Treatment for neurological diseases caused by MORC2 mutations

Abstract

The present invention relates to a pharmaceutical composition for treating and preventing Charcot-Marie-Tooth type 2Z (CMT2Z) caused by MORC2 mutations. The pharmaceutical composition according to the present invention can efficiently deliver a therapeutic gene for CMT2Z to the nervous system, and can improve clinical symptoms over a long period with only a single treatment.

Description

Treatment of neurological disorders caused by MORC2 mutations

[0001] The present invention relates to a pharmaceutical composition for the treatment and prevention of Charcot-Marie-Tooth type 2Z (CMT2Z) caused by MORC2 mutation.

[0002]

[0003] Microrchidia CW-Type Zinc Finger 2 (MORC2) is involved in various biological processes, including DNA damage repair, lipid homeostasis, and epigenetic silencing. It plays a role in transcriptional regulation, DNA damage response, and transposable element silencing, and dysregulation of these functions has been associated with neurodegenerative disorders. Single nucleotide polymorphisms (SNPs) in MORC2 are associated with the development of Charcot-Marie-Tooth disease. SNPs in MORC2 cause Charcot-Marie-Tooth type 2Z (CMT2Z), which causes peripheral neuropathy and reduced motor and sensory action potentials. In particular, SNPs in the MORC2 GHKL (Gyrase, Heat-shock protein 90; Hsp90, histidine kinase, MutL) ATPase module affect MORC2 dimerization and lead to hyperactivation of the human silencing hub (HUSH) complex. Patients with mutations in the MORC2 GHKL ATPase module have been shown to have developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysplasia, leading to the developmental delay, growth retardation, dysmorphic face, and axonal neuropathy (DIGFAN) syndrome, in addition to CMT2Z (omim.org; # 619090).

[0004] Surgery, physical therapy, and occupational therapy are utilized to alleviate the clinical symptoms of CMT and DIGFAN. However, because CMT is a genetic disease, the ultimate treatment is to correct the gene or restore gene function. Gene therapy has been attempted by delivering therapeutic genes or siRNA (small interfering RNA) into the body in various CMT animal models, such as CMT1A, CMT1X, CMT2A, CMT2D, and CMT2S. However, the cause of neuropathy in MORC2 mutants remains unclear due to various characteristics of MORC2, such as dimerization, deduplication by ATP hydrolysis, PARylation, and low ATPase activity. Therefore, elucidating the disease pathogenesis is essential for establishing a treatment strategy.

[0005] The mouse paralogs of MORC2 are Morc2a and Morc2b, and Morc2a has genetic similarity to MORC2. The production of transgenic mice with the Morc2ap.S87L gene mutation has been reported, and in that report, heterozygous Morc2ap.S87L mice (Morc2a S87L / + ) showed axonal nerve damage, cerebellar ataxia, and motor neuron damage. This shows complex DIGFAN including central nervous system neuropathy as well as the typical peripheral neuropathy of CMT2Z. Morc2a S87L / + In , an increase in apoptosis was observed in the central nervous system and muscles such as the quadriceps femoris through the accumulation of DNA damage. A decrease in Morc2a gene expression was observed in Morc2ap.S87L mutant mice, but the mechanism of neuronal apoptosis induction remains unclear.

[0006]

[0007] In order to solve the problems of the prior art, the present inventors, while studying the etiology and treatment of CMT2Z, discovered MORC2 and MORC2 GHKL ATPase as treatment candidates for CMT2Z and confirmed that their expression has a CMT2Z treatment effect, thereby completing the present invention.

[0008] Therefore, the purpose of the present invention is to provide a pharmaceutical composition for treating or preventing CMT2Z.

[0009]

[0010] To achieve the above purpose, the present invention provides a pharmaceutical composition for treating or preventing Charcot-Marie-Tooth type 2Z (CMT2Z), comprising as an active ingredient an AAV-PHP.eB vector comprising a Morc2a CDS represented by the amino acid sequence of SEQ ID NO: 1 or a Morc2a GHKL ATPase domain CDS represented by the amino acid sequence of SEQ ID NO: 2.

[0011] In the present invention, the composition is characterized by reducing hydroxyl radicals.

[0012] Additionally, in the present invention, the composition is characterized by increasing cellular ATPase activity.

[0013] Additionally, in the present invention, the composition is characterized by reducing atrophy and cell death of the cerebellum.

[0014] Additionally, in the present invention, the composition is characterized by reducing cell death of the quadriceps femoris.

[0015] In addition, in order to achieve the above another object, the present invention provides a pharmaceutical composition for treating or preventing CMT2Z, comprising Nicaraven as an active ingredient.

[0016]

[0017] The pharmaceutical composition of the present invention can efficiently deliver a therapeutic gene for CMT2Z to the nervous system, and can improve clinical symptoms over a long period of time with a single treatment.

[0018]

[0019] Figure 1 shows the cell death of Microrchidia CW-Type Zinc Finger 2A (Morc2a) p.S87L mediated by reactive oxygen species (ROS).

[0020] Figure 2 shows the defect in Morc2a protein synthesis and high hydroxyl radical-mediated cell death of p. S87L.

[0021] Figure 3 shows the induction of weakened ATPase activity due to suppression of Morc2a gene expression in p. S87L.

[0022] Figure 4 shows cell death caused by protein synthesis defects and inhibition of MORC2p.S87L and p.R252W.

[0023] Figure 5 shows that the Morc2aGHKL ATPase domain regulates cell death induced by hydroxyl radicals.

[0024] Figure 6 shows the effect of gene therapy using the Morc2aGHKL ATPase domain or Morc2a sequence by adeno-associated virus (AAV)-PHP.eB on improving central and peripheral nervous system neuropathy.

