP2017123840A - Adeno-associated virus vector for expression of glucose transporter 1 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a novel means for gene therapy of GLUT 1 (glucose transporter 1) deficiency.SOLUTION: According to the present invention, there is provided a recombinant adeno-associated virus vector comprises a gene (SLC2A1) that expresses GLUT1 in a neural cell or a pharmaceutical composition containing this vector. The composition is provided for gene delivery to neural cells in the brain of an individual, for example, by peripheral blood administration or intrathecal administration, and is capable of, for example, alleviating, ameliorating, treating the symptoms of GLUT1 deficiency. According to the present invention, the pharmaceutical composition may be intracerebrally, intrathecally, or peripherally administered or may be used in combination with ketone diet therapy.SELECTED DRAWING: None

Description

  The present invention relates to a recombinant adeno-associated virus (rAAV) virion for the treatment of genetic abnormalities in nervous system cells. More specifically, the present invention relates to rAAV for gene therapy of glucose transporter type 1 deficiency syndrome (GLUT-1DS: OMIM 606777).

Glucose transporter type 1 deficiency syndrome
syndrome; GLUT1 DS) is caused by impaired glucose transport to the brain tissue due to dysfunction of the glucose transporter (GLUT) protein. The haplo of the SLC2A1 (solute carrier family 2 (facilitated glucose transporter), member 1) gene -Insufficiency takes the form of onset. This GLUT1 protein, as well as many other neurogenetic diseases, is widely distributed in the brain. Symptoms of this disease include intractable convulsions from early infants, intellectual disability, and cerebellar ataxia. As a specific treatment for GLUT1 deficiency, ketogenic diet is used for some convulsions, but no curative treatment has been established at present.

  Methods for treating various inherited diseases by gene therapy by delivering therapeutic genes to nervous system cells have been studied. As means (vector) for delivering a therapeutic gene to such nervous system cells, means using a genetically modified adeno-associated virus (rAAV) is known. Examples of such rAAV include those described in International Publications 2012/057363, 2008/124724, 2003/093479, and the like.

International Publication No.2012 / 057363 International Publication 2008/124724 International Publication 2003/093479

De Giorgis, V. et al., Seizure 22 (2013) 803-811

  There is a need for new medicaments for treating glucose transporter 1 deficiency syndrome by gene therapy. Furthermore, it is desirable to be advantageous in actual treatment, such as low side effects, possible with simpler administration, and high persistence.

  As a result of diligent efforts aimed at gene therapy for glucose transporter 1 deficiency syndrome, the present inventors prepared a recombinant adeno-associated virus vector containing the nucleotide sequence of the gene encoding the glucose transporter 1 protein (SLC2A1 gene). Thus, when this vector was administered to a living body, it was found that the symptoms of glucose transporter 1 deficiency syndrome improved, and the present invention was completed.

That is, the present application provides the following recombinant adeno-associated virus (rAAV) vector, a pharmaceutical composition containing the same, and the like for the treatment of diseases of glucose transporter 1 deficiency syndrome described below. .
[1] A recombinant adeno-associated virus vector for the treatment of glucose transporter 1 deficiency syndrome, comprising a polynucleotide encoding a glucose transport protein.
[2] The polynucleotide includes a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or 4, or an amino acid sequence having about 90% identity with these sequences and having the ability to transport glucose [1] A recombinant adeno-associated virus vector described in 1.
[3] The recombinant adeno-associated virus vector is a protein having a mutant 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, and 445 in the amino acid sequence of the wild-type AAV2 capsid protein. A protein having a mutant amino acid sequence in which the tyrosine at position is substituted with phenylalanine, or a protein having a mutant amino acid sequence in which the tyrosine at position 446 in the amino acid sequence of the wild-type AAV9 capsid protein is substituted with phenylalanine [1 ] Or adeno-associated virus recombinant vector according to any one of [2].
[4] The polynucleotide comprises synapsin I promoter sequence, myelin basic protein promoter sequence, neuron specific enolase promoter sequence, calcium / calmodulin-dependent protein kinase II (CMKII) promoter sequence, tubulin αI promoter sequence, platelet-derived growth Factor β chain promoter sequence, glial fibrillary acidic protein (GFAP) promoter sequence, L7 promoter sequence (cerebellar Purkinje cell-specific promoter), glial fibrillary acidic protein (hGfa2) promoter sequence, glutamate receptor delta2 promoter (cerebellar Purkinje cell specific) Promoter) sequence, glutamate decarboxylase (GAD65 / GAD67) promoter sequence, polynucleotide sequence SEQ ID NO: 17, polynucleotide sequence SEQ ID NO: 18, and sequence SEQ ID NO: 19 Comprising a promoter sequence selected from the group consisting of nucleotide sequences, recombinant adeno-associated virus vector according to any one of [1] to [3].
[5] The polynucleotide according to any one of [1] to [4], wherein the polynucleotide comprises an inverted terminal repeat (ITR) selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV8, and AAV9. Recombinant adeno-associated virus vector.
[6] A pharmaceutical composition comprising the recombinant adeno-associated virus recombinant vector according to any one of [1] to [5].
[7] The pharmaceutical composition according to [6], which is administered into the brain.
[8] The pharmaceutical composition according to [6], which is administered intrathecally.
[9] The pharmaceutical composition according to [6], for peripheral administration.
[10] The pharmaceutical composition according to [6] to [9], for use in combination with a ketone diet.
[11] A method for treating glucose transporter 1 deficiency syndrome, comprising administering the pharmaceutical composition according to any one of [6] to [10] above.
[12] The method according to [11], which is used in combination with a ketone diet.
[13] The method of [11], further comprising using an antiepileptic drug in combination.

  Enhancing the glucose uptake function in the central nervous system according to the present invention by gene therapy is useful as a treatment method for glucose transporter 1 deficiency syndrome.

FIG. 1 shows the results of confirming the expression of normal and mutant GLUT1 proteins (wild type (wt), mutants R333W, A405D, V303fs) in HEK293 cells. FIG. 1a shows the detection result by Western blotting using anti-myc antibody. In addition, * shows the nonspecific band considered to be endogenous myc. FIG. 1b shows Hoechst staining of anti-myc antibody. FIG. 2 shows the results of a 2-deoxyglucose (2DG) uptake test in normal and mutant GLUT1 protein-introduced cells of FIG. FIG. 3 shows a schematic diagram of nucleotides incorporated into the recombinant adeno-associated virus (rAAV) used in this example. Each description in the figure is as follows. ITR: inverted terminal repeat; Synapsin I: neuron-specific synapsin I promoter; WPRE: woodchuck hepatitis virus post-transcriptional regulatory element; SV40polyA: SV40 polyA addition signal. FIG. 4 shows the results of detection of GLUT1 protein expression after introduction of AAV9-SLC2A1 in human nervous system cultured cells SH-SY5Y using anti-myc antibody or anti-GLUT1 antibody. In particular, a part of the stained part using anti-myc antibody is indicated by an arrow. FIG. 5 shows the results of detecting GLUT1 protein expression in mice after administration of AAV9-SLC2A1 using an anti-myc antibody. The frame in each brain schematic diagram shows the display range of the corresponding image. The locations indicated by arrows in FIGS. 5a to 5c are locations where AAV9-derived GLUT1 expression is observed by intraperitoneal administration. In the case of intraventricular administration in FIG. 5d, strong expression is observed in many places. Each abbreviation is as follows. Cx: cerebral cortex, CC: corpus callosum, H: hippocampus, LV: lateral ventricle. FIG. 6 shows the detection results using anti-myc antibody after intraventricular administration, as in FIG. 5d. The frame in the brain schematic diagram shows the display range (sagittal cross-sectional view) of the corresponding image. FIG. 7 shows the detection results of mRNA expression levels of rAAV-derived GLUT1 and endogenous GLUT1. FIG. 7a shows the detection result by RT-PCT, and FIG. 7b shows the result of quantifying the result of FIG. 7a. Abbreviations in the figure are as follows. icv: Intraventricular, ip: Intraperitoneal. The number of test individuals is indicated by n. FIG. 8 shows the experimental results for recovery of motor function (Rota rod test) in mice after administration of AAV9-SLC2A1. The result of the residence time (seconds) in the acceleration cylinder of 4 to 40 rpm is shown. Measurements were taken 3 days a day, 2 days after practice for 2 consecutive days. The number of test individuals is indicated by n. FIG. 9-1 shows the results of confirming the expression of SLC2A1 linked to each promoter in MO3.13 cells (glial cells; non-neural system). FIG. 9-2 shows the results of confirming the expression of SLC2A1 linked to each promoter in SH-SY5Y cells (human neuroblastoma cells: nervous system). FIG. 9-3 shows the results of confirming the expression of SLC2A1 linked to each promoter in HUVEC cells (human umbilical vein endothelial cells: vascular endothelial system).

