Pharmaceutical composition for treatment or prevention of rhobtb2 associated neurodevelopmental disorder

Abstract

An object of the present invention is to provide a pharmaceutical composition for treating or preventing RhoBTB2 neurodevelopmental disorder.SOLUTION: The present invention also provides a pharmaceutical composition for treating or preventing RhoBTB2 associated neurodevelopmental disorders, comprising an agent that suppresses or inhibits RhoBTB2 gene function as an active ingredient; a method for treating or preventing RhoBTB2 associated neurodevelopmental disorders in animals other than humans, wherein epileptic seizures are suppressed by suppressing or inhibiting RhoBTB2 gene function; and a method for treating or preventing RhoBTB2 associated neurodevelopmental disorders in animals other than humans, wherein epileptic seizures are suppressed by suppressing or inhibiting RhoBTB2 gene function.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a pharmaceutical composition for treating or preventing a rare disease, RhoBTB2-associated neurodevelopmental disorder. [Background technology]

[0002] RhoBTB2-associated neurodevelopmental disorders are autosomal dominant genetic disorders caused by mutations in RhoBTB2. Since 2018, 99 cases have been reported worldwide and 6 cases in Japan, making the number of patients rare (Non-Patent Document 1). In addition to intellectual disability and developmental delay, patients with these genetic disorders are also prone to a variety of neurological symptoms, including epilepsy, paralysis, encephalopathy, and movement disorders such as dystonia, which can occur episodically or suddenly (Non-Patent Documents 2 and 3). Currently, only symptomatic treatments for epilepsy and other conditions are available, and no effective treatment has yet been established. RhoBTB2-associated neurodevelopmental disorders are therefore severe, rare, and intractable diseases with no cure.

[0003] RhoBTB2 is a protein containing a GTPase and two BTB domains, and in patients with RhoBTB2-associated neurodevelopmental disorders, specific amino acid mutations are concentrated in the BTB domain region, suggesting that the effects of specific mutations are due to these mutations (Non-Patent Document 4). On the other hand, in addition to mutations in regions other than the BTB domain, cases with biallelic loss-of-function mutations have also been found (Non-Patent Document 5), and it has been reported that complete loss of function can also cause the disease.

[0004] To develop treatments, a step-by-step strategy is required to elucidate the molecular pathogenesis of each causative gene and develop treatments based on that knowledge. However, very little research has been reported to elucidate the molecular pathogenesis of RhoBTB2-associated neurodevelopmental disorders. In addition, conventional small molecule drugs require long development times and enormous costs, making the development of small molecule drugs for ultra-rare diseases such as RhoBTB2-associated neurodevelopmental disorders difficult from a cost-effectiveness perspective.

[0005] Meanwhile, in recent years, the development of nucleic acid drugs has been active. Nucleic acid drugs can target mRNA, which cannot be targeted with small molecule drugs. In Japan, siRNA has been approved as a treatment for hereditary ATTR amyloidosis and acute hepatic porphyria, and is being used in the field of rare disease treatment. Nucleic acid drugs generally have fewer side effects than small molecule drugs, and also have the advantage of lower manufacturing and quality control costs than antibody drugs. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Belal et al, Human Mutation, 2018, vol.39, p.1070-1075. [Non-patent document 2] Straub et al, American Journal of Human Genetics, 2018, vol.102, p.1-14. [Non-patent document 3] Zagaglia et al, Neurology, 2021, vol.96(11), article: e1540. [Non-patent document 4] Ji and Rivero, Cells, 2016, vol.5, article: 28. [Non-Patent Document 5] Langhammer et al., Genetics in Medicine, 2023, vol.25(8), article:100885. [Non-patent document 6] Aoto et al, Nature Communications, 2021, vol.12(1), article: 2107. [Non-Patent Document 7] Kobayashi et al,Cells,2020,vol.9(4), article: 957. [Non-patent document 8] Racine, Electroencephalography and Clinical Neurophysiology, 1972, vol.32(3), p.281-294. Summary of the Invention [Problem to be solved by the invention]

[0007] A primary object of the present invention is to provide a pharmaceutical composition for treating or preventing RhoBTB2-associated neurodevelopmental disorders. [Means for solving the problem]

[0008] Through analysis using RhoBTB2 gene knock-in mice and expressing cells into which the RhoBTB2 gene has been introduced, the inventors have revealed that RhoBTB2 mutations observed in patients commonly result in reduced Na / K-ATPase (NKA) α1, and that mutations in the BTB domain region increase RhoBTB2 protein expression.They have also found that by reducing RhoBTB2 gene expression by RNA interference, NKAα1 increases in model mice in which a mutation in the BTB domain region (R489Q) has been knocked in, and that therapeutic effects such as suppression of epileptic seizures and weight loss can be expected, thereby completing the present invention.