[0025] Figure 7 Morc2a by gene therapy S87L / + It shows the recovery of peripheral nerve cell and muscle dysfunction in mice.

[0026] Figure 8 is Morc2a of Nicaraven S87L / + It showed an effect of improving mouse neuropathy.

[0027] Figure 9 is a schematic diagram showing the mechanism of neuropathy and recovery according to mutation in Morc2a.

[0028]

[0029] Hereinafter, the present invention will be described in detail through examples, but the scope of the present invention is not limited to the following examples.

[0030]

[0031] Experimental method

[0032]

[0033] Histological analysis

[0034] After anesthetizing the mice with 2.5% avertin, total perfusion was performed using PBS and 4% paraformaldehyde (PFA) solutions. The collected tissues were then fixed in 4% PFA for 24 h and precipitated in 15% and 30% sucrose for 6 and 24 h, respectively. The precipitated tissues were then embedded at -80°C using optimal cutting temperature compound (OTC; Sakura Finetek, Tokyo, Japan). Cryosection slides were prepared at 15 μm thickness using a cryostat microtome (Leica, Wetzler, Germany). Apoptosis detection was performed using cryofixed tissues incubated with FITC-conjugated Annexin-V antibody (Trevigen, Gaithersburg, MD, USA) at room temperature for 1 h. FITC signals were measured using Cytation 5 (BioTek, Winooski, VT, USA). For H&E staining, frozen-fixed tissues were stained with 0.1% Mayer's H&E solution. Immunohistochemical staining for VDAC and cleaved Caspase 3 was performed in the following order: control serum blocking, primary and secondary antibody incubation, and signal detection using 3,3'-diaminobenzidine. The clones, hosts, and dilution conditions of the antibodies used are listed in Table 1.

[0035] Total RNA was extracted from WT and Morc2a S87L / +Total RNA was extracted from spinal cords and mouse embryonic fibroblasts (MEFs) of mice (n = 3 for WT-spinal cord (SC), S87L-SC, and WT-mouse embryonic fibroblasts (SCs); n = 2 for S87L-MEFs) using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). After quality assessment and RNA quantification, libraries were constructed from total RNA using the NEBNext Ultra II Directional RNA Library Prep Kit (New England BioLabs, Ipswich, MA, USA). mRNA was isolated using the poly(A) RNA selection kit (Lexogen, Wien, Austria), followed by cDNA synthesis and shearing according to the manufacturer's instructions. After indexing using Illumina indices 1–12, the average fragment size of the library was evaluated using an Agilent 2100 bioanalyzer (DNA High Sensitivity kit), and quantification was performed using a library quantification kit and a StepOne Real-Time PCR system (Life Technologies, Carlsbad, CA, USA). High-throughput sequencing was performed using an Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA) with paired-end sequencing (2 × 101 bp). Adapter sequences (TruSeq universal and indexed adapters) were removed from the resulting read sequences for each sample using Trimmomatic software (version 0.39). The resulting reads were aligned to the Mus musculus reference genome (GRCm38) using HISAT aligner (ver. 2.2.1) with default options. After filtering out duplicate reads using Picard Tools (version 2.25.5; https: / broadinstitute.github.io / picard / ), we used HTSeq (version 0.11.0) was used to calculate the mapped reads for gene functions (GTF file of GRCm38). Fragments per kilobase of transcript per million fragments mapped (FPKM) was calculated by dividing the number of mapped reads by the gene length and the total number of reads mapped to protein-coding genes.

[0036]

[0037] Identification of differentially expressed genes

[0038] Differentially expressed genes (DEGs) were identified for the two comparisons of S87L-SC vs. WT-SC and S87L-MEF vs. WT-MEF. For each comparison, we first identified 'expressed' genes with FPKM > 1 in more than half of the samples in at least one of the two conditions. For these expressed genes, the FPKM values ​​were converted to log2-FPKM by adding 1. The log2-FPKM values ​​were then normalized using the quantile normalization method. For each gene, the t-statistic and log2-fold change were calculated, and the empirical null distribution was estimated by performing random permutations (1,000 times) across all samples used in each comparison. Using the estimated empirical distribution, we calculated the adjusted p-value for the two tests for each gene, and then combined these p-values ​​using Stouffer's method. Finally, DEGs were identified by combining the mean 2.5 and 97.5 percentiles of the empirical distribution for p-value < 0.05, t-test p-value < 0.05, absolute log2-fold-changes > cutoff value, and log2-fold-changes (log2-fold-changes = 0.33 for S87L-SC vs. WT-SC). To identify cellular processes enriched by DEGs, enrichment analysis of Gene Ontology Biological Processes (GOBPs) was performed for DEGs using DAVID software, and GOBPs with p-values ​​< 0.05 and ≥ 5 genes were selected as processes.

[0039]

[0040] Overexpression or suppression of Morc2a, MORC2, and Acly

[0041] Fetuses were collected from the uterus of 13.5-day-old female mice, minced, and treated with collagenase IV (250 U / mg) for 30 minutes to dissociate the cells. After stopping the enzymatic reaction by adding fetal bovine serum (FBS), the cells were washed and seeded onto culture dishes. After subculture and genotyping, WT and Morc2a cells were isolated. S87L / + Mouse embryonic fibroblasts were used for further studies. Human fibroblasts were collected and established using human tissue harboring WT MORC2 or MORC2 p.R252W. Human tissue collection and cell experiments were approved by the Institutional Review Board of Samsung Medical Center, Sungkyunkwan University (IRB No. 2021-04-053).

