Treatment methods for WWOX-related diseases

Abstract

The present disclosure provides methods and compositions for the treatment of WW domain-containing oxidoreductase (WWOX)-associated CNS disorders. In various embodiments, the invention involves expressing a heterologous WWOX gene in the brain of a subject, and in various embodiments, in neurons to treat or ameliorate conditions such as WOREE syndrome and SCAR12.

Description

[Technical Field]

[0001] This disclosure includes methods for treating WWOX-related diseases. [Background technology]

[0002] Germ cell mutations in WW domain-containing oxidotriductase (WWOX) have been reported in patients with epilepsy, ataxia, and developmental disorders of sexuality (DSD). More recently, a gene meta-analysis of diagnosed Alzheimer's disease has identified the WWOX gene as a novel risk locus. Several pieces of evidence strongly suggest that WWOX expression is necessary for the normal development and function of the central nervous system (CNS), and that mutations in WWOX cause the infant neurological disorder now known as WWOX-associated epileptic encephalopathy (WOREE) syndrome.

[0003] In WOREE, autosomal recessive WWOX nonsense mutations, partial and complete deletions are associated with extremely severe disease and very early death. Heterozygous parents with a single mutant allele of WWOX do not exhibit phenotypic symptoms. It is still unclear whether these carriers are susceptible to adult epilepsy. Most patients with WOREE carry compound heterozygous mutations in WWOX, making it difficult to target each mutation individually. A milder form of the disease is associated with WWOX missense mutations and is called autosomal recessive spinocerebellar ataxia 12 (SCAR12). Little is known about the mechanisms by which WWOX regulates CNS homeostasis. It is also unclear whether WWOX is downstream of other major effectors that antagonize epilepsy and other forms of neurological disorders.

[0004] Furthermore, recent evidence links minor mutations in WWOX to autism spectrum disorder (ASD). Copy number variants (CNVs) overlapping with WWOX have been reported in many ASD patients with milder phenotypes and IQ levels, close to the normal range defining WWOX as a candidate ASD gene. CNVs inheriting WWOX were identified as low-penetrance ASD risk factors. In addition, megaanalysis of multiple sclerosis patient samples revealed over 200 autosomal susceptibility variants, including WWOX variants. Therefore, WWOX perturbations extend beyond a single neurological disorder, suggesting that WWOX is a key player in several neurological disorders. [Overview of the project]

[0005] This disclosure provides methods and compositions for treating WWOX-related CNS disorders. In various embodiments, the present invention includes expressing heterologous WWOX genes in the brain of a subject, and in various embodiments, expressing heterologous WWOX genes in neurons to treat or improve conditions such as WOREE syndrome and SCAR12.

[0006] In some embodiments, the WWOX gene includes one or more regulatory elements, including a promoter that directs the expression of the WWOX gene in neurons. For example, the promoter may be a universal promoter or a neuron-specific promoter. An exemplary neuron-specific promoter is the synapsin I promoter. In various embodiments, the WWOX gene is either a wild-type gene or a functional derivative and includes an untranslated sequence (e.g., within the 3'-UTR) that enhances mRNA stability.

[0007] In some embodiments, the individual being treated is a pediatric or neonatal patient (e.g., a patient with WOREE or SCAR12). In some embodiments, early treatment prevents the manifestation of several clinical parameters of the disease. In some embodiments, the individual is an adult patient (e.g., with WOREE or SCAR12), and the treatment can improve one or more clinical parameters, such as epileptic seizures. In various embodiments, the treatment substantially reduces the frequency and / or severity of epileptic seizures.

[0008] For example, in some embodiments, the present invention provides a method for treating WOREE syndrome or SCAR12, the method comprising administering an AAV9 gene delivery system containing a WWOX wild-type gene under the control of the synapsin I promoter to the brain of a patient requiring such treatment. The AAV9 delivery system substantially comprises the nucleotide sequences described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5.

[0009] In other embodiments, the disclosure provides expression constructs comprising the WWOX wild-type gene or a functional derivative thereof under the expression control of a neuron-specific promoter. An exemplary neuron-specific promoter is the synapsin I promoter. In some embodiments, the nucleotide sequence substantially comprises the sequence described in SEQ ID NOs: 1, 3, 4, and / or 5. In various embodiments, the expression construct is a viral vector, such as an adeno-associated virus (AAV) delivery system. In some embodiments, the expression construct is an AAV9 delivery system.

[0010] Other aspects and embodiments of this disclosure will become apparent from the following detailed description and examples. [Brief explanation of the drawing]