[0009] That is, the present invention is as follows. [1] A pharmaceutical composition for treating or preventing RhoBTB2-associated neurodevelopmental disorders, containing as an active ingredient a substance that suppresses or inhibits the function of the RhoBTB2 gene. [2] The pharmaceutical composition according to [1] above, wherein the substance that suppresses or inhibits the function of the RhoBTB2 gene is a nucleic acid that targets the RhoBTB2 gene and suppresses its expression by RNA interference. [3] The pharmaceutical composition according to [2] above, wherein the substance that suppresses or inhibits the function of the RhoBTB2 gene is an siRNA that targets the RhoBTB2 gene. [4] The pharmaceutical composition according to [2] above, wherein the substance that suppresses or inhibits the function of the RhoBTB2 gene is an adeno-associated virus vector carrying shRNA that targets the RhoBTB2 gene. [5] The pharmaceutical composition according to [2], wherein the nucleic acid is a nucleic acid that targets a region in the RhoBTB2 gene consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 4 and suppresses expression by RNA interference. [6] The pharmaceutical composition according to any one of [1] to [5] above, which is administered by intraventricular administration, intraspinal fluid administration, or intravenous administration. [7] The pharmaceutical composition according to any one of [1] to [6] above, which is administered to a human having one or more mutations in the RhoBTB2 gene selected from the group consisting of D92H mutation, W217C mutation, R461H mutation, R485C mutation, and R489Q mutation. [8] A method for treating or preventing RhoBTB2-associated neurodevelopmental disorders in animals other than humans, which comprises suppressing epileptic seizures by suppressing or inhibiting the function of the RhoBTB2 gene. [9] The method according to [8] above, wherein the function of the RhoBTB2 gene is suppressed or inhibited by inhibiting the expression of the RhoBTB2 gene through RNA interference. [Effects of the Invention]

[0010] The present invention can suppress epileptic seizures and weight loss caused by increased RhoBTB2 protein expression due to RhoBTB2 gene mutations, and is therefore useful for treating or preventing RhoBTB2-associated neurodevelopmental disorders. [Brief explanation of the drawings]

[0011] [Figure 1] This is a schematic diagram showing the state of genetic modification in Rhobtb2R489QKI mice and Rhobtb22×HA-R489Q double knock-in (DKI) mice, which have a 2×HA tag inserted to enable highly sensitive detection of Rhobtb2. [Figure 2]FIG. 1 shows changes over time in body weight and survival rate (%) of Rhobtb2R489QKI mice and Rhobtb22×HA-R489QDKI mice. [Figure 3] FIG. 3 shows the results of immunoblotting for HA-RhoBTB2 in DKI mice (homozygous and heterozygous) and wild-type mice on embryonic day 14, postnatal day 7, and postnatal day 21. [Figure 4] Figure 4 shows the results of measuring the average number of seizures per day (Figure 4(A)) and the seizure score (Figure 4(B)) for homozygous KI mice and wild-type mice. [Figure 5] FIG. 1 shows the results of measuring the relative expression levels of NKAα1 and NKAα3 (wild-type expression level is set to 1) in DKI mice (homozygous and heterozygous) and wild-type mice on day 21 after birth. [Figure 6] Figure 6(A) shows a schematic diagram of the location of each mutation in the RhoBT2 protein of each mutant used in the cell model analysis. Figure 6(B) shows the results of measuring the ratio of NKAα1 to GAPDH expression ([NKAα1 expression level] / [GAPDH expression level]) in cells expressing the mutant Flag fusion proteins by doxycycline (Dox). Figure 6(C) shows the results of measuring ATP levels in cells expressing the wild-type, Y284D mutant, and R489Q mutant of RhoBTB2. [Figure 7] Figure 7 shows the results of measuring the expression level of RhoBTB at the mRNA level (Figure 7(A)) and protein level (Figure 7(B)) 48 hours after siRNA administration in cells expressing wild-type or R489Q mutant Flag fusion proteins of RhoBTB2. [Figure 8] Figure 8(A) shows the relationship between RhoBTB2 protein expression levels and body weight (Figure 8(A)) and NKAα1 levels (Figure 8(B)) in Rhobtb22×HA-R489Q homozygous mice and Rhobtb22×HA-WT homozygous mice administered intracerebroventricularly with an AAV9 viral vector carrying #357. [Figure 9] FIG. 10 shows time-dependent changes in survival rates of Rhobtb22×HA-R489Q homozygous mice and Rhobtb22×HA-WT homozygous mice administered intracerebroventricularly with AAV9 viral vector carrying #357. DETAILED DESCRIPTION OF THE INVENTION