[0042] pMorc2aWT (CAG-mMorc2a / Hibit-CMV-mCherry), pMorc2ap.S87L (CAG-mMorc2ap.S87L / Hibit-CMV-mCherry), pMORC2WT (CMV-hMORC2 / Hibit), pMORC2p.S87L (CMV-hMORC2p.S87L / Hibit), and pMORC2p.R252W (CMV-hMORC2p.R252W / Hibit) were obtained from a commercial DNA synthesis service (VectorBuilder, Chicago, IL, USA). The synthesized plasmids were transfected into mouse embryonic fibroblasts or human fibroblasts using Neon Transfection (Life Technologies). In summary, 2.5 × 10 5 Dog cells were suspended in 10 μL R buffer from the Neon Transfection kit. Then, the 1,350 V / 30 ms / 1 pulse condition of the Neon system was used for plasmid electroporation of the cells. siRNAs for Morc2a, MORC2, or Acly were purchased from Bioneer (Daejeon, Korea) and transfected with Lipofectamine TMTransfection was performed using RNAiMAX (Thermo Fisher Scientific). Transfected cells were used for experiments 2 days after siRNA transfection. Brief information about siRNA is listed in Table 1.

[0043]

[0044] Measurement of ROS, superoxide, hydroxyl radicals, and Mitotraker

[0045] For ROS analysis, cell culture supernatants were collected and ROS levels were measured using a commercial kit (Cat No. KTE71621, AbbKine, Wuhan, China). To detect mitochondrial superoxide, cells were seeded in black-bottomed 96-well plates and labeled with MitoSOX Red superoxide indicator (Cat No. M36008, Thermo Fisher Scientific) at a final concentration of 5 μM for 30 minutes. Fluorescence intensity was detected using Cytation (BioTek). Hydroxyl radical concentrations were measured using a commercial kit (Cat No: ab219931, Abcam, Cambridge, UK). Cells were seeded in clear-bottomed 96-well plates and cultured until confluence. After washing with DPBS, cells were treated with 100 μL of OH580 probe at 37°C for 1 hour and then incubated with assay buffer for 24 hours. Fluorescence signals were measured using Cytation. Then, the cells were incubated with 100 nM MitoTracker Red (Cat No:9082, Cell Signaling Technology, Danvers, MA, USA) for 10 minutes and the cellular processes were analyzed.

[0046]

[0047] quantitative reverse transcription PCR

[0048] WT and Morc2a were assayed using TRIzol reagent (Thermo Fisher Scientific).S87L / + Total RNA was extracted from mouse cells and tissues, and cDNA was synthesized using a cDNA synthesis kit (Thermo Fisher Scientific). Quantitative reverse transcription-PCR (qRT-PCR) was performed using PowerUp SYBR Green Master Mix and the StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). All reactions were performed in triplicate, and the average value of each sample was used for further analysis. Target gene expression was normalized to that of GAPDH and Actin. The primer sequences used in the experiments were obtained from Primer Bank (https: / / pga.mgh.harvard.edu / primerbank / ) and are listed in Table 1.

[0049]

[0050] Western blot

[0051] Tissues or transfected cells were collected and lysed in NP-40 buffer (Cat No: 85125, Thermo Fisher Scientific) and T-PER buffer containing protease inhibitors (Cat No: 78510, Thermo Fisher Scientific) at 4°C for 30 min. After SDS-PAGE, proteins were electro-transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated for 1 h in blocking buffer (1x TBS and 5% w / v non-fat milk) at 25°C. After incubation with primary antibodies for 24–48 h at 4°C, the membranes were washed with Tris-phosphate-buffered saline. Immunoreactive proteins were detected using enhanced chemiluminescence (ECL kit, Abclon, Seoul, Korea) after incubation with peroxidase-conjugated secondary antibodies for 1 h at 25°C. Information on the antibodies used in this experiment is listed in Table 2.

[0052]

[0053] Protein quantification

[0054] 1 × 10 4 HEK293 cells (American Type Culture Collection; ATCC, Manassas, VA, USA) or NIH-3T3 (ATCC) at cell density were seeded in clear-bottom 96-well plates and treated with Lipofectamine TM Each well was transfected with 0.1 μg of plasmid using a 3000 (Thermo Fisher Scientific). NIH-3T3 cells were transfected with Hibit-tagged WTMorc2a or Morc2ap.S87L expression plasmids, and HEK293 cells were transfected with Hibit-tagged WTMORC2, MORC2p.R252W, or p.S87LMORC2 plasmids. Approximately 18 or 24 h after transfection, protein synthesis or degradation was inhibited using 100 μg / mL cycloheximide (Cat No. C1988; Sigma Aldrich, Saint Louis, MO, USA) or 50 ng / mL bafilomycin (Cat No. B1793, Sigma Aldrich). Then, the amount of Morc2a or MORC2 protein synthesized by the transfected plasmids was quantified using Nano-Glo® HiBiT lysis assay (Promega, Madison, WI, USA) at 0, 6, 12, and 24 h after cycloheximide or Bafilomycin treatment.

[0055]

[0056] Flow cytometry-based cell death frequency analysis

[0057] Cells were overexpressed, inhibited, or chemically treated. The number of apoptotic cells was measured by flow cytometry using the TACS Annexin V-FITC Apoptosis Detection Kit (#4830-250-K; R&D Systems, Minneapolis, MN, USA). Cells were stained with Annexin V-FITC and propidium iodide (PI) for 15 min at 25°C, and the frequency of FITC-positive cells (early or late apoptotic cells) was measured.