  In this application, a recombinant adeno-associated virus vector for the treatment of glucose transporter 1 deficiency syndrome is provided comprising a polynucleotide encoding a protein that enhances glucose uptake in the central nervous system of a living organism.

1. As used in this application for the glucose transporter in the central nervous system, the protein that improves glucose uptake in the central nervous system of the organism preferably refers to the glucose transporter protein, more preferably the glucose transporter 1 protein.

  The glucose transporter (GLUT) protein typically includes GLUT1 to GLUT7. GLUT1, GLUT3 and GLUT5 are widely distributed in almost all tissues of mammals, while GLUT2, GLUT4 and GLUT7 show tissue-specific expression. These GLUTs are considered to be proteins having 12 transmembrane regions based on their amino acid sequences, but GLUT proteins containing 14 transmembrane regions are also considered to exist.

  As the amino acid sequence of the GLUT1 protein used in the present invention, known amino acid sequences can be used. Examples of such amino acid sequences include Genbank accession numbers NP_006507 (human), NP_035530 (mouse), and NP_620182 (rat). Other animal species that can be used include those derived from mammals such as monkeys, dogs, pigs, cows, and horses. The amino acid sequences of human, mouse and rat glucose transporter 1 proteins are set forth in SEQ ID NOs: 2, 4, 5 and 6, respectively. Preferably, the vector of the present invention comprises an amino acid sequence of SEQ ID NO: 2, 4, 5 or 6, or a polynucleotide encoding an amino acid sequence having about 90% identity with these sequences and having a glucose transporting ability. Including. More preferably, the vector of the present invention comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2, 4, 5 or 6. More preferably, the vector of the present invention comprises a polynucleotide encoding an amino acid sequence consisting of SEQ ID NO: 2.

  The GLUT1 protein used in the present invention is about 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% with respect to the amino acid sequence of SEQ ID NO: 2, 4, 5 or 6. More than 96%, 97% or more, 98% or more, 99% or more, 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, A protein having an amino acid sequence having an identity of 99.9% or more and having a glucose transporting ability under physiological conditions is included. In general, the larger the value, the better. In the present invention, the mutant protein and the original protein function to the same extent (for example, exhibit the same binding ability), for example, the specific activity is about 0.01 to 100, preferably about 0.5 to 20, More preferably, it means within a range of about 0.5 to 2, but is not limited thereto.

  Furthermore, in the GLUT1 protein used in the present invention, one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2, 4, 5 or 6 or the amino acid sequence having the above identity. A protein containing an amino acid sequence and having a glucose transport ability under physiological conditions is included. Two or more of the above amino acid deletions, substitutions, insertions and additions may occur simultaneously. Examples of such proteins include, for example, 1 to 50, 1 to 40, 1 to 39, 1 to 38, 1 to 37, and 1 to 36 in the amino acid sequence of SEQ ID NO: 2 or 4. , 1-35, 1-34, 1-33, 1-32, 1-31, 1-30, 1-29, 1-28, 1-27, 1-26 1-25, 1-24, 1-23, 1-22, 1-21, 1-20, 1-19, 1-18, 1-17, 1-16 , 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7 Including amino acid sequences in which one, six, one to five, one to four, one to three, one to two, or one amino acid residue has been deleted, substituted, inserted and / or added And proteins having the ability to transport glucose under physiological conditions. In general, the smaller the number of amino acid residue deletions, substitutions, insertions and / or additions, the better.

  Examples of amino acid residues that can be substituted for each other in the protein (polypeptide) of the present invention are shown below. Amino acid residues contained 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; Group F: serine, threonine, homoserine; Group G: phenylalanine, tyrosine. The glucose transporter 1 protein in which an amino acid residue is substituted can be produced according to a method known to those skilled in the art, such as a normal genetic engineering technique. 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, etc. Can do.

  Moreover, as a preferable polynucleotide used for this invention, in polynucleotide sequence of sequence number 1 or 3, for example, 1 or more (for example, 1-50 pieces, 1-40 pieces, 1-30 pieces, 1-25 pieces) 1-20 pieces, 1-15 pieces, 1-10 pieces, 1-9 pieces (1 to several pieces), 1-8 pieces, 1-7 pieces, 1-6 pieces, 1-5 pieces, 1- A polynucleotide comprising a deletion, substitution, insertion and / or addition of 4 nucleotides, 1 to 3, 1 to 2, 1 and the like), comprising the amino acid sequence of SEQ ID NO: 2 or 4; Alternatively, it includes a polynucleotide encoding a protein having an amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2 or 4, and having a glucose transporting ability. A combination of two or more of these deletions, substitutions, insertions and additions may be included simultaneously. In general, the smaller the number of nucleotide deletions, substitutions, insertions and / or additions, the better. A preferred polynucleotide in the present invention is a polynucleotide that can hybridize under stringent hybridization conditions to its complementary sequence of SEQ ID NO: 1 or 3, for example, and encodes the amino acid sequence of SEQ ID NO: 2 or 4 Or a polynucleotide encoding a protein having the ability to transport glucose, comprising the amino acid sequence in which one or more amino acids are deleted, substituted, inserted and / or added in the amino acid sequence of SEQ ID NO: 2 or 4 .

Hybridization may be performed by a known method or a method similar thereto, for example, molecular cloning (Molecular Cloning 3rd Edition, J. Sambrook et al., Cold Spring).
Harbor Lab. Press. 2001). Moreover, when using a commercially available library, it can carry out according to the method as described in the instruction manual provided by the manufacturer. Here, the “stringent conditions” may be any of low stringent conditions, medium stringent conditions, and high stringent conditions. “Low stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 32 ° C. The “medium stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, and 42 ° C. “High stringent conditions” are, for example, conditions of 5 × SSC, 5 × Denhardt's solution, 0.5% SDS, 50% formamide, 50 ° C. Under these conditions, it can be expected that DNA having higher homology can be efficiently obtained as the temperature is increased. However, multiple factors such as temperature, probe concentration, probe length, ionic strength, time, and salt concentration can be considered as factors that affect hybridization stringency. Those skilled in the art will select these factors as appropriate. It is possible to achieve similar stringency.