[0011] [Figure 1A] This document outlines studies demonstrating the phenotype of conditional deletion of mouse Wwox in brain cells. Representative images of Wwox null (KO) and wild-type (WT) mice are shown on page 18. [Figure 1B]This document outlines a study demonstrating the phenotype of conditional deletion of mouse Wwox in brain cells. The graph shows the increase in weight (in grams, g) as a function of time. The delayed growth of Wwox null mice is evident from day 4 onwards compared to wild-type mice. Data points represent the mean body weight of mice (WT, n=3; KO, n=3). Error bars represent ±SEM (**P<0.01, ***P<0.001, Student's t-test). [Figure 1C] This document outlines a study demonstrating the phenotype of conditional deletion of Wwox in mouse brain cells. It presents Kaplan-Meier survival curves showing the postnatal lethality of Wwox null mice up to 3-4 weeks of age (WT, n=10; KO, n=11). (P=0.0023, Log-rank Mantel-Cox test). [Figure 1D] This document outlines studies demonstrating the phenotype of conditional deletion of mouse Wwox in brain cells. Conditional deletion of the Wwox gene in neural stem cells / neural progenitor cells shows overall developmental delay in N-KO mice compared to control mice (N-controls). Representative images of these mice are shown on page 17. [Figure 1E] This report outlines studies demonstrating the phenotype of conditional deletion of mouse Wwox in brain cells. N-KO mice exhibit reduced body weight compared to N-controls. Data points represent the average body weight of four mice per genotype. Error bars represent ±SEM (**P<0.01, ***P<0.001, Student's t-test). [Figure 1F] This document outlines a study demonstrating the phenotype of conditional deletion of Wwox in mouse brain cells. It presents Kaplan-Meier survival curves (n=14) showing mortality rates up to 3-4 weeks of age in N-KO compared to N-controls (n=12). (P=0.0002, Log-rank Mantel-Cox test). [Figure 1G]This is an overview of a study demonstrating the phenotype of conditional deletion of mouse Wwox in brain cells. Representative images of mice (P17) with conditional excision of Wwox in neurons (S-KO) showing (G) delayed growth and (H) weight loss (data points represent the average weight of mice (4 mice per genotype). Error bars represent ±SEM **P<0.01,***P<0.001, Student's t-test) and (I) early death (S-control n=13, S-KO n=15). (P-value 0.0001, Log-rank Mantel-Cox test). [Figure 1H] This is an overview of a study demonstrating the phenotype of conditional deletion of mouse Wwox in brain cells. Representative images of mice (P17) with conditional excision of Wwox in neurons (S-KO) showing (G) delayed growth and (H) weight loss (data points represent the average weight of mice (4 mice per genotype). Error bars represent ±SEM **P<0.01,***P<0.001, Student's t-test) and (I) early death (S-control n=13, S-KO n=15). (P-value 0.0001, Log-rank Mantel-Cox test). [Figure 1I] This is an overview of a study demonstrating the phenotype of conditional deletion of mouse Wwox in brain cells. Representative images of mice (P17) with conditional excision of Wwox in neurons (S-KO) showing (G) delayed growth and (H) weight loss (data points represent the average weight of mice (4 mice per genotype). Error bars represent ±SEM **P<0.01,***P<0.001, Student's t-test) and (I) early death (S-control n=13, S-KO n=15). (P-value 0.0001, Log-rank Mantel-Cox test). [Figure 1J]This paper outlines studies demonstrating the phenotypic effects of conditional deletion of mouse Wwox in brain cells. Conditional removal of Wwox in oligodendrocytes (O-KO) or astrocytes (G-KO) did not result in phenotypic abnormalities such as developmental delay (J and M, shown on P17), weight loss (K and N), and postnatal lethality (L and O) in either O-KO or G-KO mice compared to the corresponding control groups (G-control, O-control) (K and L: O-control n=11 and O-KO n=10, P-value 1.0, not significant, Log-rank Mantel-Cox test) (N and M: G-control n=8 and G-KO n=9 mice were used, P-value 1.0, not significant, Log-rank Mantel-Cox test). [Figure 1K] This paper outlines studies demonstrating the phenotypic effects of conditional deletion of mouse Wwox in brain cells. Conditional removal of Wwox in oligodendrocytes (O-KO) or astrocytes (G-KO) did not result in phenotypic abnormalities such as developmental delay (J and M, shown on P17), weight loss (K and N), and postnatal lethality (L and O) in either O-KO or G-KO mice compared to the corresponding control groups (G-control, O-control) (K and L: O-control n=11 and O-KO n=10, P-value 1.0, not significant, Log-rank Mantel-Cox test) (N and M: G-control n=8 and G-KO n=9 mice were used, P-value 1.0, not significant, Log-rank Mantel-Cox test). [Figure 1L]This paper outlines studies demonstrating the phenotypic effects of conditional deletion of mouse Wwox in brain cells. Conditional removal of Wwox in oligodendrocytes (O-KO) or astrocytes (G-KO) did not result in phenotypic abnormalities such as developmental delay (J and M, shown on P17), weight loss (K and N), and postnatal lethality (L and O) in either O-KO or G-KO mice compared to the corresponding control groups (G-control, O-control) (K and L: O-control n=11 and O-KO n=10, P-value 1.0, not significant, Log-rank Mantel-Cox test) (N and M: G-control n=8 and G-KO n=9 mice were used, P-value 1.0, not significant, Log-rank Mantel-Cox test). [Figure 1M] This paper outlines studies demonstrating the phenotypic effects of conditional deletion of mouse Wwox in brain cells. Conditional removal of Wwox in oligodendrocytes (O-KO) or astrocytes (G-KO) did not result in phenotypic abnormalities such as developmental delay (J and M, shown on P17), weight loss (K and N), and postnatal lethality (L and O) in either O-KO or G-KO mice compared to the corresponding control groups (G-control, O-control) (K and L: O-control n=11 and O-KO n=10, P-value 1.0, not significant, Log-rank Mantel-Cox test) (N and M: G-control n=8 and G-KO n=9 mice were used, P-value 1.0, not significant, Log-rank Mantel-Cox test). [Figure 1N]This paper outlines studies demonstrating the phenotypic effects of conditional deletion of mouse Wwox in brain cells. Conditional removal of Wwox in oligodendrocytes (O-KO) or astrocytes (G-KO) did not result in phenotypic abnormalities such as developmental delay (J and M, shown on P17), weight loss (K and N), and postnatal lethality (L and O) in either O-KO or G-KO mice compared to the corresponding control groups (G-control, O-control) (K and L: O-control n=11 and O-KO n=10, P-value 1.0, not significant, Log-rank Mantel-Cox test) (N and M: G-control n=8 and G-KO n=9 mice were used, P-value 1.0, not significant, Log-rank Mantel-Cox test). [Figure 10] This paper outlines studies demonstrating the phenotypic effects of conditional deletion of mouse Wwox in brain cells. Conditional removal of Wwox in oligodendrocytes (O-KO) or astrocytes (G-KO) did not result in phenotypic abnormalities such as developmental delay (J and M, shown on P17), weight loss (K and N), and postnatal lethality (L and O) in either O-KO or G-KO mice compared to the corresponding control groups (G-control, O-control) (K and L: O-control n=11 and O-KO n=10, P-value 1.0, not significant, Log-rank Mantel-Cox test) (N and M: G-control n=8 and G-KO n=9 mice were used, P-value 1.0, not significant, Log-rank Mantel-Cox test). [Figure 2A] This section outlines studies demonstrating neocortical hyperexcitability in the S-KO neocortex. It presents in vivo recordings (P13-P17) from S-control, S-HT, and S-KO genes, showing burst activity in S-KO (shown in red) compared to heterozygotes (S-HT, shown in blue) and S-control (shown in black). [Figure 2B]This section outlines studies demonstrating neocortical hyperexcitability in the S-KO neocortex. In vitro recordings (P13-P17) derived from the superficial neocortex show spontaneous neocortical bursts in isolated neocortical section specimens. An inset shows an enlarged trace of a burst event from an S-KO example. A whole-brain inset shows the location of recording electrodes in vivo. Of all data, including both spontaneous activity and electrical stimulation artifacts, obtained from 11 S-control, 7 S-control, 14 S-HT, and 24 S-KO animal sections (11 S-control, 23 S-HT, and 42 S-KO sections), 0 S-control sections showed bursts (0%), 4 sections from 3 S-HT animals showed bursts (approximately 20%), and 36 sections from 20 S-KO animals showed bursts (approximately 84%). [Figure 2C] This document outlines studies demonstrating neocortical hyperexcitability in the S-KO neocortex. It also presents a second example of in vivo recordings and their time-frequency spectrograms. [Figure 2D]This document outlines studies demonstrating neocortical hyperexcitability in the S-KO neocortex. Power spectral analysis of in vivo and in vitro datasets of S-control, S-HT, and S-KO obtained from stimulus artifact-free data. Gray bars indicate significant regions identified at each frequency. In vivo, increased power is observed in S-KO compared to S-HT at 12–20 Hz, and in S-KO compared to S-control at 7–15 Hz. In vitro, increased power is observed in S-KO compared to S-HT and S-control at 3–13 Hz. Z-scores of in vitro data normalized to the mean and standard deviation at 320–400 Hz are shown. Box plots show power normalized across the frequency bands indicated on the horizontal axis. At 0-4.9 Hz and 5-9 Hz, the power of S-KO was significantly increased compared to S-control alone (in vivo, S-control, n=5 subjects; S-KO, n=7 subjects; S-HT, n=7 subjects, *P<0.05; in vitro *P<0.05; **P<0.01 Student's t-test, S-KO compared to S-control and S-HT, S-control, n=11 sections, 7 animals, S-HT, n=11 sections, 8 animals, S-KO, n=34 sections, 20 subjects). [Figure 2E] This document outlines studies demonstrating neocortical hyperexcitability in the S-KO neocortex. The data shows responses to electrical stimulation from layer V, recorded in layers II / III of the neocortex. Arrows indicate the termination and onset of the first peak response, and carets indicate the termination and onset of the second peak response at a 100uA stimulation intensity. [Figure 2F] This section outlines