[0058]

[0059] Quantitative ATPase activity assay

[0060] Morc2a or MORC2ATPase activity was quantified using the QuantiChrom ATPase / GTPase Assay Kit (BioAssay Systems, Hayward, CA, USA). Briefly, 20 mg of tissue was homogenized with 200 μl lysis buffer (T-PER, Thermo Fisher Scientific) and a protease inhibitor cocktail (Cat No; 11697498001, Roche, Basel, Switzerland). The supernatant was collected after centrifugation at 14,000 × g for 10 min. ATPase reactions were measured by mixing 1–10 μl of the supernatant, 20 μl of assay buffer, and 10 μl of 4 nM ATP (Sigma-Aldrich, St Louis, MI, USA) in a 96-well plate at 25°C for 30 min. After the reaction was terminated at 25°C for 30 minutes using 200 μl of stop solution, ATPase activity was measured by absorbance at 620 nm and calculated as free phosphate per milligram of tissue.

[0061]

[0062] AAV preparation and in vivo transduction

[0063] AAV vectors were prepared in the form of ITR-CMV promoter-Morc2a or Morc2aGHKL ATPase-pA-ITR by DNA synthesis. Then, 70 μg of AAV-donor vector, AAV PHP.eB-rep / cap plasmid, and helper plasmid were diluted to 1 × 10 using calcium phosphate. 8 HEK 293T cells were co-transfected. Three days after co-transfection, viral particles were collected and purified by cesium chloride centrifugation. AAV titration was performed using qRT-PCR, and the vectors were stored at -80°C until use. Vector preparation and recombinant AAV production were performed using Vector Builder (Chicago, IL, USA).

[0064] Morc2a S87L / + Mating females with WT males, and producing WT or Morc2a S87L / + Mice were included in the experimental groups after genotyping and simple randomization according to the ARRIVE guidelines. Mice were anesthetized with 2.5% Avertin and injected with 6.0 × 10 ng / ml into the subarachnoid space between L4 and L5 of 8-week-old mice using a HAMILTON microsyringe (Sigma Aldrich). 12 vg / kg AAV particles were slowly injected. To analyze the therapeutic effect, control and AAV-transduced mice were sacrificed 6 months after AAV injection.

[0065]

[0066] Semi-thin film and electron microscopy analysis

[0067] Sciatic nerves collected from control and AAV-transduced mice were examined pathologically using light and electron microscopy. Each specimen was individually fixed in a solution containing 2% glutaraldehyde in 25 mM cacodylate buffer. Semithin sections were stained with toluidine blue, and ultrathin sections were stained with uranyl acetate and lead citrate. The fixed specimens underwent a series of steps, including incubation in 1% OsO4 for 1 hour, dehydration in a series of ethanol solutions, passage through propylene oxide, and embedding in epoxy resin (Epon 812, Oken, Nagano, Japan). For semithin section pathological examination, three samples from each group were selected and analyzed by bright-field microscopy. For electron microscopy, ultrathin sections (60 nm in diameter) were collected on 200-mesh nickel grids and stained with 1% uranyl acetate and Reynolds' lead citrate for 10 minutes. Pathological evaluation was performed using an HT7700 electron microscope (Hitachi, Tokyo, Japan) at 80 kV. The distribution of myelinated fibers was analyzed by measuring their size using the Zeiss Zen2 program (Carl Zeiss, Oberkochen, Germany). The number of myelinated fibers in each mouse was analyzed by examining a montage consisting of at least 20 photographs taken at 600× magnification using semithin sections.

[0068]

[0069] Nerve conduction velocity analysis

[0070] The hair was completely removed from the posterior hindlimb of the mice. NCV analysis was performed using a Nicolet Viking Quest device (Natus Medical). For motor NCV analysis, the stimulating cathode was placed at two locations: the sciatic notch and 6 mm distal to the sciatic notch. The recording electrode was placed on the ventral aspect of the gastrocnemius muscle. A ground electrode was also placed on the back of each animal. To ensure unbiased results, an independent examiner, blinded to genotype and treatment group, determined motor nerve conduction velocity (MNCV) and compound muscle action potential (CMAP) amplitude. CMAP was measured using a maximal stimulus. For sensory NCV analysis, the stimulating cathode and recording electrodes were placed on the tail, specifically 30 mm proximal to each other. Similarly, a ground electrode was placed on the back of each animal. An independent examiner determined sensory nerve conduction velocity (SNCV) and sensory nerve action potential (SNAP) amplitude.

[0071]

[0072] Evil power analysis

[0073] Briefly, mice grasped the T-bar of a grip dynamometer (BIO-GS3, Bioseb, France) with their hind paws and pulled backwards in a horizontal plane. The force exerted on the bar at the point where the animal released the bar was recorded as the peak tension. Two tests were performed for each mouse, 10 minutes apart, and the average value was determined as the grip strength.

[0074]

[0075] In vivo AAV quantification

[0076] 6 x 10 12AAV-PHP.eB-Morc2a was injected intrathecally into WT mice, and the mice were sacrificed 3 weeks after AAV transduction. Tissue samples from the brain, spinal cord, quadriceps, and liver were collected, and genomic DNA was extracted. Next, AAV DNA was quantified by qPCR using a primer set for ITR detection. Primer sequences are listed in Table 2.

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] Example 1. Induction of reactive oxygen species-mediated cell death by Morc2ap.S87L

[0083] Morc2ap.S87L is a mutation occurring in the GHKL ATPase domain (Figure 1A). Morc2ap.S87L homozygous mice are embryonic lethal, and Morc2apS87L heterozygous mice show sublethal characteristics. S87L / + Only about 25% of mice were born at 15 months of age. Morc2a S87L / + A greater number of apoptotic cells were observed in the cerebral cortex, cerebellar Purkinje cells, spinal cord ganglia, and quadriceps fasciculus of Morc2a compared to WT (Fig. 1B). S87L / + To analyze the etiology of neuropathy, we performed functional annotation analysis by clustering genes whose expression significantly changed in the spinal cord and mouse embryonic fibroblasts (MEFs).