As a hybridizable polynucleotide, when calculated by homology search software such as FASTA, BLAST using default parameters, the nucleotide sequence of SEQ ID NO: 7, 9, or 11 and, 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, 99.1% or more, 99.2% Above, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more, 99.7% or more,
A polynucleotide having 99.8% or more and 99.9% or more of identity can be exemplified. In general, the larger the homology value, the better.

  The identity or homology of amino acid sequences and polynucleotide sequences can be determined by the algorithm BLAST (Proc. Natl. Acad. Sci. USA 872264-2268, 1990; Proc. Natl. Acad. Sci USA 90: 5873, 1993). ). Programs called BLASTN and BLASTX based on the BLAST algorithm have been developed (Altschul SF, et al: J Mol Biol 215: 403, 1990). When analyzing a base sequence using BLASTN, parameters are set to, for example, score = 100 and wordlength = 12. In addition, when an amino acid sequence is analyzed using BLASTX, the parameters are, for example, score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used.

2. About glucose transporter 1 deficiency syndrome (GLUT1-DS) The glucose transporter 1 deficiency syndrome in which the vector of the present invention is used specifically includes epileptic seizure, abnormal eye movement seizure, apnea seizure, Doose syndrome. , Hypotonia, cerebellar ataxia, spastic paralysis, dystonia, cognitive impairment, learning disorder, mental retardation, acquired cerebellar dysfunction, ataxia, mental confusion, lethargy / somnolence, hemiplegia, systemic paralysis, sleep disorder, headache, vomiting Various symptoms are observed. Heterozygosity and homozygosity of the SLC2A1 gene (1p34.2) encoding GLUT1 is associated with symptoms.

  For the treatment of glucose transporter 1 deficiency, ketogenic diet or general antiepileptic drugs (for example, valoproic acid, clonazepam, and combinations thereof) are selected so that ketone is an energy source instead of glucose. Is done.

3. Recombinant adeno-associated virus (rAAV) vector of the present invention In the present invention, a vector for delivering a gene encoding a protein having a glucose transport function in a cell to a nervous system cell is described in WO 2012/057363. A recombinant adeno-associated virus vector (also referred to herein as a vascular administration vector) that can efficiently deliver a gene to nerve cells even by peripheral administration, or those described in WO 2008/124724 or the like can be used. The rAAV vector of the present invention is capable of passing through the blood-brain barrier of a living body, and thus is delivered to the brain through the blood-brain barrier, for example, peripheral administration to the patient, intrathecal administration, etc. The desired therapeutic gene can be introduced into neural cells such as the spinal cord. It can also be administered directly to a target site in the brain.

  The rAAV vector of the present invention is preferably a natural adeno-associated virus type 1 (AAV1), type 2 (AAV2), type 3 (AAV3), type 4 (AAV4), type 5 (AAV5), type 6 (AAV6) ), 7 type (AAV7), 8 type (AAV8), 9 type (AAV9), etc., but is not limited thereto. The nucleotide sequences of these adeno-associated virus genomes are known, and GenBank accession numbers: AF063497.1 (AAV1), AF043303 (AAV2), NC_001729 (AAV3), NC_001829.1 (AAV4), NC_006152.1 (AAV5), respectively. , AF028704.1 (AAV6), NC_006260.1 (AAV7), NC_006261.1 (AAV8), AY530579 (AAV9) can be referred to. Of these, type 2, type 3, type 5 and type 9 are human. In the present invention, it is particularly preferable to use a capsid protein (VP1, VP2, VP3, etc.) derived from AAV1, AAV2 or AAV9. AAV1 and AAV9 have been reported to have a relatively high efficiency in infecting nerve cells among human-derived AAV (Taymans, et al., Hum Gene Ther 18: 195-206, 2007, etc.).

The capsid protein contained in the rAAV vector used in the present invention is preferably a protein having at least one tyrosine other than phenylalanine as described in WO 2012/057363, WO 2008/124724, etc. It is a mutant protein having an amino acid sequence substituted with the amino acid. For example, in the amino acid sequence of the wild type AAV1 capsid protein, the tyrosine at position 445 is substituted with phenylalanine (SEQ ID NO: 9), and in the amino acid sequence of the wild type AAV2 capsid protein, the tyrosine at position 444 is substituted with phenylalanine. The amino acid sequence (SEQ ID NO: 10), or the amino acid sequence (SEQ ID NO: 11) in which the tyrosine residue at position 446 in the amino acid sequence of the wild-type AAV9 capsid protein is substituted with a phenylalanine residue, can form a capsomere The mutant protein is (WO 2012/057363, WO 2008/124724). Further, 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% with respect to any one of the amino acid sequences 9 to 11 above. More than 96%, 97% or more, 98% or more, 99% or more, 99.1% or more, 99.2% or more, 99.3% or more, 99.4% or more, 99.5% or more, 99.6% or more,
A protein having an amino acid sequence having an identity of 99.7% or more, 99.8% or more, or 99.9% or more and capable of forming a capsomer is included. In addition, for example, in any one of the amino acid sequences of SEQ ID NOs: 9 to 11, for example, 1 to 50, 1 to 40, 1 to 35, 1 to 30, 1 to 25, 1 to 20, 1 -15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9 (1 to several), 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2 or 1 amino acid residues deleted, substituted, inserted and / or added amino acid sequence, and Examples include proteins capable of forming capsomeres. A combination of two or more of these deletions, substitutions, insertions and additions may be included simultaneously. In another embodiment, the capsid protein used in the present invention has a function of forming a capsomere alone or together with other capsid protein members (eg, VP2, VP3, etc.). In the capsomere, a polynucleotide containing a target therapeutic gene to be delivered to a nervous system cell is packaged.

  When administered into the bloodstream, the rAAV vector of the present invention can pass through the blood-brain barrier of living organisms including adults and fetuses. In the present invention, the nervous system cell as a gene transfer target includes at least a nerve cell contained in the central nervous system such as the brain and spinal cord, and further includes a glial cell, a microglia, an astrocyte, an oligodendrocyte, Ventricular ependymal cells, cerebral vascular endothelial cells and the like may be included. The ratio of nerve cells among the nervous system cells into which genes are introduced 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 more, 96% or more, 97% or more, 98% or more, 99% or more, 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% Above, 99.9% or more, or 100%.

  The Rep protein used in the present invention has a function of recognizing an ITR sequence and performing genome replication depending on the sequence, a function of recruiting and packaging a wild type AAV genome (or rAAV genome) into a viral vector, As long as it has a known function such as the function of forming the rAAV vector of the present invention to the same extent, it may have the same number of amino acid sequence identities as described above, or a deletion of the same number of amino acid residues as described above. Substitutions, insertions and / or additions may be included. Examples of the functionally equivalent range include the ranges described in the explanation regarding the specific activity. In the present invention, a known Rep protein derived from AAV3 is preferably used.