[0012] The pharmaceutical composition of the present invention contains as an active ingredient a substance that suppresses or inhibits the function of the RhoBTB2 gene and is used for the treatment or prevention of RhoBTB2-associated neurodevelopmental disorders. RhoBTB2-associated neurodevelopmental disorders are diseases in which mutant proteins produced in the brain due to RhoBTB2 gene mutations cause epileptic or sudden movement disorders such as epilepsy, paralysis, encephalopathy, and dystonia. RhoBTB2 mutations identified in patients with neurodevelopmental disorders include D92H, W217C, R461H, R485C, and R489Q. The pharmaceutical composition of the present invention reduces the amount of RhoBTB2 mutant proteins in the brain, thereby reducing the frequency and severity of movement disorders such as epilepsy and providing therapeutic or preventive effects. The inventors of the present invention were the first to discover that reducing the expression level of RhoBTB2 can improve the pathology of RhoBTB2-associated neurodevelopmental disorders and potentially provide therapeutic benefits.

[0013] Suppression or inhibition of the function of the RhoBTB2 gene can be achieved by inhibiting the expression of the RhoBTB2 gene. Specifically, substances that suppress or inhibit the function of the RhoBTB2 gene, which are active ingredients of the pharmaceutical composition of the present invention, include substances that suppress the expression of the RhoBTB2 gene itself through RNA interference or other mechanisms. Examples of such substances include small interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA), which have a double-stranded structure consisting of the sense and antisense strands of a partial region of the cDNA of the RhoBTB2 gene (the RNAi (RNA interference) target region). Alternatively, the substance may be an RNAi-inducing vector that can produce siRNA or other nucleotides in target cells in the brain. siRNA, shRNA, miRNA, and RNAi-inducing vectors can be designed and produced by standard methods based on the cDNA sequence of the target RhoBTB2 gene. Alternatively, RNAi-inducing vectors can be prepared by inserting the nucleotide sequence of the RNAi target region into the nucleotide sequence of various commercially available RNAi vectors, such as those using adeno-associated virus vectors.

[0014] When the active ingredient of the pharmaceutical composition of the present invention is a substance that suppresses the expression of the RhoBTB2 gene itself by RNA interference, the target region of the RhoBTB2 gene for RNA interference is preferably a region consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4, and a region consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 1 or 2 is particularly preferred because it provides a particularly high expression-suppressing effect. These nucleotide sequences are shown in Table 1 below. By using as an active ingredient a nucleic acid that targets a region in the RhoBTB2 gene consisting of the nucleotide sequence represented by any one of SEQ ID NOs: 1 to 4 and suppresses expression by RNA interference, a high therapeutic effect against RhoBTB2-associated neurodevelopmental disorders can be expected.

[0015] The active ingredient of the pharmaceutical composition of the present invention may be a substance that suppresses or inhibits the decrease in ATP and Na / K-ATPase (NKA) caused by RhoBTB2 protein by directly or indirectly binding to the RhoBTB2 protein. Such a substance is not particularly limited and may be any of a nucleic acid, a peptide, a protein, or a low-molecular-weight compound.

[0016] An example of a protein that binds to RhoBTB2 protein and suppresses or inhibits the decrease in NKA caused by RhoBTB2 protein is an anti-RhoBTB2 antibody. Such an antibody may be a monoclonal antibody or a polyclonal antibody. It may also be an artificially synthesized antibody such as a chimeric antibody, a single-chain antibody, or a humanized antibody. These antibodies can be produced by conventional methods.

[0017] The active ingredient of the pharmaceutical composition of the present invention may be a substance that degrades the RhoBTB2 protein. Degradation of the RhoBTB2 protein itself inhibits its function. The substance that degrades the RhoBTB2 protein may be a degrading enzyme that directly degrades the RhoBTB2 protein, or may be a substance that labels the RhoBTB2 protein with various labels, such as polyubiquitin, so that it becomes a substrate for the protease.

[0018] The pharmaceutical composition of the present invention may contain various additives in addition to the active ingredient, a substance that suppresses or inhibits the function of the RhoBTB2 gene. Examples of such additives include excipients, binders, lubricants, wetting agents, solvents, disintegrants, solubilizers, suspending agents, emulsifiers, isotonicity agents, stabilizers, buffers, preservatives, antioxidants, flavoring agents, and coloring agents. These additives can be appropriately selected from pharmaceutically acceptable substances that are used in pharmaceutical formulations.

[0019] The pharmaceutical composition of the present invention can be formulated by conventional methods into dosage forms suitable for various administration routes, such as liquids, suspensions, injections, sprays, tablets, powders, granules, capsules, chewable tablets, and syrups.