[0084] WT and Morc2a from mRNA analysis data S87L / + We identified 1,154 differentially expressed genes (DEGs, p < 0.05) between mice. Functional enrichment analysis revealed that Morc2a S87L / +Both MEFs and spinal cord tissues showed activation of the MAP kinase cascade, oxidative stress response, p53 signaling, and apoptosis-related pathways (Fig. 1C). The brain and nervous system are vulnerable to oxidative stress due to high oxygen demand, which leads to excessive production of reactive oxygen species (ROS), resulting in DNA damage in the central nervous system. Morc2a was also detected. S87L / + Mice exhibited higher ROS production in the brain, which was more severe in the cerebellum than in WT mice (Fig. 1D), and ROS was induced by Morc2a S87L / + It suggests that it is involved in neuronal cell death in mice.

[0085] To ensure the convenience of cell experiments, additional experiments were performed using MEFs. Morc2a S87L / + MEFs exhibited approximately 15% higher ROS levels than WT MEFs (Fig. 1E), and this increase in ROS increased cell death (Fig. 1F). Therefore, we analyzed the characteristics of mitochondria, the main site of ROS production. The amount of active mitochondria measured using MitoTracker was significantly different between WT and Morc2a MEFs. S87L / + There was no significant difference between MEFs (Fig. 1G). Among ROS such as superoxide (O2·-), hydrogen peroxide (H2O2), and hydroxyl radical (·OH), Morc2a S87L / + The increase in ROS levels in mice was primarily due to hydroxyl radicals rather than superoxide (Fig. 1H). High levels of ROS can induce cell death or necrosis, and hydroxyl radicals are the most aggressive ROS due to their high reactivity with biomolecules. In the ROS-induced mitochondrial cell death pathway, VDAC plays a key role in the release of ROS and cytochrome c, and Morc2a S87L / +In the brain, cerebellum, sciatic nerve, and quadriceps femoris, all of which exhibited high apoptosis, VDAC expression was activated compared to WT. In particular, the expression sites of VDAC and cleaved caspase-3, a marker of apoptosis, were similar, suggesting that Morc2ap.S87L would induce increased ROS production and apoptosis.

[0086]

[0087] Example 2. Induction of hydroxyl radical-mediated cell death due to protein synthesis defect of Morc2a p.87L.

[0088] Decreased Morc2a gene expression was confirmed in the cerebellum, cerebrum, spinal cord, sciatic nerve, and limb tissues of Morc2ap.S87L mutant mice (Fig. 2A). Next, we analyzed the correlation between Morc2a expression and high hydroxyl radical-mediated cell death in MEFs. In Morc2ap.S87L MEFs, Morc2a gene expression was reduced by approximately 15%, whereas Morc2a protein was reduced by approximately 45% (Fig. 2B). This suggests that Morc2ap.S87L causes protein translation defects or instability. To confirm this, NIH-3T3 cells were transfected with WTMorc2a or Morc2ap.S87L plasmids containing a C-terminal Hibit tag (Morc2a / Hibit or Morc2a p.S87L / Hibit), and the proteins were quantified.

[0089] Morc2a p.S87L / Hibit produced a lower amount of Morc2a protein than WTMorc2a / Hibit. The amount of Morc2a protein was measured by Hibit quantitation 24 hours after plasmid transfection, and WTMorc2a / Hibit produced 4-5 times more protein than Morc2a p.S87L / Hibt (p<0.0001). Normalizing the transfection efficiency using the mCherry reporter, we confirmed that Morc2a p.S87L protein was produced approximately 71% less than WT Morc2a protein (Fig. 2C). MORC2 is primarily degraded by autophagy, and cycloheximide inhibits protein synthesis. Twenty-four hours after transfection with WTMorc2a / Hibit or Morc2a p.S87L / Hibit plasmids, Morc2a protein was quantified using an autophagy inhibitor (Bafilomycin A1) and cycloheximide treatment. There was no significant change in the slope of protein abundance between WT and Morc2a p.S87L. This suggests that the reduced Morc2a in p.S87L is due to a defect in protein synthesis rather than protein degradation.

[0090] We analyzed whether suppression of Morc2a expression was associated with high cellular hydroxyl radical concentrations. When the Morc2a gene was suppressed using siRNA (siMorc2a) against Morc2a in WT MEFs (Figure 2D), mitochondrial activity increased (Figure 2E). Notably, Morc2a suppression significantly increased cellular ROS production, such as hydroxyl radicals (Figures 2F and 2G). Hydroxyl radicals can cause DNA damage, and Morc2a suppression increased cell death by generating high levels of hydroxyl radicals (Figure 2H). Next, we investigated whether the functional enhancement of Morc2ap.S87L induced high hydroxyl radical production. Overexpression of Morc2ap.S87L in WT MEFs did not affect mitochondrial activity, cellular hydroxyl radical levels, or other ROS production (Figures 2I, 2J, and 2K). In contrast, WTMorc2a overexpression significantly reduced the production of cellular hydroxyl radicals and other ROS (Figures 2J and 2K). While lower cell death was expected with WTMorc2aCDS overexpression, no difference was observed between groups due to the basal cell death rate of WT MEFs (Figure 2L). In summary, the high cellular hydroxyl radical content of Morc2ap.S87L was due to loss of function, not gain of function.

[0091]

[0092] Example 3. Attenuation of ATPase activity, HUSH activity, and PARylation due to decreased expression of Morc2ap.S87L.