  The polynucleotide encoding the Rep protein used in the present invention has a function of recognizing an ITR sequence and performing genome replication depending on the sequence, and packaging a wild type AAV genome (or rAAV genome) into a viral vector. As long as it encodes a Rep protein having the same level of known functions such as the function and 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. Examples of the functionally equivalent range include the ranges described in the explanation regarding the specific activity. In the present invention, a rep gene derived from AAV2 or AAV3 is preferably used.

  In one embodiment of the present invention, the capsid protein VP1 and the like (VP1, VP2 and / or VP3) encoded in the internal region of the wild-type AAV genome described above, and the Rep protein, the polynucleotide encoding the same is an AAV helper plasmid. It is incorporated into and used. The capsid protein (VP1, VP2 and / or VP3) and Rep protein used in the present invention may be incorporated into one, two, three or more plasmids as necessary. In some cases, one or more of these capsid proteins and Rep proteins may be included in the AAV genome. In the present invention, preferably the capsid protein (VP1, VP2 and / or VP3) and the Rep protein are all encoded by one polynucleotide and provided as an AAV helper plasmid.

  The polynucleotide packaged in the rAAV vector of the present invention (ie, polynucleotide) is an internal region located between the ITRs located 5 ′ and 3 ′ of the wild type genome (ie, rep gene and cap A polynucleotide of one or both of the genes) can be prepared by replacing with a gene cassette containing a polynucleotide encoding a protein of interest (therapeutic gene) and a promoter sequence for transcription of the polynucleotide. Preferably, ITRs located 5 'and 3' are located at the 5 'and 3' ends of the AAV genome, respectively. Preferably, in the rAAV genome of the present invention, the ITRs located at the 5 ′ end and the 3 ′ end include a 5 ′ ITR and a 3 ′ ITR included in the genome of AAV1, AAV2, AAV3, AAV4, AAV8 or AAV9. . Generally, since the ITR portion takes a sequence in which complementary sequences are easily replaced (flip and flop structure), the ITR contained in the rAAV genome of the present invention may be reversed in the 5 ′ and 3 ′ directions. . In the rAAV genome of the present invention, the length of the polynucleotide (ie, therapeutic gene) replaced with the internal region is preferably about the same as the length of the original polynucleotide. That is, the rAAV genome of the present invention is preferably about the same length as the wild type full length of 5 kb, for example, about 2 to 6 kb, preferably about 4 to 6 kb. The length of the therapeutic gene integrated into the rAAV genome of the present invention is preferably subtracted from the length of the transcriptional regulatory region including the promoter, polyadenylation, etc. (for example, assuming about 1 to 1.5 kb). The length is about 0.01 to 3.7 kb, more preferably about 0.01 to 2.5 kb, and even more preferably about 0.01 to 2 kb, but is not limited thereto.

  In general, a polynucleotide packaged in a recombinant adeno-associated virus vector may require time (several days) to express a target therapeutic protein when it is single-stranded. In this case, it is possible to design the introduced therapeutic gene so as to be a sc (self-complementary) type having self-complementarity, and to show the effect in a shorter time. Specific details are described in, for example, Foust KD, et al. (Nat Biotechnol. 2009 Jan; 27 (1): 59-65). The polynucleotide packaged in the rAAV vector of the present invention may be non-sc type or sc type.

  In one embodiment, an rAAV vector of the invention preferably comprises a polynucleotide comprising a neural cell specific promoter sequence and a therapeutic gene operably linked to the promoter sequence (ie, such a poly Nucleotides are packaged). As a promoter sequence used in the present invention, a promoter sequence specific to a nervous system cell is derived from, for example, a nerve cell, a glial cell, an oligodendrocyte, a cerebral vascular endothelial cell, a microglia, a ventricular epithelial cell, etc. However, it is not limited to these. Specific examples of such promoter sequences include synapsin I promoter sequence, glutamate decarboxylase (GAD65 / GAD67) promoter sequence, Tie2 promoter sequence (brain capillary specific promoter), myelin basic protein promoter sequence, neuron Promoters including specific enolase promoter sequences, glial fibrillary acidic protein promoter sequences, L7 promoter sequences (cerebellar Purkinje cell specific promoters), glutamate receptor delta2 promoters (cerebellar Purkinje cell specific promoters), It is not limited. In the rAAV vector of the present invention, promoter sequences such as calcium / calmodulin-dependent protein kinase II (CMKII) promoter, tubulin αI promoter, platelet-derived growth factor β chain promoter and the like can also be used. Furthermore, as a promoter used in the present invention, a tissue promoter containing nerve cells, for example, a promoter containing a polynucleotide sequence described in any of SEQ ID NOs: 17 to 19 relating to the enhancer / promoter of SLC2A1 can be used. These promoter sequences may be used alone or in any combination. Further, it may be a commonly used strong promoter sequence such as CMV promoter and CAG promoter. Particularly preferred promoter sequences in the present invention include 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), and Examples include promoters comprising the polynucleotide sequences set forth in SEQ ID NOs: 17-19. Furthermore, a known sequence such as an enhancer sequence that assists transcription of mRNA, translation into protein, Kozak sequence, an appropriate polyadenylation signal sequence and the like may be included.

  The therapeutic gene of interest incorporated into the rAAV genome of the present invention is delivered to the nervous system cell with high efficiency and is incorporated into the genome of that cell. When using the rAAV vector of the present invention, the number of nerves 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 with the case of using the conventional rAAV vector. It is possible to introduce a gene into a cell. The number of neurons into which the gene has been introduced can be determined by, for example, preparing an rAAV vector that packages an rAAV vector genome incorporating an arbitrary marker gene, then administering this rAAV vector to a test animal, and adding the rAAV vector genome to the rAAV vector genome. It can be measured by counting the number of neural cells that express the incorporated marker gene (or marker protein). Known marker genes can be selected. Examples of such marker genes include LacZ gene, green fluorescent protein (GFP) gene, photoprotein gene (firefly luciferase, etc.) and the like.

4). Other or additional means for improving the expression of glucose transporter 1 protein for other therapeutic genes include, for example, other proteins that control the expression and function of endogenous glucose transporter 1 protein, such as glucose on the membrane Nedd4 etc. that internalizes and inactivates transporter 1 may also be useful.

  For example, in order to suppress the function of Nedd4 that internalizes and inactivates glucose transporter 1 on the membrane, therapeutic genes incorporated into the rAAV genome of the present invention include antisense molecules, ribozymes, interfering RNA (iRNA), and microRNA. a polynucleotide for altering (eg, disrupting or reducing) the function of a target endogenous Nedd4 gene, such as (miRNA), or a poly for altering (eg, reducing) the expression level of an endogenous Nedd4 protein It may be a nucleotide. For example, in order to effectively inhibit the expression of a target gene using an antisense sequence, the length of the antisense nucleic acid is preferably 10 bases or more, 15 bases or more, 20 bases or more, and 100 bases or more. More preferably 500 bases or more. Usually, the length of the antisense nucleic acid used is shorter than 5 kb, preferably shorter than 2.5 kb.

By using a ribozyme, the mRNA of the target protein can be specifically cleaved to suppress the expression of the protein. For the design of such a ribozyme, various known literatures can be referred to (for example, FEBS Lett. 228: 228, 1988; FEBS Lett. 239: 285,
1988; Nucl. Acids. Res. 17: 7059, 1989; Nature 323: 349, 1986, etc.).