[0020] The method of administration of the pharmaceutical composition of the present invention is not particularly limited as long as it is in a dosage form suitable for an administration method that allows the active ingredient to be delivered so that it functions in target cells in the brain. As the method of administration of the pharmaceutical composition of the present invention, particularly preferred are administration methods in which the active ingredient is administered directly into the brain, such as intracerebrospinal fluid administration (spinal fluid administration) and intraventricular administration, but intravenous injection (intravenous administration) is also acceptable. Even when administered intravenously, the active ingredient can be delivered into the brain by utilizing transporters that pass through the blood-brain barrier, such as insulin and transferrin.

[0021] By administering the pharmaceutical composition of the present invention to humans or non-human animals and suppressing or inhibiting the function of the RhoBTB2 gene, movement disorders such as epilepsy caused by increased levels of RhoBTB2 mutant protein in these animals can be reduced, weight loss can be suppressed, and this is useful for treating or preventing RhoBTB2-associated neurodevelopmental disorders. The animal in question is not particularly limited, and may be a human or a non-human animal. Non-human animals include mammals such as cows, pigs, horses, sheep, goats, monkeys, dogs, cats, rabbits, mice, rats, hamsters, and guinea pigs, and birds such as chickens, quails, and ducks.

[0022] The pharmaceutical composition of the present invention is preferably administered to humans having one or more mutations in the RhoBTB2 gene selected from the group consisting of D92H mutation, W217C mutation, R461H mutation, R485C mutation, and R489Q mutation, for the treatment or prevention of neurodevelopmental disorders, and is particularly preferably administered to humans having the R489Q mutation for the treatment or prevention of neurodevelopmental disorders. [Example]

[0023] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples. All experiments were conducted in accordance with the ethical guidelines of the Japan Neuroscience Society and the Ministry of Education, Culture, Sports, Science and Technology. The animal experiment protocols were approved by the Animal Experiment Committee of Hamamatsu University School of Medicine (Permit Numbers 2020019 and 2020093).

[0024] <Cell line> Flp-In® T-REx® 293 cells (Invitrogen) were cultured in Dulbecco's minimum essential medium (Fujifilm Wako Pure Chemical Industries, Ltd.) supplemented with 10% fetal bovine serum (Tet-System Approved FBS, Thermo Fisher Scientific), 100 units / mL penicillin, and 100 μg / mL streptomycin (all from Fujifilm Wako Pure Chemical Industries, Ltd.) at 37°C in a 5% CO2 incubator. To generate doxycycline (Dox)-inducible cells, Flp-InT-REx 293 cells on a 10 cm dish were co-transfected with 1 μg pcDNA5 / FRT / TO-Flag-RHOBTB2 (an expression vector containing the Flag-RHOBTB2 gene in the pcDNA5 / FRT / TO vector (Invitrogen)) and 9 μg pOG44 plasmid using a transfection reagent (Lipofectamine® LTX Reagent, Thermo Fisher Scientific). 48 h after transfection, the cells were split into four plates and cultured in fresh medium containing hygromycin B. Five to ten hygromycin-resistant colonies were selected and further expanded. Induction of Flag-RHOBTB2 expression was confirmed by immunoblotting after 24 to 48 h of culture with Dox (1 ng / mL).

[0025] <Plasmid construction> Using the BLOCK-iT® RNAi Designer, we selected four highly specific sequences from the RHOBTB2 gene sequence shared between humans and mice, as shown in Table 1.

[0026] [Table 1]

[0027] Oligo-RNAs formed by annealing two RNAs having the base sequences listed in Table 2 were introduced into a plasmid using an RNAi expression vector kit (BLOCK-iT® Pol II miR RNAi Expression Vector Kits, Invitrogen) according to the manufacturer's protocol. In Table 2, the underlined portions in the base sequences of "#584_bottom," "#357_bottom," "#375_bottom," and "#376_bottom" indicate the same regions as the base sequences of "#584," "#357," "#375," and "#376," respectively.

[0028] [Table 2]

[0029] <siRNAトランスフェクション> For siRNA transfection, siRNAs consisting of the base sequences listed in Table 1 were used. These were synthesized by Invirtogen. Tetracycline was added to the cells 24 hours before siRNA transfection. For siRNA transfection using a transfection reagent (Lipofectamine RNAiMax, Invitrogen), 10 nM siRNA (Invirtogen) or control siRNA (Invirtogen) was dissolved in 1 mL of transfection medium. Cells were harvested 48 hours after transfection.

[0030] <rt-qpcr> cDNA was extracted from the siRNA-transfected cells using an extraction kit (SuperPrep® II Cell Lysis & RT Kit, Toyobo Co., Ltd.) according to the manufacturer's protocol. The target-specific primers shown in Table 3 and 4 μL of cDNA were used for PCR. The cDNA obtained by reverse transcription was subjected to real-time quantitative PCR using a real-time PCR master mix (GeneAce SYBR® qPCR Master Mix α (319-07683), manufactured by TAKARA). The relative expression of each gene was normalized by the GAPDH expression level and quantified using a standard curve.