[0093] Mitochondrial cell death induced by hydroxyl radicals by Morc2a S87L / + Although involved in neuropathy, the predicted cause of human CMT2Z in MORC2p.S87L is low GHKL ATPase activity. Human MORC2p.S87L had low GHKL ATPase activity and higher HUSH activity. Similarly, Morc2a S87L / +MEFs exhibited lower ATPase activity than WT MEFs (Fig. 3A). There was a positive correlation between Morc2a expression and ATPase activity (Figs. 2B and 3A). Morc2a S87L / + MEFs also showed increased expression of Transgene Activation Suppressor (Tasor) and M-Phase Phosphoprotein 8 (Mpp8) (Fig. 3B). In addition, MORC2 is involved in DNA repair, and its ATPase activity promotes chromatin remodeling, enhancing the accessibility of repair factors during DNA damage and increasing the stability of Poly (ADP-ribose) polymerase 1 (PARP1). Morc2ap.S87L showed lower PARP1 expression in MEFs, indicating greater susceptibility to ROS-induced DNA damage. Morc2a S87L / + This coincides with increased expression of γ histone family member X (γH2AX) and p53 in MEFs (Fig. 3B).

[0094] Morc2a S87L / + We analyzed whether inhibition of Morc2a expression or gain of function in the mouse model decreased ATPase activity. Morc2a inhibition significantly decreased cellular ATPase activity (Fig. 3C). Morc2a S87L / +Consistent with our observations in MEFs, Morc2a expression and cellular ATPase activity were positively correlated in several tissues (Figs. 2A, 3D, and 3E). Overexpression of Morc2ap.S87L in WT MEFs resulted in lower cellular ATPase activity than that of WTMorc2a and similar to that of the control (Fig. 3F). A previous study on transient expression of MORC2p.S87L in HeLa cells showed lower ATPase activity than that of WT MORC2 overexpression and no significant difference compared to the control, which is similar to our results. Therefore, the lower ATPase activity or HUSH complex activation of Morc2ap.S87L was expected to be due to loss of function rather than gain of function.

[0095]

[0096] Example 4. MORC2p.S87L and Morc2ap.S87L share similar MORC2 protein synthesis defects.

[0097] Morc2a S87L / +The primary cause of apoptosis in mice can be summarized as low Morc2a gene expression and protein translation defects, resulting in high levels of cellular hydroxyl radicals and low ATPase activity. Therefore, we analyzed whether human pathogenic MORC2 variants shared the same pathogenesis observed in mice. MORC2p.R252W and MORC2p.S87L are mutations in the GHKL ATPase domain and exhibit lower ATPase activity, raising the possibility that they share the pathogenesis observed in Morc2ap.S87L. We obtained and analyzed human fibroblasts harboring the p.R252W mutation and, as expected, human MORC2p.R252W fibroblasts showed reduced MORC2 protein expression (Figure 4A). MORC2p.R252W showed similar results to Morc2ap.S87L, with elevated levels of cellular hydroxyl radicals and ROS, decreased ATPase activity, and increased apoptosis (Figures 4B, 4C, 4D, and 4E).

[0098] The p.S87 and p.R252W residues of Morc2a and MORC2 are located within a loop of the GHKL ATPase module, and mutations within this loop can affect protein synthesis or stability (Fig. 4F). Therefore, we analyzed the changes in protein abundance of MORC2 p.S87L and p.R252W. Expression plasmids for MORC2, MORC2 p.S87L, and MORC2 p.R252W with Hibit tags were constructed, and newly synthesized MORC2 was transfected into human embryonic kidney 293 cells (HEK293) and quantified by Hibit analysis. MORC2 p.S87L and p.R252W showed lower MORC2 synthesis than WT MORC2. Furthermore, the slopes of changes in MORC2 abundance under conditions that inhibited autophagy or protein synthesis were similar between groups. It can be inferred that the p.S87L and p.R252W variants did not significantly affect the stability of MORC2 after production (Fig. 4G). Notably, MORC2p.S87L showed lower protein synthesis than p.R252W, which may explain why the MORC2p.S87L variant exhibits more severe neuropathy than p.R252W in CMT2Z patients. The protein synthesis defects observed in MORC2p.S87L and p.R252W imply loss of function. Accordingly, we analyzed changes in hydroxyl radical levels and ATPase activity after MORC2 inhibition (Fig. 4H). Reduction of MORC2 expression increased the levels of hydroxyl radicals, decreased ATPase activity, and increased cell death (Figs. 4I, 4J, and 4K), which is consistent with the observations of Morc2ap.S87L (Figs. 2E, 2F, 2G, and 2H).

[0099]

[0100] Example 5. Relationship between the Morc2aGHKL ATPase domain and cellular hydroxyl radical concentration.

[0101] To date, hydroxyl radicals are not known to be involved in the pathogenesis of CMT2Z through MORC2 or Morc2a variants. ROS are involved in cell death after DNA damage, and conversely, DNA damage induces ROS production through the γH2ax-Nox1 / Rac1 pathway. Therefore, we investigated whether DNA damage accumulation through HUSH activation or hydroxyl radicals is involved in the cell death process in Morc2ap.S87L. In WT MEFs, electroporation-induced DNA damage increased the number of generated hydroxyl radicals (Fig. 5A). Therefore, Morc2a S87L / + In mice, the activation of the HUSH complex and the reduction in PARylation were predicted to compromise genome integrity and lead to the accumulation of DNA damage, which in turn contributes to elevated cellular hydroxyl radical levels. Because hydroxyl radicals also induce DNA damage, it was hypothesized that increased DNA damage and hydroxyl radical levels would independently or synergistically increase cell death.