  “RNAi” is a phenomenon in which the expression of the introduced foreign gene and target endogenous gene is both suppressed when double-stranded RNA having the same or similar sequence as the target gene sequence is introduced into the cell. Point to. As RNA used here, for example, double-stranded RNA that causes RNA interference of 21 to 25 bases in length, such as dsRNA (double strand RNA), siRNA (small interfering RNA), shRNA (short hairpin RNA), or miRNA (microRNA). Such RNA can be locally delivered to a desired site by a delivery system such as a liposome, and can be locally expressed using a vector capable of generating the double-stranded RNA. Methods for preparing and using such double-stranded RNA (dsRNA, siRNA, shRNA or miRNA) are known from many literatures (Japanese Patent Publication No. 2002-516062; US Publication No. 2002 / 086356A); Nature Genetics, 24 (2), 180-183, 2000 Feb. etc.).

  In order to use these other therapeutic genes, for example, a known internal ribosome entry site (IRES) sequence can be interposed in the polynucleotide contained in the vector of the present invention. When the rAAV genome of the present invention is non-sc type, it is possible to select a promoter and a target gene having a wider range of length, and it is also possible to use a plurality of target genes. The total length of the polynucleotide packaged in the rAAV vector of the present invention is preferably about 5 kb or less (about 4.7 kb or less excluding the ITR region).

5. Preparation of rAAV vector of the present invention A general method can be used for preparing the rAAV vector of the present invention. For example, (a) a first polynucleotide encoding a capsid protein (generally, an AAV helper plasmid) And (b) transfecting the cultured cells with a second polynucleotide (including the therapeutic gene of interest) packaged in the rAAV vector of the present invention. The preparation method of the present invention further includes (c) a step of transfecting a cultured cell with a plasmid encoding an adenovirus-derived factor called an adenovirus (AdV) helper plasmid, or a step of infecting the cultured cell with adenovirus. Can be included. Furthermore, the step of culturing the above-described transfected cultured cells and the step of collecting the recombinant adeno-associated virus vector from the culture supernatant can also be included. Such a method is already known and used in the examples of the present 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. As such a promoter sequence, for example, cytomegalovirus (CMV) promoter, EF-1α promoter, SV40 promoter and the like can be appropriately used. Furthermore, a known enhancer sequence, Kozak sequence, poly A addition signal sequence and the like may be included as appropriate.

  The second polynucleotide (b) contains a therapeutic gene at a position operable with a neural cell specific promoter. Furthermore, a known enhancer sequence, Kozak sequence, poly A addition signal sequence and the like may be included as appropriate. This first polynucleotide may further comprise a cloning site cleavable by various known restriction enzymes downstream of the nervous system cell specific promoter sequence. A multi-cloning site containing a plurality of restriction enzyme recognition sites is more preferred. One skilled in the art can incorporate a therapeutic gene of interest downstream of a neural cell-specific promoter according to known genetic engineering procedures. See, for example, Molecular Cloning (Molecular Cloning 3rd Edition, J. Sambrook et al., Cold Spring Harbor Lab. Press. 2001) for such genetic manipulation procedures.

  In preparing the rAAV vector of the present invention, a helper virus plasmid (eg, adenovirus, herpes virus or vaccinia) can be used and introduced into cultured cells simultaneously with the first and second polynucleotides. Preferably, the preparation method of the present invention further comprises a step of introducing an adenovirus (AdV) helper plasmid. In the present invention, preferably, the AdV helper is derived from the same type of virus as the cultured cells. For example, when using human cultured cells 293T, a helper virus vector derived from human AdV can be used. As such an AdV helper vector, a commercially available one (for example, AAV Helper-Free System (catalog number 240071) manufactured by Agilent Technologies) can be used.

  In preparing the rAAV vector of the present invention, various known methods such as the calcium phosphate method, the lipofection method, and the electroporation method can be used as a method for transfection of one or more plasmids into cultured cells. it can. Such a method is described in, for example, Molecular Cloning 3rd Ed., Current Protocols in Molecular Biology, John Wiley & Sons 1987-1997.

6). Pharmaceutical Composition Comprising the rAAV Vector of the Present Invention The rAAV vector of the present invention can contain a gene that is useful for the treatment of neurological diseases, particularly diseases associated with abnormal protein function at synapses (eg, etc.). These genes can be incorporated into nerve cells of the brain, spinal cord, retina, for example, across the blood brain barrier. An rAAV vector containing such a therapeutic gene is included in the pharmaceutical composition of the present invention. As such a therapeutic gene, for example, a polynucleotide encoding the antibody, neurotrophic factor (NGF), growth factor (HGF), acidic fiber cell growth factor (aFGF) and the like as described above can be selected. It can be expected to treat such neurological diseases by peripheral administration of such an rAAV vector to a subject.

  The active ingredients of the pharmaceutical composition of the present invention may be blended singly or in combination. However, a pharmaceutically acceptable carrier or a pharmaceutical additive may be blended with the active ingredient and provided in the form of a pharmaceutical preparation. it can. In this case, for example, the active ingredient of the present invention can be contained in the preparation in an amount of 0.1 to 99.9% by weight.

  Examples of pharmaceutically acceptable carriers or additives include excipients, disintegrants, disintegration aids, binders, lubricants, coating agents, dyes, diluents, solubilizers, solubilizers, isotonic agents. Agents, pH adjusters, stabilizers and the like can be used. For oral administration, excipients such as microcrystalline cellulose, sodium citrate and calcium carbonate, disintegrants such as starch and alginic acid, granulating binders such as polyvinylpyrrolidone, lubricants, etc. are commonly used in the art. Can be used with excipients. When an aqueous suspension and / or elixir is desired for oral administration, the active ingredient is used in combination with various sweeteners or flavors, colorants or dyes, and if necessary, an emulsifier and / or suspending agent is also used. And can be used with water, ethanol, propylene glycol, glycerin, and the like, and diluents that combine them.

  Examples of preparations suitable for oral administration include powders, tablets, capsules, fine granules, granules, liquids or syrups. Examples of preparations suitable for parenteral administration include injections, intramedullary injections, suppositories and the like. In the case of parenteral administration, a solution in which the active ingredient of the present invention is dissolved in either sesame oil or peanut oil or dissolved in an aqueous propylene glycol solution can be used. The aqueous solution should be appropriately buffered as necessary (preferably pH 8 or more), and the liquid diluent must first be made isotonic. As such a liquid diluent, for example, physiological saline can be used. The prepared aqueous solution is suitable for intravenous injection, while the oily solution is suitable for intra-articular, intramuscular and subcutaneous injection. All these solutions can be prepared aseptically by standard pharmaceutical techniques well known to those skilled in the art. Furthermore, the active ingredient of the present invention can also be administered locally such as on the skin. In this case, topical administration in the form of creams, jellies, pastes, ointments is desirable according to standard pharmaceutical practice.

The dose of the pharmaceutical composition of the present invention is not particularly limited, and an appropriate dose is selected according to various conditions such as the type of disease, the age and symptoms of the patient, the route of administration, the purpose of treatment, the presence or absence of a concomitant drug, etc. Is possible. 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 (for example, body weight 60 kg), but is not limited thereto. These daily doses may be administered in two to four divided doses. When vg (vector genome) is used as a dosage unit, for example, administration in the range of 10 ⁹ to 10 ¹⁴ vg, preferably 10 ¹⁰ to 10 ¹³ vg, more preferably 10 ¹⁰ to 10 ¹² vg per kg body weight. The amount can be selected, but is not limited thereto.