studies demonstrating neocortical hyperexcitability in the S-KO neocortex. The amplitudes of the first and second peak responses are shown (first peak: *P=0.0127; second peak: **P=0.008, *P=0.0202; Wilcoxon rank-sum test; S-control, n=6 intercepts, 3 subjects; S-HT, n=8 intercepts, 5 subjects; S-KO, n=8 intercepts, 6 subjects). [Figure 3A] This report outlines studies demonstrating that neuronal deletion in Wwox impairs myelin formation and oligodendrocyte maturation. Quantification of the total fluorescence intensity of CNPs and MBPs from three identical fragments of the cortical region shows a decrease in intensity in S-KO compared to S-control (n=3) (n=3). [Figure 3B] This paper outlines studies demonstrating that Wwox neuronal deletion impairs myelination and oligodendrocyte maturation. Representative images of cerebellar brain sections immunostained with anti-CNP and anti-MBP are shown. [Figure 3C] This report outlines studies demonstrating that neuronal deletion in Wwox impairs myelin formation and oligodendrocyte maturation. Quantification of CNP and MBP fluorescence intensity shows lower intensity in S-KO compared to S-control. [Figure 3D] This document outlines studies demonstrating that Wwox neuronal deletion impairs myelination and oligodendrocyte maturation. The images show sagittal sections of brain tissue immunostained for CC1 and anti-PDGFRα. The image (marked with a white dotted line and enlarged with a white square) shows a decrease in the number of mature oligodendrocytes in the corpus callosum in S-KO compared to S-controls at P17. [Figure 3E] This report outlines studies demonstrating that neuronal deletion in Wwox impairs myelination and oligodendrocyte maturation. It includes quantification of CC1 and PDGFRα-positive cells within the corpus callosum, showing a significant decrease in CC1-positive cells and an increase in PDGFRα-positive cells in SK-O mice compared to S-controls. Data points represent the number of cells counted within an area (0.5 mm²) from three independent sections of S-control (n=3) and S-KO mice (n=3). Error bars represent ±SEM (*P≦0.01, **P≦0.001). Scale bars: A) 50 μm, B) 2 μm, and C) 50 μm. [Figure 4A] This document outlines studies demonstrating that Wwox neuronal deletion reduces myelin formation and axonal conduction. The image shows electron micrographs (EM) from the mid-sagittal section of the corpus callosum, showing fewer myelinated axons and more unmyelinated axons in the optic nerve of S-KO (n=3) compared to S-control (n=3) at P17. [Figure 4B]This document outlines studies demonstrating that Wwox neuronal deletion reduces myelination and axonal conduction. It quantifies the number of myelinated corpus callosum axons and unmyelinated optic nerve axons per visual field in S-controls and S-KOs. The graphs represent myelinated corpus callosum axons (S-controls, n=2500 and S-KO, n=1200) and unmyelinated optic nerve axons (S-controls, n=500 and S-KO, n=3000), counted from EM images per FOV (field of view). Error bars represent ±SEM (***P<0.001, Student's t-test). [Figure 4C] This document outlines a study demonstrating that Wwox neuronal deletion reduces myelination and axonal conduction. It presents g-ratio analysis showing the reduction in axonal myelin thickness in the corpus callosum and optic nerve in S-KO (n=3) compared to S-controls (n=3). Axonal diameter and myelin thickness were calculated from electron micrographs of the corpus callosum (n=300 for each genotype) and optic nerve (n=300 for each genotype) (***P≦0.001, Student's t-test). [Figure 4D] This document outlines studies demonstrating that Wwox neuronal deletion reduces myelination and axonal conduction. Representative examples of evoked responses from corpus callosum stimulation are presented: (i) recorded at a vertical distance of 250 μm from the stimulator. The inset shows the electrode and stimulation configuration. (N1 *P=0.0104; N2=0.5737 Wilcoxon rank-sum test) (ii) Latency from the start of stimulation to N1 and N2. (ii) Ratio of the amplitude of N1 to the amplitude of N2. (N1 / N2). (**P=0.0030; Wilcoxon rank-sum test n=8 intercepts, 5 subjects; S-control; n=8 intercepts, 6 subjects, S-KO). [Figure 5] This report outlines a study demonstrating the non-cellular autonomous function of WWOX in OPC differentiation. Quantification of oligodendrocyte counts before, after, and after myelination (covering a 14 mm diameter region). Box plots show the results of two independent experiments (WT-DRGs + WT-OPCs, n=4; KO-DRGs + WT-OPCs, n=4). (**P<0.01. No significant difference in ns). Scale bars: A) 100 μm, D) 50 μm. [Figure 6A]This document outlines a study demonstrating that WWOX-deficient oligocortical spheroids exhibit hyperexcitability and myelin sheathing. A schematic diagram of the oligocortical spheroid section setup is shown. One electrode is used for recording local collective potentials (LFPs), and a second electrode is used for whole-cell patch recording. The electrodes are positioned 150 μm and 10–15 μm away from the edge of the section. [Figure 6B] This document outlines a study demonstrating that WWOX-deficient oligocortical spheroids exhibit hyperexcitability and myelin sheathing. Cellular resting membrane potentials were measured at 15 weeks in whole-cell patch-clamp recordings of OS-WT and OS-WWOX-KO organoids (OS-WT n=3, OS-WWOX-KO n=4; OS-WT RMP=-52.1±0.90mV, OS-WWOX-KO RMP=-21.28±6.91mV). (*P<0.01, Student's t-test). [Figure 6C] This document outlines a study demonstrating that WWOX-deficient oligocortical spheroids exhibit hyperexcitability and myelin sheathing. Mean spectral powers of OS-WT and OS-WWOX-KO organoids at week 15 under baseline conditions were presented (n=9 intersections, 3 organoids for OS-WT; n=6 intersections, 2 organoids for OS-WWOX-KO; OS-WT AUC=0.0285±0.0097, OS-WWOX-KO AUC=0.0541±0.0093. (*P<0.05, Student's t-test)). [Figure 6D] This document outlines studies demonstrating that WWOX-deficient oligocortical spheroids exhibit hyperexcitability and myelin sheathing. The area under the curve for the mean spectral power of (C) in the δ and θ range (0.5–7.9 Hz) is shown. (*P<0.05, Student's t-test). [Figure 6E] This document outlines a study demonstrating that WWOX-deficient oligocortical spheroids exhibit hyperexcitability and myelin sheathing. The image shows OS at 30 weeks, stained for CNP(OLs) and MBP(OLs). The image on the right is a magnified view of the area enclosed by the rectangle on the left. (OS-WT, n=5; OS-WWOX-KO, n=5). Scale bar 100 μm. [Figure 6F]This document outlines a study demonstrating that WWOX-deficient oligocortical spheroids exhibit hyperexcitability and myelin sheathing. Representative electron micrographs show more myelinated axons (OS-WT) (n=3) compared to OS-WWOX-KO (n=3) organoids at 37 weeks. Enlarged area indicated in the box. Scale bar 100 μm. [Figure 6G] This report summarizes studies demonstrating that WWOX-deficient oligocortical spheroids exhibit hyperexcitability and myelin sheathing defects. The bar graphs represent the proportion of myelinated and unmyelinated axons in OS-WT (n=2) and OS-WWOX-KO (n=3). Scale bar: 40 μm. [Figure 7A] This document outlines a study demonstrating that restoring WWOX in synapsin I-positive neurons improves growth and extends the lifespan of Wwox null mice. The diagram shows a plasmid vector construct containing the mouse Wwox gene under a human synapsin I promoter. The Wwox gene sequence is followed by the IRES promoter and the EGFP gene sequence. [Figure 7B] This paper outlines a study showing that restoring WWOX in synapsin I-positive neurons improves growth and extends the lifespan of Wwox null mice. The image shows the physical appearance of wild-type (WT) and Wwox null mice injected with either AAV9-hSynI-GFP (control virus) or AAV9-hSynI-mWwox-IRES-GFP virus at P17. [Figure 7C] This document outlines a study showing that restoring WWOX in synapsin I-positive neurons improves growth and extends the lifespan of Wwox null mice. The graph shows mouse weight on a specified day. Error bars represent ±SEM (n=4 mice per genotype). [Figure 7D] This document outlines a study showing that restoring WWOX in synapsin I-positive neurons improves growth and extends the lifespan of Wwox null mice. The graph shows the blood glucose levels of mice on a specified day. Error bars represent ±SEM (n=4 mice per genotype). [Figure 7E]This report outlines a study demonstrating that WWOX restoration in synapsin I-positive neurons improves growth and extends the lifespan of Wwox null mice. The Kaplan-Meier survival graph shows the extended lifespan of Wwox knockout mice injected with AAV9-hSynI-mWwox (n=18) compared to mice injected with AAV9-hSynI-GFP (n=6) or without injection (n=10) (p-value < 0.0001, Log-rank Mantel-Cox test). [Figure 7F] This report outlines a study demonstrating that WWOX restoration in synapsin I-positive neurons improves growth and extends the lifespan of Wwox null mice. The Kaplan-Meier survival graph shows the extended lifespan of Wwox knockout mice injected with AAV9-hSynI-hWWOX (n=6) [median 92 days] compared to mice injected with AAV9-hSynI-GFP (n=4) or not injected (n=8) (p-value = 0.0001, Log-rank Mantel-Cox test). [Figure 8A] This document outlines a study demonstrating that WWOX-induced neuronal recovery reduces epileptic activity in the neocortex. Representative traces of cell adhesion recordings at P20–21 days are shown from WT, KO, and KO[KO+A-Wwox] pups treated with AAV9-hSynI-mWwox. Traces represent spontaneous neocortical activity (showing action potentials). Panels represent 12-second recordings with insets showing zoom-ins at 0.5-second intervals. Clear hyperactivity in the KO brain is observed in these representative traces, with KO showing bursts of action potentials. Graphs show the mean of 20 neurons from WT pups (n=2), 20 neurons from KO+A-Wwox pups (n=2), and 30 neurons from KO pups (n=2) (****p-value < 0.0001, Student's t-test). [Figure 8B]This document outlines a study demonstrating that neuronal recovery in WWOX reduces epileptic activity in the neocortex. Representative traces of cell adhesion recordings performed in WT and KO+A-Wwox adult mice (6 months old) are shown. Traces represent spontaneous neocortical activity (action potentials). The panel shows a 12-second recording with insets showing zoom-ins at 0.5-second intervals. The graph shows the mean of 60 neurons from WT adult mice (n=3) and 60 neurons from KO+A-Wwox adult mice (n=3). No significant difference was observed in the mean firing rates between WT and KO+A-Wwox adult mice. [Figure 9A] This document outlines a study demonstrating that WWOX recovery in neurons improves myelination by promoting OPC differentiation in WWOX nulls. Images of whole-brain sagittal sections immunolabeled with anti-MBP at P17 from mice are shown (n=3 per group). MBP staining in the cortex, hippocampus, and cerebellum is shown (magnified from the top panel). [Figure 9B] This document outlines a study demonstrating that WWOX recovery in neurons improves myelination by promoting OPC differentiation in Wwox nulls. The image shows sagittal sections of brain (P17) tissue immunostained for CC1 and PDGFRα. It also shows an increase in the number of mature oligodendrocytes in the corpus callosum after treatment with AAV9-hSynI-mWwox compared to Wwox nulls injected with AAV9-hSynI-GFP virus. [Figure 9C] This report outlines a study demonstrating that WWOX recovery improves myelination in neurons by promoting OPC differentiation in Wwox null mice. The graph shows the quantification of CC1 and PDGFRα-positive cells counted from three similar sagittal brain sections per mouse genotype in the corpus callosum (area 0.5 mm²) of WT (n=3), KO (n=3), and KO+A-Wwox (n=3) mice. Error bars represent ±SEM (*p value < 0.01, **p value < 0.001, ***p value < 0.0001). Scale bars: (A) 2 mm (upper panel), 250 μm (middle and lower panels), (B) 250 μm (upper panel), 50 μm (middle and lower panels). [Figure 10A]This document outlines a study demonstrating that WWOX recovery in neurons reverses the abnormal behavioral phenotype in Wwox null mice. Representative images (periphery and center) of an open-field study showing the follow-up patterns of WT (females, n=7, males, n=6) and AAVmWwox-injected KO mice (females, n=7, males, n=5) at 8–10 weeks. [Figure 10B] This report outlines a study demonstrating that WWOX recovery in neurons reverses the abnormal behavioral phenotype in Wwox null mice. The graphs show movement speed and distance (cm) from open-field tracking. [Figure 10C] This report outlines a study demonstrating that WWOX recovery in neurons reverses the abnormal behavioral phenotype in Wwox null mice. The graphs show movement speed and distance (cm) from open-field tracking. [Figure 10D] This document outlines a study demonstrating that WWOX recovery in neurons reverses the abnormal behavioral phenotype in Wwox null mice. The image shows mouse tracking images from an elevated cusp maze test using WT and KO+AAV-Wwox mice (8-10 weeks old). The left mouse tracking image shows OA (open arm) and CO (closed arm) as white dotted boxes. The graph shows the time spent in the closed arm (i) and open arm (ii) for WT (female, n=7, male n=6) and KO+AAV-Wwox (female n=7, male n=5) mice (seconds). [Figure 10E] This paper outlines a study demonstrating that WWOX recovery in neurons reverses the abnormal behavioral phenotype in Wwox null mice. The graphs show the latency (seconds) of mice falling from a rotarod in various tests (WT and mice rescued at 8-10 weeks). Females (WT, n=7; KO+-mWwox, n=5) and males (WT, n=5; KO+-mWwox, n=5) are shown separately (***p-value < 0.0001). [Figure 11A]This document outlines research on the generation and characterization of WWOX knockout brain organoids. CO at week 10 was stained with WWOX, the panradicular glial marker SOX2, and the ventricular radial glial (vRG) marker CRYAB. The image on the right shows a magnified view of the area enclosed by the rectangle. The dashed line defines the ventricular space (WT: n=8, derived from 3 batches; KO: n=8, derived from 3 batches). Scale = 50 μm (left), 25 μm (right). [Figure 11B] This document outlines research on the generation and characterization of WWOX knockout brain organoids. It includes quantification of markers representing various populations from the ventricular zone (VZ), subventricular zone (SVZ), and cortical plate (CP). NeuN represents mature neurons, TBR2 represents intermediate progenitor cells (IP), and SOX2 represents radial glial cells (vRG). Box plots represent the first and third quartiles, with whiskers indicating minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WT: n=8, 3 batches; KO: n=8, 3 batches; W-AAV: n=4, 1 batch). [Figure 11C] This report outlines research on the generation and characterization of WWOX knockout brain organoids. qPCR analysis was performed to assess the expression levels of various neuronal markers in 15 weeks of CO: SOX2 and PAX6 (progenitor cells), TUBB3 (panneurons), SLC17A6 and SLC17A7 (VGLUT2 and VGLUT1; glutamatergic neurons), and GAD1 and GAD2 (GAD67 and GAD65; GABAergic neurons). The y-axis represents the change in relative expression levels. Data are expressed as mean SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WT: n=4, from one batch; KO: n=4, from one batch; and W-AAV: n=4, from one batch). [Figure 11D]This document outlines a study on the generation and characterization of WWOX knockout brain organoids. Immunofluorescence (IF) staining of the glutamatergic neuron marker VGLUT1 and the GABAergic neuron marker GAD67 (GAD1) ​​was performed on CO at week 10 (WT: n=8, from 3 batches; KO: n=8, from 3 batches; and W-AAV: n=4, from 1 batch). Scale = 100 μm. Image quantification is shown. VGLUT1 and GAD67 were quantified as the surface area covered by staining and normalized to the number of nuclei in each image. Box plots represent the first and third quartiles, with whiskers indicating minimum and maximum points and a central band representing the median. Because the samples were not normally distributed, statistical significance was determined using the Kruskal-Wallis test with Dunn's multiple comparison test. Outliers were excluded by the ROUT test (Q=1%). Data information: ns (not significant), *P ≤ 0.05, and ****P ≤ 0.0001. [Figure 12A] This document outlines a study demonstrating that WWOX-KO brain organoids exhibited hyperexcitability and epileptic-like activity. The study includes sample recordings from 7-week-old hESC-derived brain organoids (CO). Sample tracing results show visible differences in local collective potentials; WWOX-KO CO showed increased activity compared to WT under baseline conditions (left) and in the presence of 100 μM 4-AP (right). [Figure 12B] This report outlines studies demonstrating hyperexcitability and epileptic-like activity in WWOX-KO brain organoids. Sample recordings are from 7-week-old hESC-derived brain organoids (CO). Mean spectral powers of WT and KO CO at 7 weeks under baseline conditions are shown. Lines indicate the frequency range of 0.25–1 Hz. Statistical significance was determined using a two-sided independent Welch's t-test (WT: n=14 intercepts, 5 organoids, and 3 batches; KO: n=14 intercepts, 8 organoids, and 3 batches). [Figure 12C]This document outlines a study demonstrating hyperexcitability and epileptic-like activity in WWOX-KO brain organoids. Sample recordings are from 7-week-old hESC-derived brain organoids (CO). The normalized area under the curve of mean spectral power in the frequency range of 0.25–1 Hz is shown. Data are represented by mean SEM. Statistical significance was determined using a two-sided independent Welch's t-test. The numbers on each bar indicate the number of intercepts and organoids (i.e., intercepts (organoids)) analyzed. [Figure 12D] The WWOX coding sequence was reintroduced into 6-week-old WWOX-KO organoids (lenti-WWOX) using lentiviral transduction. Immunofluorescence staining shows WWOX expression in different populations of WWOX-KO organoids after lentiviral infection. NT = untreated. Scale = 50 μm. [Figure 12E] Lentiviral transduction was used to reintroduce the WWOX coding sequence into WWOX-KO CO strains at 6 weeks (lentiviral-WWOX). Area under the normalized curve for the mean spectral power of the WT strain, the two KO strains, and the two KO strains infected with lentiviral-WWOX at 7 weeks under baseline conditions, over the frequency range of 0.25–1 Hz. Data are represented by mean SEM. The numbers in each bar indicate the number of intercepts and organoids analyzed (i.e., intercepts (organoids)). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test. Data information: ***P ≤ 0.001 and ****P ≤ 0.0001. [Figure 13A] This report outlines studies demonstrating impaired astrogenesis and DNA damage response in WWOX-KO brain organoids. CO at weeks 15 and 24 was stained for the astrocyte and radial glial marker GFAP, and the astrocyte-specific marker S100b (WT W15: n=9 from 3 batches; KO W15: n=16 from 3 batches; W-AAV W15: n=4 organoids from 1 batch; WT W24: n=10 from 4 batches; KO W24: n=9 from 3 batches; and W-AAV W24: n=4 from 1 batch). Scale = 100 μm. [Figure 13B]This report outlines a study demonstrating impaired astrogenesis and DNA damage response in WWOX-KO brain organoids. The image shows qPCR analysis of astrocyte markers in CO at week 15. The y-axis represents the change in relative expression levels. Data are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WT: n=4, from one batch; KO: n=4, from one batch; and W-AAV: n=4, from one batch). [Figure 13C] This report outlines studies demonstrating impaired astrogenesis and DNA damage response in WWOX-KO brain organoids. The image shows qPCR analysis of astrocyte markers in CO at week 24. The y-axis represents changes in relative expression levels. Data are expressed as mean ± SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WT: n=4 from two batches; KO: n=3 from two batches; and W-AAV: n=3 from one batch). [Figure 13D] This document outlines a study demonstrating impaired astrogenesis and DNA damage response in WWOX-KO brain organoids. It shows IF staining of CO at week 6 for astrocyte markers around the VZ (WT: n=6, 2 batches, and KO: n=6, 2 batches). Scale = 100 μm (left) and 50 μm (right). [Figure 13E] This document outlines