[0031] [Table 3]

[0032] <Mouse> To generate the Rhobtb2 R489Q mutant, fertilized one-cell eggs were collected from ICR female mice (purchased from SLC Japan) and transfected with a genome editing agent by in vitro electroporation, as described by Aoto et al. (Non-Patent Document 6). The sequences of the guide RNA (gRNA) and single-stranded ODN (ssODN) are shown in Table 4.

[0033] [Table 4]

[0034] N-terminal HA-tagged WT mice (Rhobtb2 2×HA-WT ) and R489Q mutant mice (Rhobtb2 2×HA-R489Q To generate the Rhobtb2 chromosomes, female ICR WT mice were translocated into male Rhobtb2 chromosomes. R489Q The mice were crossed with Rhobtb2 / + mice and subjected to i-GONAD in the same manner as in the method of Kobayashi et al. (Non-Patent Document 7). 2×HA-R489Q / WT ) and a line with a 2xHA tag but without the R489Q mutation (Rhobtb2 2×HA-WT / WT ) and crossed them to generate Rhobtb2 2×HA-R489Q / 2×HA-WT We obtained Rhobtb2 mice. 2×HA-R489Q / 2×HA-WT Male and female mice were crossed to express Rhobtb2 2×HA-WT Homozygous for Rhobtb2 2×HA-WT and Rhobtb2 2×HA-R489Q Heterozygous and Rhobtb2 2×HA-R489Q Homozygous mice were obtained. The insertion of the HA tag was confirmed by immunoblotting or immunoprecipitation and PCR assays.

[0035] One verified mutant founder mouse from each strain was used for the experiment after mating with ICR mice (purchased from SLC Japan) for at least three generations. Total Rhobtb2 mRNA from the mutant mice was verified by Sanger sequencing. For genotyping, genomic DNA from ear samples was analyzed by high-resolution melting analysis to distinguish between group 1 (wild-type or homozygous mice) and group 2 (heterozygous mice) using an Eco® 48 real-time PCR system (PCRmax, Illumina). In group 1, PCR products were analyzed for Rhobtb2 to distinguish between wild-type and homozygous mice. R489Q In the case of , digestion was performed with SexAI. The sizes of the PCR products and the primers are shown in Table 5.

[0036] [Table 5]

[0037] <Behavioral assessment of spontaneous seizures> 21-day-old Rhobtb2 WT / WT Mice and Rhobtb2 R489Q / R489Q Three mice were individually placed in transparent square acrylic cages, and their activities were recorded over three days, including overnight, using a video camera with an infrared camera. Spontaneous seizures were manually scored from the recorded video using a modified Racine scale (8) (0: no abnormalities, 1: immobility, 2: head nodding, brief myoclonic seizures, 3: facial clonus, myoclonus, and tail rigidity, 4: forelimb / hindlimb clonus and clonic seizures, 5: standing up and falling, running and jumping, 6: tonic-clonic seizures).

[0038] <Immunoblotting> Cells and tissues collected from at least three age-matched mice of each genotype were homogenized in ice-cold RIPA lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1 mM EDTA pH 8.0, 0.1% sodium deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (11836153001, Roche) and a phosphatase inhibitor (for tissue, 04906837001, Roche). Subsequently, the homogenized cells were sonicated and centrifuged at 10,000 × g for 10 minutes at 4°C. Protein concentrations were measured using a BCA kit (Pierce). An equal volume of 2× SDS sample buffer was added to the supernatant, boiled for 5 minutes as needed, and then directly subjected to immunoblotting. Samples were separated on an SDS-polyacrylamide gel and transferred to a PVDF membrane. After transfer, the membrane was blocked in TBST supplemented with 3% (w / v) BSA or 0.5-5% (w / v) nonfat milk for 30 minutes to 1 hour at room temperature, and then incubated overnight at 4°C with primary antibodies diluted in blocking buffer.

[0039] The primary antibodies used and their dilutions were as follows: flag (KO602-L, TransGenic, 1:1000), GAPDH (60004-1, Proteintech, 1:50000), Gapdh (AB_2107436, 1:10,000), HRP-conjugated GAPDH (AB_2737588, 1:10,000), HA.11 (AB_2565335, 1:1,000), NKAα1 (AB_626713, 1:1,000), NKAα2 (AB_2061149, 1:1,000), N KAα3 (AB_10848453, 1:1,000), Cul3 (AB_10638777, 1:1,000), AMPKα1 (AB_722764, 1:1,000).