7). Administration of the rAAV vector of the present invention The rAAV of the present invention is capable of passing through the body's blood brain barrier (including the unfinished fetal and neonatal blood brain barrier, and the established adult blood brain barrier). By peripheral administration to adults and fetuses or neonates), the rAAV of the present invention can be delivered to nervous system cells such as the brain and spinal cord. Furthermore, the rAAV vector used in the present invention can target nerve cells contained in the adult brain, spinal cord and the like by peripheral administration. In the present specification, peripheral administration means peripheral administration to those skilled in the art, such as intravenous administration, intraarterial administration, intraperitoneal administration, intracardiac administration, intramuscular administration, and intravascular administration of umbilical cord (for example, when targeting a fetus). Refers to the route of administration normally understood as administration. In addition to blood, administration to a site fluidly connected to the brain, such as intrathecal administration, can also be used for the rAAV vector of the present invention. In another embodiment, the rAAV vector of the invention can be administered locally to a target site in the brain, such as the hippocampus. For example, when the rAAV of the present invention is administered into the cerebrospinal fluid by intrathecal administration or into the blood by peripheral administration, a simpler administration means can be provided as compared with intracerebral administration.

8). Preparation kit of rAAV vector of the present invention In another embodiment, the present invention provides a kit for producing the rAAV of the present invention. Such a kit can include, for example, (a) a first polynucleotide for expressing capsid protein VP1 and the like, and (b) a second polynucleotide packaged in an rAAV vector. For example, the first polynucleotide comprises a polynucleotide encoding the amino acid of SEQ ID NO :. For example, the second polynucleotide may or may not contain the therapeutic gene of interest, but preferably may contain various restriction enzyme cleavage sites for incorporating such therapeutic gene of interest. .

  The kit for producing the rAAV vector of the present invention can further include any configuration described herein (for example, AdV helper and the like). The kits of the present invention may also further comprise instructions describing a protocol for generating rAAV vectors using the kits of the present invention.

9. About the chemotherapeutic agent used together with rAAV of this invention The rAAV vector which concerns on this invention can also be used together with the conventional treatment method. Examples of such treatment methods include ketogenic diets or common antiepileptic drugs (eg, valproproic acid, clonazepam, and combinations thereof). For example, after administration of the rAAV of the present invention, it can be expected that the above-mentioned ketogenic diet is unnecessary or that the type and dosage of antiepileptic drugs are greatly reduced.

10. Regarding measurement of therapeutic effect The therapeutic effect of the rAAV vector of the present invention is determined by using various known measuring means related to the action of GLUT1. Such publicly known means include, for example, motor function evaluation and WISC by Gross Motor Function Classification System, coarse motor function classification system, etc.
(Wechsler Intelligence Scale for Children) and the like, and examples include, but are not limited to, improvement of blood / cerebral spinal fluid glucose concentration ratio.

11. The meaning of each term used in the present specification for the term in the present specification is as follows. In the present specification, terms that are not particularly explained are intended to indicate the meanings of terms that are normally understood by those skilled in the art.

  As used herein, unless otherwise stated, the terms “viral vector”, “viral virion”, and “viral particle” are used interchangeably.

  As used herein, “nervous system” refers to an organ system composed of neural tissue. In addition, as used herein, “neural cells” include at least neurons contained in the central nervous system such as the brain and spinal cord, and further include glial cells, microglia, astrocytes, It may include oligodendrocytes, ventricular ependymocytes, cerebral vascular endothelial cells and the like.

  As used herein, the term “polynucleotide” is used interchangeably with “nucleic acid”, “gene” or “nucleic acid molecule” and is intended to be a polymer of nucleotides. As used herein, the term “nucleotide sequence” is used interchangeably with “nucleic acid sequence” or “base sequence” and refers to the sequence of deoxyribonucleotides (abbreviated A, G, C, and T). As shown. For example, “a polynucleotide comprising a nucleotide sequence of SEQ ID NO: 1 or a fragment thereof” intends a polynucleotide comprising the sequence represented by each deoxynucleotide A, G, C and / or T of SEQ ID NO: 1 or a fragment thereof. Is done.

  Each of the “viral genome” and “polynucleotide” according to the present invention may exist in the form of DNA (eg, cDNA or genomic DNA), but in some cases may be in the form of RNA (eg, mRNA). As used herein, viral genomes and polynucleotides can each be double-stranded or single-stranded DNA. In the case of 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). Unless otherwise stated, in this specification, when the arrangement on a gene such as a promoter, target gene, polyadenylation signal, etc. encoded by the rAAV genome is described, when the rAAV genome is a sense strand, the strand itself In the case of an antisense strand, the complementary strand is described.

  In the present specification, “protein” and “polypeptide” are used interchangeably, and a polymer of amino acids is intended. The polypeptide used in the present specification has the N-terminus (amino terminus) at the left end and the C-terminus (carboxyl terminus) at the right end in accordance with the convention of peptide designation. The partial peptide of the polypeptide of the present invention (in the present specification, sometimes abbreviated as the partial peptide of the present invention) is the partial peptide of the polypeptide of the present invention described above, preferably the polypeptide of the present invention described above. It has the same properties as a peptide.

  In the present specification, the term “plasmid” means various known genetic elements such as plasmids, phages, transposons, cosmids, chromosomes and the like. A plasmid can replicate in a particular host and transport gene sequences between cells. As used herein, a plasmid includes various known nucleotides (DNA, RNA, PNA and mixtures thereof) and may be single-stranded or double-stranded, but preferably is double-stranded. . For example, as used herein, the term “rAAV vector plasmid” is intended to include the duplex formed by the rAAV vector genome and its complementary strand, unless otherwise specified. The plasmid used in the present invention may be linear or circular.

  As used herein, the term “packaging” includes events that include preparation of a single-stranded viral genome, assembly of the coat protein (capsid), encapsidation of the viral genome, and the like. Say. When an appropriate plasmid vector (usually multiple plasmids) is introduced into a cell line that can be packaged under appropriate conditions, recombinant viral particles (ie, viral virions, viral vectors) are assembled and put into culture. Secreted.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited to the following examples.

(1) Creation of a system that demonstrates that the causative gene SLC2A1 causes disease
For evaluation of SLC2A1 gene expression, normal SLC2A1 gene expression vector and mutant vector were introduced into human fetal kidney cells (HEK293 cells), and protein expression and subcellular localization were observed by cell immunostaining and Western blotting 48 hours after gene introduction did. As a normal SLC2A1 gene expression vector, a myc-DDK (FLAGR) tagged SLC2A1 expression vector (RC222696; OriGene Technologies, Rockville, MD) was used. Mutant vectors were selected from known missense mutations as mild (A405D) and moderate (R333W) (Leen WG et al., Brain 133 (2010) 655-670). (Nakamura S. et al., Molecular Genetics and Metabolism 116 (2015) 157-162). In addition, an empty vector (SLC2A1 (-)) was prepared by removing the SLC2A1 gene and the tag sequence from the normal SLC2A1 gene expression vector and used as a control. As primary antibodies, c-myc antibody (9E10; Santa Cruz Biotechnology, Santa Cruz, CA) and GLUT1 antibody (N-terminus; OriGene Technologies, Rockville, MD) were used. Furthermore, as a function evaluation after gene introduction, the intracellular glucose uptake capacity via GLUT was evaluated using a 2-deoxyglucose (2DG) metabolic rate measurement kit (COSMO BIO Co. Ltd., Tokyo, Japan). (Saito K, et al., Anal. Biochem. 412 (2011) 9-17).