studies demonstrating impaired astrogenesis and DNA damage response in WWOX-KO brain organoids. Staining of the DNA damage markers cH2AX and 53BP1 in cell nuclei at 6 weeks CO under physiological conditions, along with the panradicular glial marker SOX2 (WT: n=8 from 3 separate batches; KO: n=12 from 3 separate batches; and W-AAV: n=4 from 1 batch). Scale = 50 μm (left), 25 μm (right). [Figure 13F]This document outlines studies demonstrating impaired astrogenesis and DNA damage response in WWOX-KO brain organoids. The images show quantifications of cH2AX (top) and 53BP1 lesions (bottom) in the nuclei of cells constituting the innermost layer of the VZ, normalized to the total number of nuclei in this layer. Box plots represent the first and third quartiles, with whiskers indicating minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WT: n=8 organoids from 3 batches; KO: n=12 organoids from 3 batches; and W-AAV: n=4 from 1 batch). Data information: ns (not significant), *P≦0.05, **P≦0.01, and ****P≦0.0001. [Figure 14A] This report outlines a study demonstrating that brain organoid RNA sequencing revealed major differentiation defects. RNA sequencing (RNA-seq) and transcriptome analysis (WT: n=2, KO: n=4) of CO at week 15 are included. qPCR analysis of selected Wnt target genes validates the RNA sequencing results. The y-axis represents changes in relative expression levels. Data are expressed as mean_SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WT: n=4 from one batch; KO: n=4 from one batch; and W-AAV: n=4 from one batch). [Figure 14B] This document outlines a study demonstrating that RNA sequencing of brain organoids revealed major differentiation defects. RNA sequencing (RNA-seq) and transcriptome analysis (WT: n=2, KO: n=4) of CO at week 15 were performed. CO at week 16 was subdivided into cytoplasmic (C) and nuclear (N) fractions. The experiment was performed twice, using a total of two WT organoids and four KO organoids (two for each KO lineage). KAP-1 represents the nucleus, and HSP90 represents the cytoplasm. The numbers at the bottom are quantifications of the nuclear fraction with β-catenin band intensity normalized to the cytoplasmic fraction [bC(N / C)]. [Figure 14C]This document outlines a study demonstrating that RNA sequencing of brain organoids revealed major differentiation defects. It includes RNA sequencing (RNA-seq) and transcriptome analysis (WT: n=2, KO: n=4) of CO organoids at 15 weeks. A heatmap shows the expression levels of markers from six different layers of the human cortex, from the deepest to the most superficial, in organoids at 15 weeks: TBR1, BCL11B (CTIP2), SATB2, POU3F2 (BRN2), CUX1, and RELN. [Figure 14D] This document outlines a study demonstrating that brain organoid RNA sequencing revealed major differentiation defects. It includes RNA sequencing (RNA-seq) and transcriptome analysis (WT: n=2, KO: n=4) of CO at week 15. IF staining of CO at week 15 examines decreased levels of deep cortical markers CTIP2 (BCL11B) and TBR1, as well as the superficial marker SATB2 (WT: n=3; KO: n=4; and W-AAV: n=4). Scale = 50 μm. [Figure 14E] This document outlines a study demonstrating that RNA sequencing of brain organoids reveals major differentiation defects. RNA sequencing (RNA-seq) and transcriptome analysis of CO at week 15 (WT: n=2, KO: n=4). Quantification of cortical markers observed in G, normalized to the total number of nuclei. The y-axis shows the scalar change compared to the mean of WT CO. Box plots represent the first and third quartiles, with whiskers indicating minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WT: n=9 from 3 batches; KO: n=16 from 3 batches; and WAAV: n=4 organoids from 1 batch). Data information: *P≦0.05, **P≦0.01, ***P≦0.001, and ****P≦0.0001. [Figure 15A]This document outlines a study demonstrating that WWOX-associated epileptic encephalopathy brain organoids reproduce neuronal abnormalities. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with WOREE syndrome and their healthy parents, reprogrammed into iPSCs, and then differentiated into COs. WSM COs at 10 weeks of age (WSM F1: n=2 from one batch; WSM M2: n=2 from one batch; WSM S2: n=2 from one batch; WSM S5: n=2 from one batch; WSM S5 W-AAV3: n=2 from one batch; and WSM S5 W-AAV6: n=2 from one batch) were stained for the progenitor cell marker SOX2, the neuronal marker b3-tubulin, and WWOX. Scale = 50 μm. [Figure 15B] This document outlines a study demonstrating that brain organoids associated with WWOX-associated epileptic encephalopathy (WWOX) reproduce neuronal abnormalities. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with WOREE syndrome and their healthy parents, reprogrammed into iPSCs, and then differentiated into COs. Representative traces of spontaneous spikes recorded from 7-week-old CO neurons in WSM P, WSM S, and WSM S W-AAV patients. Each recording is 12 seconds long, with a 0.5-second zoom (right-hand box). [Figure 15C] This report outlines a study demonstrating that brain organoids associated with WWOX-associated epileptic encephalopathy (WWOX) reproduce neuronal abnormalities. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with WOREE syndrome and their healthy parents, reprogrammed into iPSCs, and then differentiated into CO organoids (COs). Mean firing rates are shown for 24 neurons derived from WSM P CO (4 organoids), 41 neurons (3 organoids) derived from WSM S CO, and 40 neurons derived from WSM S W-AAV organoids (3 organoids). Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test. Bars represent mean SEM. [Figure 15D]This document outlines a study demonstrating that brain organoids from WWOX-associated epileptic encephalopathy (WWOX) reproduce neuronal abnormalities. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with WOREE syndrome and their healthy parents, reprogrammed into iPSCs, and then differentiated into CO cells. GAD67 and VGLUT1 immunostaining was performed on WSM CO cells at 10 weeks (WSM F1: n=2 from one batch; WSM M2: n=2 from one batch; WSM S2: n=2 from one batch; WSM S5: n=2 from one batch; WSM S5 W-AAV3: n=2 from one batch; and WSM S5 W-AAV6: n=2 from one batch). Scale = 50 μm. [Figure 15E] This document outlines a study demonstrating that brain organoids from WWOX-associated epileptic encephalopathy (WWOX) reproduce neuronal abnormalities. Peripheral blood mononuclear cells (PBMCs) were isolated from patients with WOREE syndrome and their healthy parents, reprogrammed into iPSCs, and then differentiated into COs. Quantification of the data is shown in Figure 15D. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WSM F1: n=2, 1 batch; WSM M2: n=2, 1 batch; WSM S2: n=2, 1 batch; WSM S5: n=2, 1 batch; WSM S5 W-AAV3: n=2, 1 batch; and WSM S5 W-AAV6: n=2, 1 batch). Data information: ns (not significant), *P≦0.05, **P≦0.01, ***P≦0.001, and ****P≦0.0001. [Figure 16A] This document outlines a study demonstrating that WWOX-associated epileptic encephalopathy exhibits similar molecular abnormalities to those seen in complete WWOX loss. WSM CO2 at week 15, stained for the astrocyte markers GFAP and S100b (WSM F1: n=2 from one batch; WSM M2: n=3 from one batch; WSM S2: n=2 from one batch; WSM S5: n=2 from one batch; WSM S5 W-AAV3: n=2 from one batch; and WSM S5 W-AAV6: n=2 from one batch). Scale = 50 μm. [Figure 16B]This document outlines studies demonstrating that WWOX-associated epileptic encephalopathy exhibits similar molecular abnormalities to those seen in complete WWOX loss. The study also shows impaired DNA damage response in WOREE-derived organoids. SOX2 exhibits radial glial formation in the VZ (WSM F1: n=4 from one batch; WSM M2: n=4 from one batch; WSM S2: n=4 from one batch; WSM S5: n=4 from one batch; WSM S5 W-AAV3: n=4 from one batch; and WSM S5 W-AAV6: n=4 from one batch). Scale = 50 μm (left) and 25 μm (right). [Figure 16C] This document outlines a study demonstrating that WWOX-associated epileptic encephalopathy exhibits similar molecular abnormalities to those seen in complete WWOX loss. The image shows quantification of DNA damage lesions in the nuclei of cells within the VZ of WSM CO at week 6, indicated by cH2AX (left) and 53BP1 (right). Box plots represent the first and third quartiles, with whiskers indicating minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WSM F1: n=4, 1 batch; WSM M2: n=4, 1 batch; WSM S2: n=4, 1 batch; WSM S5: n=4, 1 batch; WSM S5 W-AAV3: n=4, 1 batch; and WSM S5 W-AAV6: n=4, 1 batch). [Figure 16D] This report outlines a study demonstrating that WWOX-associated epileptic encephalopathy exhibits similar molecular abnormalities to those observed in complete WWOX loss. The study includes qPCR analysis of selected Wnt target genes in WSM CO at week 10. The y-axis represents changes in relative expression levels. Data are presented as mean SEM. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WSM P: n=6, derived from one batch; WSM S: n=4, derived from one batch; and WSM S W-AAV: n=4, derived from one batch). [Figure 16E]This document outlines a study demonstrating that WWOX-associated epileptic encephalopathy exhibits similar molecular abnormalities to those seen in complete WWOX loss. WSM CO2 at week 15 stained for cortical markers CTIP2 and SATB2 (WSM F1: n=2 from one batch; WSM M2: n=3 from one batch; WSM S2: n=2 from one batch; WSM S5: n=2 from one batch; WSM S5 W-AAV3: n=2 from one batch; and WSM S5 W-AAV6: n=2 from one batch). Scale = 50 μm. [Figure 16F] This document outlines studies demonstrating that WWOX-associated epileptic encephalopathy exhibits similar molecular abnormalities to those seen in complete WWOX loss. Quantification of staining is shown in E. Box plots represent the first and third quartiles, with whiskers indicating minimum and maximum points and a central band representing the median. Statistical significance was determined using one-way ANOVA with Tukey's multiple comparison test (WSM F1: n=2, 1 batch; WSM M2: n=3, 1 batch; WSM S2: n=2, 1 batch; WSM S5: n=2, 1 batch; WSM S5 W-AAV3: n=2, 1 batch; and WSM S5 W-AAV6: n=2, 1 batch). Data information: *P≦0.05, **P≦0.01, ***P≦0.001, and ****P≦0.0001. [Modes for carrying out the invention]