[0040] After washing three times with TBST, the membrane was incubated with anti-mouse / rabbit horseradish peroxidase-conjugated secondary antibody (Jackson Immuno Research Labs, 1:10,000 dilution) for 1 hour at room temperature. Chemiluminescence was detected using Clarity Western ECL Substrate (BioRad) and imaged using an imaging system (Fusion FX). Band intensity of the imaged blots was quantified using Image J software (v.1.8.0, NIH).

[0041] <Immunoprecipitation> For immunoprecipitation of endogenous HA-Rhobtb2 protein, mouse brains were homogenized in RIPA lysis buffer, and 2000 μg of protein lysate was immunoprecipitated with 20 μL of washed anti-HA magnetic beads (88836, Thermo Fisher Scientific). The immunoprecipitated proteins were eluted with 2× SDS sample buffer and subjected to immunoblotting.

[0042] <Production of adeno-associated virus (AAV)> AAV production plasmids were constructed by inserting the short hairpin sequence of the BLOCK-iT Pol II miR RNAi expression vector into an adeno-associated virus serotype 9 (AAV9) vector containing the CAG and human synapsin I promoters. The CAG promoter is a ubiquitous and highly expressible promoter, while the human synapsin I promoter is a neuron-specific promoter. RhoBTB2 miR-expressing serotype AAV9 (miR_357, 584) and the corresponding miR-negative control virus (miR_neg) were prepared by transfection into the AAVPro® 293T cell line (632273, TAKARA). The AAV virus was purified using a purification kit (AAVpro® Purification kit Maxi, 6666, TAKARA). The viral titer was measured using a titration kit (AAV Pro® Titration Kit, 6233, TAKARA).

[0043] [Example 1] We analyzed knock-in mice (KI mice) carrying the R489Q mutation identified in patients, and double knock-in mice (DKI mice) carrying the R489Q mutation and 2xHA, to elucidate the molecular pathogenesis of RhoBTB2-associated neurodevelopmental disorders. Rhobtb2 R489Q KI mice, Rhobtb2 2×HA-R489Q DKI mice were generated as described above. Figure 1 shows a schematic representation of the genetic modification status of each mouse.

[0044] The changes in body weight and survival rate (%) of each mouse over time are shown in Figure 2. R489Q KI mice and Rhobtb2 2×HA;R489Q The changes in body weight over time in DKI mice are shown in Figure 2(A). R489Q KI mice and Rhobtb2 2×HA;R489Q The time course of survival rates of DKI mice is shown in Figure 2(B). In the figure, "WT" indicates wild-type mice, and "Het KI" indicates heterozygous Rhobtb2 mice. R489Q KI mice, "Homo KI," are homozygous for Rhobtb2 R489Q KI mice, "Het DKI," are heterozygous for Rhobtb2 2×HA;R489Q DKI mice, "Homo DKI," are homozygous for Rhobtb2 2×HA;R489Q These are the results for DKI mice. R489Q KI mice and Rhobtb2 2×HA-R489Q In both DKI mice, heterozygous mice had normal survival and growth rates similar to wild-type mice, but homozygous mice exhibited growth retardation and epileptic seizures similar to those seen in patients, and began to die at 3 weeks of age and by 6 weeks of age.

[0045] Rhobtb2 2×HA-R489Q The expression status of HA-RhoBTB2 in DKI mice (homozygous and heterozygous) and wild-type (WT) mice on embryonic day 14, postnatal day 7, and postnatal day 21 was confirmed by immunoblotting. The results are shown in Figure 3. The results confirmed that HA-RhoBTB2 was expressed in DKI mice, and that the expression level was higher in homozygous mice than in heterozygous mice.

[0046] Spontaneous seizures (epileptic seizures) were evaluated for homozygous and wild-type KI mice. Figure 4(A) shows the average number of seizures per day for each mouse, and Figure 4(B) shows the seizure scores. Unlike wild-type mice, homozygous KI mice had a significantly increased number of epileptic seizures.

[0047] Furthermore, the expression levels of NKAα1 and NKAα3, two of the three α subunits of NKA, were examined by immunoblotting in 21-day-old DKI mice (homozygous and heterozygous) and wild-type mice (n = 4). As a control, the expression level of GAPDH was also examined. Based on the band intensity obtained by immunoblotting, the relative expression level was calculated, with the wild-type expression level set at 1. The results are shown in Figure 5. The expression levels of both NKAα1 and NKAα3 were reduced in mutant mice compared to wild-type mice, with the reduction being even greater in homozygous mice. These results indicate that increased RhoBTB2 levels result in decreased NKA levels.