SDS-PAGE and Western blotting were performed using lysates of HEK293 cells into which normal type (wild type: Wt) and each mutant SLC2A1 gene were introduced. Although SLC2A1 protein expression could be confirmed in wild type and missense mutation-introduced cells, protein expression was not observed in the frameshift mutation. Cell immunostaining with GLUT1 antibody (N-terminus) and myc antibody (C-terminus)
The cell membrane was stained in the cells into which the type and missense mutations were introduced, but the cell membrane was not stained in the frameshift mutation. Since the plasmid containing the frameshift mutation has a stop codon sequence located before the tag protein, a plasmid containing the tag protein (V303fs without stop codon) except for the stop codon was newly created. Staining was performed, but SLC2A1 protein expression was not observed, and cell membrane was not stained by cell immunostaining (FIG. 1). In the 2DG uptake test, the sugar uptake ability decreased in the order of introduction of Wild type>A405D>R333W>V303fs> empty vector, which correlated with the severity of clinical symptoms. (Figure 2)

(2) Preparation of viral vector for SLC2A1 expression As a joint research with our department of neurology, myc-DDK-tagged SLC2A1 gene (Catalog No. RC222696, ORIGENE) was incorporated into AAV vector as an insert DNA, and therapeutic AAV9- SLC2A1 was created. AAV vector is described in International Publication No. 2012/057363, type 9 vector (mutated capsid protein derived from AAV9, ITR sequence derived from AAV3) that has been confirmed to be well-transfected into the brain by intravenous administration in experimental animals Etc.) were used. GLUT1 is mainly expressed in brain tissue and erythrocytes, but has been confirmed to be expressed in vascular endothelial cells and nerve cells in mouse brain tissue (Choeori C et al, 2002). In this study, the synapsin 1 promoter (SynI promoter), which is a neuron-specific promoter, was used to increase the expression efficiency in neurons (Fig. 3).

Next, after adding AAV9-SLC2A1 to cultured cells of human nervous system, it was examined whether SLC2A1 protein was expressed. Human neuroblastoma-derived cells (SH-SY5Y cells) 24 hours after seeding with 1 × 10 ⁵ cells / well, 3 × 10 ⁹ vg of AAV9-SLC2A1 was added. Cell immunostaining was performed 40 hours after the addition of AAV9-SLC2A1. As the primary antibody, c-myc antibody (9E10; Santa Cruz Biotechnology, Santa Cruz, CA) and GLUT1 antibody (N-terminus; OriGene Technologies, Rockville, MD) were used. Cellular immunostaining confirmed SH-SY5Y cells that were positive for myc staining near the cell membrane. In co-staining with GLUT1 antibody, it was confirmed at the cell level that myc positive region and GLUT1 were consistently and strongly expressed (FIG. 4).

(3) Administration of viral vectors to disease model animals (mice)
GLUT1 knockout mice heterozygous (GLUT1 (+/-) mice) have hypoglycemia, cerebrospinal fluid sugar, ataxia, convulsive seizures, etc. and are considered GLUT1 deficiency model mice with similar symptoms to human GLUT1 deficiency (GLUT1 (-/-) mice are embryonic lethal). Mice were given a frozen fertilized egg produced in the Department of Drug Delivery, Graduate School of Pharmaceutical Sciences, Tohoku University as a joint research and bred at this hospital. As for the administration method of AAV9-SLC2A1, neonatal mice around 1 week after birth are vulnerable to the brain blood barrier, and intraperitoneal administration to neonatal mice is based on the high rate of drug transfer into the brain even when administered intraperitoneally. Selected. Day-old 7 GLUT1 (+/-) mice the AAV9-SLC2A1 administered 1.8 × 10 ¹¹ vg intraperitoneal and brain taken at 6 weeks of age, to confirm protein expression in tissue immunostaining (Fig. 5 and Fig. 6). As the primary antibody, myc-tag antibody (562; MBL CO., Ltd, Nagoya, Japan) was used. At the same time, RNA extraction and cDNA synthesis were performed from brain tissue, and expression was confirmed by RT-PCR (FIG. 7). As for RT-PCR primers, one was set on the SLC2A1 cDNA and the other on the myc tag sequence in the vector specific (SEQ ID NOs: 10 and 11, respectively). In addition, for the measurement of the total amount of the SLC2A1 transcript, primers having the sequences described in SEQ ID NOs: 12 and 13 were used. Also. GLUT1 (+/-) mice were injected with AAV9-SLC2A1 into the bilateral ventricles for protein expression evaluation and functional evaluation. In addition, electrophysiological microelectrodes (sharp glass) were placed on the bilateral ventricles of GLUT1 (+/-) mice at 6 weeks of age (corresponding to the average age at diagnosis in humans).
electrode) to inject AAV9-SLC2A1 at 9.3 x 109 vg per side (1.8 x 1010 vg on both sides). Brains are collected 8 weeks after treatment and protein expression is confirmed by tissue immunostaining and Western blotting. At the same time, cDNA is synthesized from RNA extracted from the cerebrum, RT-PCR is performed using vector-specific primers (SEQ ID NOs: 14 and 15), expression confirmation band intensity measurement and real-time PCR quantification at the mRNA level (probe for quantification) The expression level of GLUT1-RNA was compared in SEQ ID NO: 16). As functional evaluation, GLUT1 (+/-) mice have been reported to have decreased motor function (Wang D et al., 2006), so Rota-rod test and Footprint test were performed 4 and 8 weeks after treatment ( FIG. 8).

Results In tissue immunostaining, exogenous GLUT1-positive cells (GLUT1-myc-positive cells) were strongly expressed in the cerebral cortex and hippocampus around the lateral ventricle, which is the tissue around the administration (FIGS. 5 and 6). GLUT1-myc positive cells are β-III-Tubulin positive, a neuronal marker, and GFAP, an astrocyte marker, and CD31, a vascular endothelial cell marker, are negative. Confirmed to do. Expression and expression level of mRNA levels were confirmed by intraperitoneal and intraventricular administration by vector-specific RT-PCR using RNA extracted from mouse cerebrum (FIG. 7). Quantitative expression of GLUT1 mRNA in real-time PCR and GLUT1 +/- mouse expression levels of 1.048 ± 0.22 (n = 3), whereas GLUT1 +/- mice after intraventricular administration were 2.428 ± 0.36 (n = 3, P <0.05) ) And increased.

In addition, the Rota-rod test was performed in the GLUT1 +/- mice after intraventricular administration, compared to 116 ± 26 seconds (n = 6) in the control GLUT1 +/- mice at 10 weeks of age, 4 weeks after treatment. (Ht-AAV icv), 164 ± 28 seconds (n = 7, P = 0.24). Furthermore, assuming clinical application, untagged AAV9-hSLC2A1 was prepared, and a Rota-rod test was performed 4 weeks after intraventricular administration (Ht-AAV ^* icv), 226 ± 13 seconds (n = 4 , P <0.05) and a significant improvement was observed (FIG. 8; left). As shown in FIG. 8, the wild type (Wt: GLUT1 + / +) was 188 ± 20 (n = 10). The same improvement effect was obtained by intraperitoneal administration, but the effect tended to be weaker than that by intrathecal administration (FIG. 8; right). In addition, in the case of intrathecal administration, the amount of vg to be administered was small, and the gene transfer effect could be confirmed in a shorter period.