[0012] This disclosure provides methods and compositions for the treatment of WW domain-containing oxidodriductase (WWOX)-related CNS diseases. In various embodiments, the present invention includes expressing heterologous WWOX genes in the brain of a subject, and in various embodiments, includes expressing heterologous WWOX genes in neurons to treat or improve conditions such as WOREE syndrome and SCAR12.

[0013] The term "WWOX-related CNS disorders" refers to disorders caused by or associated with mutated WWOX genes or abnormal WWOX expression. Mutations can result in complete or partial genomic deletions (nonsense mutations) that lead to protein loss or shortening, or, in milder cases, missense mutations. These disorders manifest in the CNS. Examples of such disorders include WWOX-related epileptic encephalopathy (WOREE) syndrome, spinocerebellar ataxia, autosomal recessive 12 (SCAR12), multiple sclerosis, Alzheimer's disease, West syndrome, autism, and disorders of sex development (DSD).

[0014] In certain embodiments, the present invention provides a method for treating WWOX-related CNS disorders. This method involves administering to the brain of a patient requiring such treatment a WWOX wild-type gene or a functional derivative thereof, which is under the control of regulatory elements(s) that bring about WWOX expression in the brain. In various embodiments, the WWOX-related CNS disorders are selected from WOREE syndrome, SCAR12, Alzheimer's disease, West syndrome, autism, multiple sclerosis, and DSD.