[0048] [Example 2] We used RhoBTB2-expressing cells, which can express Flag-RhoBTB2 or its mutants upon administration of Dox, to elucidate the molecular pathogenesis of RhoBTB2-associated neurodevelopmental disorders.

[0049] RhoBTB2-expressing cells were prepared as described above using Flp-InT-REx 293 cells, which contain a single copy of the expression construct and exhibit mild Dox-induced expression. The RhoBTB2 fusion constructs expressed in the wild-type, D92H, W217C, R461H, R485C, R489Q, and Y284D mutants were used.

[0050] The location of each mutation in the RhoBT2 protein is shown in Figure 6(A). In Figure 6(A), the R489Q mutant, for which a knock-in mouse was created, is shown in reverse text. The D92H mutant, W217C mutant (all GTPase domain mutants), R461H mutant, R485C mutant, and R489Q mutant (all BTB domain mutants) are mutants found in patients with neurodevelopmental disorders. The Y284D mutant is a mutant found in lung cancer tissue. RhoBTB2 functions as an adaptor protein for the Cul3 (Cullin-3)-ubiquitin ligase complex, and the Y284D mutant is unable to bind to Cul3.

[0051] The expression levels of NKAα1 and GAPDH were examined for each cell type in the same manner as described above, and the ratio of their expression levels ([NKAα1 expression level] / [GAPDH expression level]) was calculated. The results are shown in Figure 6(B). In the figure, "*" indicates p<0.05, "**" indicates p<0.01, and "ns" indicates no significant difference. Expression of the D92H, W217C, R461H, R485C, and R489Q mutants by Dox treatment reduced the expression level of NKAα1. In contrast, expression of the Y284D mutant by Dox treatment did not result in a reduction in the expression level of NKAα1.

[0052] ATP levels were measured in cells expressing the wild-type, Y284D, and R489Q mutants. The results are shown in Figure 6(C). While no decrease in ATP levels was observed in cells expressing the wild-type or Y284D mutants, a significant decrease in ATP levels was observed in cells expressing the R489Q mutant. These results suggest that the binding of RhoBTB2 to Cul3 is essential for the decrease in NKA caused by increased RhoBTB2.

[0053] Human NKAα1, α2, and α3 deficiencies are known to cause epilepsy, alternating hemiplegia in childhood, acute-onset dystonia-parkinsonism, and encephalopathy, both paroxysmal and sudden, and share many clinical similarities with RhoBTB2-associated neurodevelopmental disorders. Based on these findings, it is thought that increased RhoBTB2 due to mutations leads to decreased ATP and NKA protein, which causes epileptic seizures in mutant mice.

[0054] [Example 3] Based on the findings obtained in Example 2, we conceived of gene silencing therapy using siRNA to suppress RhoBTB2 gene expression. The sequence identity between human and mouse RhoBTB2 mRNA sequences is 90%, and by selecting siRNA candidates that target the same sequence in humans and mice, it will be possible to verify the effects in both cell models (a system in which human RhoBTB2 is expressed) and mutant mice (a system in which mouse RhoBTB2 is expressed).

[0055] Four siRNAs, #584, #357, #375, and #376, listed in Table 1, were selected as candidate siRNAs for gene silencing therapy. Each candidate siRNA was administered to cells expressing wild-type and R489Q mutant Flag-RhoBTB2 upon tetracycline treatment, as used in Example 2, and the effect on Flag-RhoBTB2 expression was examined. Specifically, the expression levels of each Flag-RhoBTB2 were examined at the mRNA and protein levels in cells transfected with 10 nM siRNA 48 hours later. As described above, mRNA levels were measured by RT-qPCR, and protein levels were measured by immunoblotting. The expression levels of Flag-RhoBTB2 in each cell were calculated relative to the expression level in cells treated with the negative control siRNA ("#neg" in the figure), which was set at 1.0.

[0056] Figure 7(A) shows the results of measuring the expression levels of each Flag-RhoBTB2 at the mRNA level in cells expressing the wild-type Flag fusion protein of RhoBTB2 (left panel) and cells expressing the Flag fusion protein of the RhoBTB2 R489Q mutant (right panel). Figure 7(B) shows the results of measuring the expression levels of each Flag-RhoBTB2 at the protein level in cells expressing the wild-type Flag fusion protein of RhoBTB2 (left panel) and cells expressing the Flag fusion protein of the RhoBTB2 R489Q mutant (right panel). As shown in Figure 7, #357 and #584 suppressed expression levels by approximately 70% at the mRNA level and more than 90% at the protein level compared to the negative control siRNA ("#neg" in the figure).

[0057] [Example 4] By using the four base sequences listed in Table 1 as shRNA instead of siRNA and incorporating this into an AAV9 viral vector with neuronal targeting, gene therapy with sustained efficacy after a single administration becomes possible. As described above, AAV9 viral vectors carrying shRNA containing #357 or #584 (AAV9-CAG-shRNA-RhoBTB2) were created and their therapeutic effects were examined.