[0102] The mechanism of increasing hydroxyl radicals was investigated. The N-terminus of MORC2 is known to promote lipogenesis through protein-protein binding with adenosine triphosphate (ATP)-citrate lyase (Acly). Acly inhibition increases ROS production and exhibits anticancer effects. In addition, Acly is phosphorylated in a MORC2 expression-dependent manner. Since the Morc2aGHKL ATPase domain is located in the N-terminal region, Morc2ap.S87L shows a decrease in Morc2a. In other words, Morc2a inhibition can activate ROS production through the Morc2a-Acly interaction. Acly inhibition significantly increased hydroxyl radicals in WT MEFs, but not in Morc2a. S87L / +There was no difference in Acly and phosphorylated Acly levels upon Morc2a inhibition in mouse cells or MEFs (Figures 5B and 5C). These results are consistent with previous analyses of ACLY activity in human fibroblasts. Therefore, ACLY was independent of hydroxyl radical generation in Morc2ap.S87L fibroblasts.

[0103] Given that DIGFAN is associated with variants of the MORC2 GHKL ATPase domain, analysis of the link between the GHKL ATPase domain and hydroxyl radicals was important. Hsp90, an evolutionarily conserved protein domain and a component of the GHKL ATPase domain, was predicted to be a scavenger of hydroxyl radicals. Accordingly, we analyzed the relationship between Hsp90 and hydroxyl radicals using the Hsp90 inhibitors Radicicol and Geldamycin. Inhibition of Hsp90 with these two chemicals in WT MEFs resulted in an increase in cellular hydroxyl radicals (Fig. 5D). Although Radicicol or Geldamycin did not selectively inhibit Hsp90 within the GHKL ATPase domain, this suggests that Hsp90 within the GHKL ATPase domain may be involved in hydroxyl radical-mediated cell death.

[0104]

[0105] Example 6. Potential of Morc2aGHKL ATPase to Treat Cell Death

[0106] Since Morc2a overexpression reduces cellular hydroxyl radicals (Figure 2K), we focused on the entire CDS of Morc2a. Furthermore, the GHKL ATPase domain has been linked to severe neuropathy, and it was predicted that it would be involved in cellular hydroxyl radical concentrations. Therefore, we selected the entire Morc2a CDS (SEQ ID NO: 1) and the Morc2aGHKL ATPase domain CDS (SEQ ID NO: 2) as candidate targets for Morc2a downregulation (Figure 5E). The Morc2aGHKL ATPase domain shares high similarity with the human MORC2GHKL ATPase domain. Therefore, we selected the Morc2a sequence corresponding to amino acids 1–265 as the GHKL ATPase domain for our experiments.

[0107] Overexpression of the Morc2a or Morc2aGHKL ATPase domain did not induce changes in mitochondrial activity (Fig. 5F), but significantly reduced the levels of hydroxyl radicals and ROS (Fig. 5G). Furthermore, ATPase activity was increased by overexpression of Morc2a or Morc2aGHKL ATPase (Fig. 5H), which was expected to alleviate genomic instability caused by HUSH activation. Overexpression of the Morc2aCDS and Morc2aGHKL ATPase domains demonstrated a decrease in apoptosis in Morc2ap.S87L MEFs (Fig. 5I). Both Morc2aCDS and Morc2aGHKL ATPase domain CDS induced similar improvements in cellular hydroxyl radical levels, ATPase activity, and cell death. This suggests that Morc2aGHKL ATPase is essential for regulating hydroxyl radical levels and ATPase activity. Additionally, both WTMorc2aCDS and the small-sized Morc2aGHKL ATPase domain CDS can be used to establish gene therapy strategies.

[0108]

[0109] Example 7. Restoration of Morc2a and Improvement of Central and Peripheral Nervous System Neuropathy by AAV-PHP.eB Transduction

[0110] Morc2a S87L / + Neural delivery of therapeutic genes is necessary to evaluate the therapeutic efficacy of neuropathy in mice. AAV serotype 9 was used for CMT1X gene therapy via intrathecal injection and showed therapeutic efficacy. Morc2a S87L / + Considering the central and peripheral neuropathy in mice, we concluded that in vivo therapeutic gene delivery targeting the brain and spinal cord is reasonable, and thus AAV-PHP.eB, which shows specificity for the central nervous system, was selected as a delivery tool for therapeutic genes (Fig. 6A). Recombinant AAV harboring Morc2aGHKL ATPase domain CDS (AAV-GHKL) and Morc2aCDS (AAV-Morc2a) were prepared and delivered to a single dose of 6.0 × 10 12 Two-month-old mice were transduced with vg / kg AAV via the intrathecal route, and the therapeutic effect was analyzed after 6 months.

[0111] Morc2a S87L / + Mice exhibited cerebellar atrophy, and the same pathology was observed in CMT2Z patients carrying the MORC2p.S87L and p.T362R variants. AAV-GHKL and AAV-Morc2a transduction improved atrophy and cell death in the cerebellum. Morc2ap.S87L had a 22% smaller cerebellum size compared to WT, and AAV-GHKL and AAV-Morc2a transduction improved Morc2a S87L / + AAV-Morc2a transduction significantly improved cerebellar atrophy in mice by 6% and 9%, respectively. AAV-Morc2a transduction further reduced the relative frequency of cell death in the cerebellum (Fig. 6B). The reduction in cell death frequency was more evident in the spinal cord. S87L / + The frequency of apoptotic cells in the spinal cord was significantly higher than in the WT (p < 0.05, 4 times higher than in the WT), but AAV treatment significantly reduced Morc2aS87L / + The frequency of spinal cord cell death was reduced to the level of WT mice (Fig. 6C). That is, gene therapy with AAV-Morc2a or AAV-GHKL clearly improved neuronal cell death in the central nervous system.