[0015] In some embodiments, the WWOX-associated CNS disorder is WOREE syndrome or SCAR12. In some such embodiments, the patient has a compound heterozygous mutation in WWOX.

[0016] The terms “treatment” or “treating” in this disclosure mean improving at least one clinical parameter associated with the disease, and further include preventing the onset of the disease (or one or more clinical parameters of the disease). The terms “treatment” or “treating” also mean improving (compared to an untreated subject) at least one symptom or aspect of the disease, such as survival rate, growth, number or frequency of epileptic seizures, cognitive function, social functioning, fertility (e.g., in autism), ataxia, retinopathy, intellectual disability, and microcephaly.

[0017] The term "WWOX wild-type gene" refers to a gene containing the WWOX coding sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2. The term "WWOX wild-type gene" further includes a cDNA sequence (represented by SEQ ID NO: 1) or a gene sequence containing one or more introns. For example, a complete gene sequence containing introns is represented by the NCBI reference sequence NC_000016.10. The term "WWOX wild-type gene" further includes native nucleotide polymorphisms or amino acid modifications in the human population (with respect to SEQ ID NO: 1 or SEQ ID NO: 2, respectively) that are not associated with disease or loss of WWOX function or expression. In various embodiments, a WWOX gene may be a functional equivalent of the WWOX wild-type gene, i.e., a WWOX gene may encode one or more amino acid modifications (e.g., 1, 2, 3, 4, or 5 amino acid modifications) that are independently selected from insertions, deletions, or substitutions and do not significantly affect WWOX activity or expression (e.g., in neurons). Generally, functional derivatives encode amino acid sequences having at least 95% sequence identity, or at least 96% sequence identity, or at least 97% sequence identity, or at least 98% sequence identity, or at least 99% sequence identity, to the amino acid sequence of SEQ ID NO: 2. The WWOX wild-type gene may further include a promoter and regulatory elements including 5' and 3' untranslated regions, but these regulatory elements are not limited to the natural WWOX gene sequence, but can instead be selected to achieve a desired level of mRNA expression or turnover and / or gene expression with desired cell specificity. In some embodiments, the WWOX wild-type gene does not contain substantial untranslated regions, i.e., it is essentially composed of or may consist of the WWOX coding sequence. According to embodiments of the present invention, the WWOX wild-type gene includes at least a heterologous promoter. As used herein, “heterologous promoter” is a promoter located at a non-natural location, for example, at a location that controls the expression of a coding sequence that is not controlled in nature.

[0018] In some embodiments, the WWOX wild-type gene encodes the amino acid sequence of SEQ ID NO: 2. In some embodiments, the WWOX wild-type gene is a cDNA sequence, and in some embodiments, the cDNA sequence includes the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3.

[0019] The term "promoter" refers to a DNA sequence that can control RNA expression, for example, the transcription of the WWOX wild-type gene. A promoter sequence contains at least a proximal element to control gene expression, and optionally may further contain more distal upstream elements, the latter of which are often called enhancers. Therefore, an "enhancer" is either a DNA sequence that can stimulate promoter activity, or a heterologous element inserted to enhance the promoter's inherent elements or its level or tissue specificity. A promoter may be entirely derived from a native gene, or it may consist of different elements derived from different promoters present in nature, or it may even contain synthetic DNA segments. Furthermore, the precise boundaries of regulatory sequences may not be fully defined, and it is recognized that several variations of DNA fragments may possess identical promoter activity.

[0020] In some embodiments, the regulatory element includes a promoter that directs the expression of the WWOX gene in neurons. For example, the promoter may be a universal promoter. Examples of universal promoters include the CMV promoter, the E2F1 promoter, and the U1snRNA promoter, or derivatives thereof. In some embodiments, the promoter is a universal promoter, and the construct is delivered specifically or selectively to neurons. In some embodiments, the regulatory element is a promoter that is specifically expressed in neurons (neuron-specific promoter). Exemplary neuron-specific promoters include the synapsin I promoter, the CamKII promoter, the MeCP2 promoter, the NSE promoter, and the Hb9 promoter, or derivatives thereof. In some embodiments, the promoter is not expressed or is expressed at a lower level in glial cells. In some embodiments, the promoter is not expressed or is expressed at a lower level in oligodendrocytes and / or astrocytes.

[0021] In some embodiments, the regulatory element is a synapsin I promoter or a functional derivative thereof for directing neuron-specific expression. In some embodiments, the promoter is, for example, the synapsin I promoter represented by GeneBank registry number NM_006950 (SEQ ID NO: 4), which confers highly neuron-specific and long-term transgene expression. The structure of the synapsin I promoter is described in Schloch et al., Neutron-specific Gene Expression of Synapsin I, J. Biol. Chem. 271(6):3317-3323 (1996). In various embodiments, the synapsin I promoter is a functional derivative containing an NRSE / RE-1 sequence that confers neuron-specific expression. In various embodiments, the synapsin I promoter contains a nucleotide sequence of at least about 250 nucleotides, or at least about 300 nucleotides, or at least about 350 nucleotides from the 3' end of SEQ ID NO: 4. The synapsin I promoter (or a portion thereof) may contain up to approximately 20%, up to approximately 10%, or up to approximately 5% nucleotide modifications, provided that neuron-specific or neuron-selective expression of the promoter is maintained.

[0022] In some embodiments, the WWOX wild-type gene under the control of a regulatory element, or a functional derivative thereof, comprises a nucleotide sequence substantially described in SEQ ID NO: 5.