[0058] Rhobtb2 2×HA-R489Q DKI mice were crossed with heterozygous mice to identify Rhobtb2 2×HA-R489Q Homozygous mice were obtained. 2×HA-WT DKI mice were crossed with heterozygous mice to identify Rhobtb2 2×HA-WT Homozygous mice were obtained. Rhobtb2 2×HA-R489Q Homozygous mice and Rhobtb2 2×HA-WT Homozygous neonatal mice were intracerebroventricularly injected with AAV9 viral vectors carrying #357, #584, or #neg at postnatal day 0–1, and then housed. Body weight changes and survival rates were monitored over time. Brains were harvested at postnatal day 21, and the levels of RhoBTB2 and NKAα1 in the brain were measured. These experiments were performed three times independently.

[0059] Figure 8(A) shows the relationship between the amount of RhoBTB2 in the brain and the body weight of each mouse that received intracerebroventricular administration of AAV9 viral vectors carrying #357, and Figure 8(B) shows the relationship between the amount of RhoBTB2 in the brain and the amount of NKAα1 in the brain. In Figure 8, "2×HA WT" indicates the amount of RhoBTB2. 2×HA-WT Homozygous mice, "2xHA R489Q", lack Rhobtb2 2×HA-R489Q "Control" indicates mice that were not administered the AAV9 viral vector, and "miR 357" indicates mice that were administered the AAV9 viral vector carrying #357.

[0060] Furthermore, the time course of survival rates of mice to which AAV9 viral vectors carrying #357 were intracerebroventricularly administered is shown in Figure 9. In Figure 9, "2xHA WT+miR357" indicates the survival rate of Rhobtb2 cells administered with AAV9 viral vectors carrying #357. 2×HA-WT Homozygous mice (n=3) were administered with AAV9 viral vector #357, and the "2×HA R489Q+miR357" group showed Rhobtb2 expression. 2×HA-R489Q Homozygous mice (n=4) were treated with Rhobtb2 2xHA R489Q, which was not administered the AAV9 viral vector. 2×HA-R489Q Homozygous mice (n=22) are shown.

[0061] As shown in Figure 8, there was a negative correlation between the amount of RhoBTB2 in the brain and body weight, with mice with higher RhoBTB2 levels experiencing reduced body weight. Also, as shown in Figure 8, there was a negative correlation between the amount of RhoBTB2 in the brain and the amount of NKAα1. Furthermore, as shown in Figure 9, there was a tendency for survival rates to improve with the administration of #357. These results demonstrate that administering a nucleic acid drug that targets RhoBTB2 and reduces its expression level through RNAi can improve the pathology of RhoBTB2-associated neurodevelopmental disorders and may be a promising therapeutic option.

Claims

1. A pharmaceutical composition for treating or preventing RhoBTB2-associated neurodevelopmental disorders, comprising as an active ingredient a substance that suppresses or inhibits the function of the RhoBTB2 gene.

2. The pharmaceutical composition according to claim 1, wherein the substance that suppresses or inhibits the function of the RhoBTB2 gene is a nucleic acid that targets the RhoBTB2 gene and suppresses its expression by RNA interference.

3. The pharmaceutical composition according to claim 2, wherein the substance that suppresses or inhibits the function of the RhoBTB2 gene is an siRNA that targets the RhoBTB2 gene.

4. The pharmaceutical composition according to claim 2, wherein the substance that suppresses or inhibits the function of the RhoBTB2 gene is an adeno-associated virus vector carrying shRNA that targets the RhoBTB2 gene.

5. The pharmaceutical composition according to claim 2, wherein the nucleic acid is a nucleic acid that targets a region in the RhoBTB2 gene consisting of a base sequence represented by any one of SEQ ID NOs: 1 to 4 and suppresses expression by RNA interference.

6. The pharmaceutical composition according to claim 1 or 2, which is administered by intraventricular administration, intraspinal fluid administration, or intravenous administration.

7. 3. The pharmaceutical composition according to claim 1 or 2, which is administered to a human having one or more mutations in the RhoBTB2 gene selected from the group consisting of D92H mutation, W217C mutation, R461H mutation, R485C mutation, and R489Q mutation.

8. 1. A method for treating or preventing a RhoBTB2-associated neurodevelopmental disorder in a non-human animal, comprising: A method for suppressing epileptic seizures by suppressing or inhibiting the function of the RhoBTB2 gene.

9. The method according to claim 8, wherein the function of the RhoBTB2 gene is suppressed or inhibited by inhibiting the expression of the RhoBTB2 gene through RNA interference.

Images