[0112] Since the pathological lesions of CMT begin in the peripheral nervous system and progress to the central nervous system, we analyzed peripheral neuropathy of the sciatic nerve. Axonal neuropathy of the sciatic nerve is the primary diagnostic indicator of CMT2Z peripheral neuropathy. S87L / + The frequency of large myelinated fibers in the sciatic nerves of mice was reduced. In contrast, AAV-Morc2a and AAV-GHKL transduction restored the frequency of large myelinated sciatic nerve fibers to WT levels. Electron microscopy confirmed the therapeutic effect of AAV gene therapy on axonal neuropathy in the sciatic nerve (Fig. 6D). This suggests that peripheral neuropathy in CMT2Z mice can be improved by intrathecal injection of AAV-Morc2a or AAV-GHKL.

[0113]

[0114] Example 8. Morc2a in AAV gene therapy S87L / + Restoration of mouse peripheral nerve and muscle dysfunction

[0115] Morc2a S87L / + Since the mice exhibited cell death in the quadriceps femoris (Fig. 1B), we analyzed the characteristics of the quadriceps femoris. Morc2a S87L / + The quadriceps femoris fiber bundle size of the mice was 20% smaller than that of WT mice (p = 0.07). AAV-Morc2a and AAV-GHKL did not improve quadriceps femoris atrophy, but significantly reduced cell death in quadriceps femoris fibers, restoring the cell death to levels similar to those of WT mice (Fig. 6E). In addition, Morc2a S87L / +The reduced quadriceps femoris muscle mass in mice was restored to levels similar to those in WT mice by AAV gene therapy. Therefore, the maintenance of quadriceps femoris mass was expected to be due to a reduction in muscle cell death (Fig. 7A). Recovery of muscle strength was also observed, consistent with the restoration of quadriceps femoris muscle mass (Fig. 7B).

[0116] The standard assay for diagnosing CMT is nerve conduction velocity analysis. Gene therapy was performed on mice with Morc2a, similar to WT mice. S87L / + Morc2a enhanced motor nerve conduction velocity and potential in the mouse sciatic nerve (Fig. 7C). Nerve conduction analysis of sensory neurons also revealed therapeutic effects similar to those observed in motor neurons (Fig. 7D). In summary, Morc2a S87L / + Morc2aGHKL or Morc2aCDS in mice induced pathological recovery and functional improvement in the central nervous system, peripheral nervous system, and muscles.

[0117] When AAV-PHP.eB was administered intrathecally, it exerted therapeutic effects not only on the brain and spinal cord but also on peripheral nerves and muscles, which led to further analysis. AAV-Morc2a was transduced intrathecally into WT mice, and the presence of AAV particles was analyzed 3 weeks later by inverted terminal repeat (ITR) quantification. AAV was detected not only in the central nervous system (CNS) of the spinal cord and brain, but also in the liver, quadriceps, and sciatic nerve (Figure 7E). This was similar to transgene expression in tissues other than the CNS following intracerebroventricular injection of AAV-PHP.eB. In summary, AAV-PHP.eB can cross the blood-brain barrier and exert systemic therapeutic effects by restoring Morc2a or Morc2aGHKL ATPase.

[0118]

[0119] Example 9. Morc2a as a hydroxyl radical scavenger S87L / + Improvement of mouse neuropathy

[0120] Nicaraven, a low-molecular-weight substance, has recently been shown to have potential as a hydroxyl radical scavenger. Morc2a is activated by the reduction of hydroxyl radicals. S87L / + To examine changes in mouse neuropathy, Nicaraven treatment was performed. Nicaraven treatment lowered the concentration of hydroxyl radicals in Morc2ap.S87L MEFs, but no changes in cellular ATPase levels were observed. In conclusion, Nicaraven did not induce changes in cellular ATPase activity, but reduced hydroxyl radicals and thus cell death (Figure 8A). After verifying the therapeutic effect at the cellular level, diseased mice were intraperitoneally injected with 100 mg / kg Nicaraven for 30 days, and improvements in grip strength, a muscle function assessment index, were observed (Figure 8B). Additional pathological analysis revealed a decrease in cell death in the quadriceps femoris (Figure 8C).

[0121]

[0122] A schematic diagram of the present invention explained through the above examples is shown in Fig. 9. The present invention confirmed that Morc2a / MORC2 mutations reduce protein stability and cause insufficiency, resulting in neuropathy. In addition, it was suggested that cell death through increased hydroxyl radicals and decreased ATPase activity of cells caused by Morc2a / MORC2 mutations is the cause of CMT2Z, and as a solution to this, it was demonstrated that treatment of CMT2Z is ​​possible through replacement of the defective gene using the entire MORC2 gene or the MORC2GHKL ATPase gene or treatment with a hydroxyl radical scavenger.

Claims

1. A pharmaceutical composition for treating or preventing Charcot-Marie-Tooth type 2Z (CMT2Z), comprising as an active ingredient an AAV-PHP.eB vector comprising a Morc2aCDS represented by the amino acid sequence of SEQ ID NO: 1 or a Morc2aGHKL ATPase domain CDS represented by the amino acid sequence of SEQ ID NO:

2.

2. In paragraph 1, A pharmaceutical composition characterized in that the composition reduces hydroxyl radicals.

3. In paragraph 1, A pharmaceutical composition characterized in that the composition increases cellular ATPase activity.

4. In paragraph 1, A pharmaceutical composition characterized in that the composition reduces atrophy and cell death of the cerebellum.

5. In paragraph 1, A pharmaceutical composition characterized in that the composition reduces cell death of the quadriceps femoris muscle.

6. A method for preventing, improving or treating CMT2Z, comprising administering a pharmaceutical composition according to any one of claims 1 to 5 to a subject in need thereof.

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