Discussion A proof of concept by treatment with rAAV for glucose transporter 1 deficiency syndrome according to the present invention was shown. It is expected that many hereditary disease treatments will be sent from Japan as a prelude to this disease treatment.

(4) Preparation of plasmid vector for SLC2A1 expression
GLUT1 is also expressed in neurons and non-neural cells such as vascular endothelial cells and glial cells that constitute the brain and central nervous system (F. Maher, SJ Vannucci, IA Simpson, Glucose transporter proteins in brain, FASEB. J. 8 (1994) 1003-1011.). Therefore, we worked on the construction of vectors that can be expressed in both types of cells.
A CMV promoter incorporated in a commercially available plasmid vector for SLC2A1 expression (catalog No. RC222696, ORIGENE) is a promoter A (SEQ ID NO: 17), promoter B (SEQ ID NO: 18) sequence or promoter A + B (SEQ ID NO: 19). A replacement for the sequence was made. The prepared plasmid was introduced into various cell lines to confirm the expression of SLC2A1. As controls, those containing the original CMV promoter and those without the promoter were used.
The above promoter A, promoter B and promoter A + B were separately cloned by the following procedure. Promoter region strongly involved in SLC2A1 expression in rats (Hwang DY, Ismail-Beigi F. Stimulation of GLUT-1 glucose transporter expression in response to hyperosmolarity. Am J Physiol Cell Physiol. 2001 Oct; 281 (4): C1365-72. ), A region on the human genome with high nucleotide sequence homology (about 70%) was identified. This identified promoter B (SEQ ID NO: 18), promoter A (SEQ ID NO: 17) and promoter A + B (SEQ ID NO: 19) further including the upstream region thereof were uniquely cloned, and each polynucleotide sequence was specified.

  The prepared SLC2A1 expression plasmid vector was used as MO3.13 cells (glia cells; non-neural system), SH-SY5Y cells (human neuroblastoma cells: nervous system), and HUVEC cells (human umbilical vein endothelial cells: vascular endothelial system). And the expression of SLC2A1 was detected using an anti-Myc tag antibody bound to SLC2A1 (FIGS. 9-1 to 3). The cells were subjected to Hoechst staining.

result
Expression of SLC2A1 promoter A, promoter B, and promoter A + B vectors was observed in MO3.13 cells and SH-SY5Y cells (FIGS. 9-1 and 9-2). Therefore, it was confirmed that combining promoter A, promoter B and promoter A + B with the GLUT1 gene resulted in expression closer to that in vivo than a vector using not only the CMV promoter but also the SynI promoter.
Therefore, the recombinant AAV vector combining promoter A, promoter B and promoter A + B and the GLUT1 gene can express GLUT1 in neurons, non-neuronal cells such as glial cells, and vascular cells. It can be expected to provide a high therapeutic effect.

  By using the rAAV vector of the present invention, glucose transporter 1 deficiency syndrome caused by genetic defects (including congenital and acquired) in nervous system cells is treated (palliated, improved, repaired, etc.) ) Can be expected.

SEQ ID NO: 1 human glutamine transporter 1 nucleotide sequence (NM_006516)
SEQ ID NO: 2: human glutamine transporter 1 amino acid sequence (NP_006507)
SEQ ID NO: 3: Nucleotide sequence of tagged human glutamine transporter 1 SEQ ID NO: 4: Amino acid sequence of tagged human glutamine transporter 1 SEQ ID NO: 5: Mouse glutamine transporter 1 amino acid sequence (NP_035530.2)
SEQ ID NO: 6 rat glutamine transporter 1 amino acid sequence (NP_620182.1)
SEQ ID NO: 7: AAV1 capsid protein Y445F variant amino acid sequence SEQ ID NO: 8: AAV2 capsid protein Y444F variant amino acid sequence SEQ ID NO: 9: AAV9 capsid protein Y446F variant amino acid sequence SEQ ID NO: 10: Detection primer 1 nucleotide sequence sequence Number 11: Detection primer 2 nucleotide sequence SEQ ID NO 12: Detection primer 3 nucleotide sequence SEQ ID NO 13: Detection primer 4 nucleotide sequence SEQ ID NO 14: Detection primer 5 nucleotide sequence SEQ ID NO 15: Detection primer 6 nucleotide sequence sequence No. 16: Detection probe nucleotide sequence SEQ ID NO: 17: SLC2A1 promoter A nucleotide sequence SEQ ID NO: 18: SLC2A1 promoter B nucleotide sequence SEQ ID NO: 19: SLC2A1 promoter A + B nucleotide sequence

Claims (10)

  A recombinant adeno-associated virus vector for the treatment of glucose transporter 1 deficiency syndrome comprising a polynucleotide encoding a glucose transport protein.   The polynucleotide according to claim 1, wherein the polynucleotide comprises the amino acid sequence of SEQ ID NO: 2 or 4, or a polynucleotide encoding an amino acid sequence having about 90% identity with these sequences and having glucose transport ability. Recombinant adeno-associated virus vector.   The recombinant adeno-associated virus vector is a protein having a mutant amino acid sequence in which tyrosine at position 445 in the amino acid sequence of wild-type AAV1 capsid protein is substituted with phenylalanine; tyrosine at position 445 in the amino acid sequence of wild-type AAV2 capsid protein A protein having a mutated amino acid sequence in which is substituted with phenylalanine, or a protein having a mutated amino acid sequence in which tyrosine at position 446 in the amino acid sequence of the wild-type AAV9 capsid protein is substituted with phenylalanine An adeno-associated virus recombinant vector according to 1.   The polynucleotide comprises synapsin I promoter sequence, myelin basic protein promoter sequence, neuron specific enolase promoter sequence, calcium / calmodulin-dependent protein kinase II (CMKII) promoter sequence, tubulin αI promoter sequence, platelet derived growth factor β chain Promoter sequence, glial fibrillary acidic protein (GFAP) promoter sequence, L7 promoter sequence (cerebellar Purkinje cell specific promoter), glial fibrillary acidic protein (hGfa2) promoter sequence, and glutamate receptor delta2 promoter (cerebellar Purkinje cell specific promoter) ) Sequence, glutamate decarboxylase (GAD65 / GAD67) promoter sequence, polynucleotide sequence of SEQ ID NO: 17, polynucleotide sequence of SEQ ID NO: 18, and sequence of SEQ ID NO: 19. Comprising a promoter sequence selected from the group consisting of nucleotide sequences, recombinant adeno-associated virus vector according to any of claims 1 to 3.   The recombinant adeno-associated virus according to any one of claims 1 to 4, wherein the polynucleotide comprises an inverted terminal repeat (ITR) selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV8, and AAV9. vector.   A pharmaceutical composition comprising the recombinant adeno-associated virus recombinant vector according to any one of claims 1 to 5.   The pharmaceutical composition according to claim 6, for administration in the brain.   7. A pharmaceutical composition according to claim 6 for intrathecal administration.   The pharmaceutical composition according to claim 6, for peripheral administration.   10. A pharmaceutical composition according to claims 6-9 for use with a ketone diet.