[0023] In other embodiments, the regulatory element is a promoter that directs the expression of the WWOX gene in oligodendrocytes. Exemplary promoters include the MBP promoter, the PLP1 promoter, and the CNP promoter, or derivatives thereof. In some embodiments, the regulatory element includes a promoter that directs the expression of the WWOX gene in astrocytes. Exemplary promoters include the GFAP promoter or the S100b promoter, or derivatives thereof.

[0024] In some embodiments, the WWOX wild-type gene or promoter further comprises one or more enhancer sequences, which may include distal portions of the synapsin I promoter or other neuron-specific promoters. In some embodiments, the promoter comprises one or more neuron-specific or neuron-selective enhancers for increasing expression levels in neurons. See, for example, Charron G. et al., Multiple Neuron-specific Enhancers in the Gene Coding for the Human Neurofilament Light Chain. J. Biol. Chem. 270(51):3064-30610 (1995).

[0025] In various embodiments, the WWOX wild-type gene or functional derivative includes an untranslated sequence (e.g., 3'-UTR) that enhances mRNA stability. For example, in some embodiments, the WWOX wild-type gene may include a β-globin mRNA 3'-UTR, or components of the β-globin mRNA 3'-UTR that confer mRNA stability. In some embodiments, the WWOX wild-type gene includes a 3' untranslated sequence derived from mRNA that exhibits low turnover in neurons. In some embodiments, the transcribed WWOX nucleotide sequence includes one or more woodchuck hepatitis posttranscriptional regulatory elements (WPREs) that can enhance stability. In some embodiments, the WPREs are contained within the 3'UTR of the WWOX gene.

[0026] In some embodiments, the WWOX wild-type gene is delivered along with one or more detectable labels, including but not limited to fluorescent proteins such as GFP or RFP, which enable visualization of the expression of a WWOX-containing expression construct.

[0027] In some embodiments, the WWOX wild-type gene or a functional derivative (or fragment thereof) is delivered together with a Cas endonuclease enzyme or a polynucleotide encoding the Cas endonuclease enzyme, and a guide RNA (gRNA) or a polynucleotide encoding the gRNA to induce or enhance the insertion of the WWOX wild-type gene or a portion thereof. In some embodiments, WWOX-associated CNS disease is characterized by known mutations in WWOX. In such embodiments, a gRNA complementary to the mutated region, or a DNA sequence encoding the gRNA, is delivered together with the Cas endonuclease. In these embodiments, the WWOX wild-type gene may be a fragment of the WWOX wild-type gene for replacing a mutated sequence cleaved by a Cas endonuclease enzyme (e.g., Cas9).

[0028] For example, if the exact mutation in the WWOX gene in an individual is known, the individual can be treated by administering a Cas endonuclease (e.g., Cas9) enzyme and a gRNA targeting the mutated sequence into the individual's brain, thereby reverting to the wild type by editing the mutated sequence, cleaving the mutated region, and / or replacing the sequence in the mutated region with the sequence in the wild-type region. In some mutations, a functional WWOX gene can be obtained simply by cleaving the mutated sequence with a Cas endonuclease. For other mutations, it is necessary not only to cleav the mutated sequence but also to replace it with the wild-type sequence (a fragment of the WWOX wild-type gene). In such cases, this method also includes administering a donor DNA sequence corresponding to the fragment of the WWOX wild-type gene to replace the mutated sequence.

[0029] In some embodiments, Cas endonucleases (e.g., Cas9) may be administered to brain cells as a protein, or as a polynucleotide encoding an enzyme expressible in brain cells (e.g., neurons). In some embodiments, Cas endonucleases are expressed via a neuron-specific promoter (e.g., synapsin I promoter) as described herein. In some embodiments, Cas endonucleases are delivered as mRNA and therefore do not require transcription in transfected cells.

[0030] When using polynucleotides encoding Cas endonucleases, the promoters and delivery vectors described herein for the WWOX gene may be used, or delivery vectors capable of delivering longer polynucleotides (which may be more suitable for delivering polynucleotides encoding Cas endonucleases), such as AAV6 and lentiviral vectors, may be used. When delivering Cas endonucleases as proteins, delivery particles, liposomes, etc., may be used for their delivery to the brain (e.g., to neurons).

[0031] Similarly, the gRNA may be delivered as an RNA molecule or as a DNA molecule encoding the gRNA using the delivery and promoter systems described herein for the WWOX gene. In some embodiments, the gRNA is expressed via a neuron-specific promoter.

[0032] WWOX polynucleotides for replacing mutated DNA can be administered as separate sequences or as part of a vector containing sequences encoding Cas endonuclease and gRNA. In such cases, donor DNA can be cleaved from the vector, for example, by using a second set of “donor-specific” guide RNAs that, with the support of Cas endonuclease, can cleave blunt-ended donors from the vector.

[0033] Cas endonuclease molecules of various species (e.g., Cas9), including S. pyogenes and S. thermophilus Cas9, can be used in the methods and compositions described herein. Other Cas endonucleases are described in U.S. Patent Publication 20160010076 (which is incorporated herein by reference in its entirety). The constructs and methods described herein may include the use of any Cas endonuclease, including the Cas9 enzyme, and their corresponding gRNAs or other compatible gRNAs. Cas9 of the Streptococcus thermophilus LMD-9 CRISPR1 system has been shown to function in human cells (see Cong et al., Science 339, 819 (2013)).

[0034] Generally, there are two distinct systems of guide RNA: System 1, which uses separate crRNAs and tracrRNAs that work together to guide Cas9 cleavage, and System 2, which uses a chimeric crRNA-tracrRNA hybrid that combines two separate gRNAs in a single system (called single guide RNA or sgRNA) (see also Jinek et al., Science 2012;337:816-821). TracrRNAs can be variably shortened and have been shown to function at various lengths in both the separate system (System 1) and the chimeric gRNA system (System 2).

[0035] Cas endonucleases can be guided to a specific 17-20 nt genomic target having an additional protospacer facile motif of the sequence NGG, using a gRNA, e.g., sgRNA or tracrRNA / crRNA, which has a 17-20 nt gRNA at its 5' end that is complementary to the complementary strand of the genomic DNA target site. Accordingly, embodiments may use a single guide RNA containing a crRNA fused to a tracrRNA that is normally transcoded, having a sequence at its 5' end that is complementary to the target sequence, e.g., a protospacer facile motif (PAM), e.g., 25-17, optionally 20 or fewer nucleotides (nt), e.g., 20, 19, 18, or 17 nt, preferably 17 or 18 nt, of the complementary strand to the target sequence, e.g., immediately 5' to the protospacer facile motif (PAM), e.g., NGG, NAG, or NNGG, e.g., a single Cas9 guide RNA described in Mali et al., Science 2013 Feb.15;339(6121):823-6. The gRNA may contain XN, which can be any sequence that does not interfere with the binding of ribonucleic acid to Cas9, and N (in RNA) can be 0-200, for example, 0-100, 0-50, or 0-20.

[0036] In some embodiments, the gRNA contains one or more adenine (A) or uracil (U) nucleotides at its 3' end. In some embodiments, the gRNA contains one or more U, e.g., 1 to 8 or more U, at its 3' end as a result of the presence of one or more T, which are used as termination signals to terminate RNA PolIII transcription.

[0037] In some embodiments, the gRNA targets a site that differs from any remaining sequence in the genome by at least three or more mismatches to minimize off-target effects. Modified RNA oligonucleotides, such as locked nucleic acids (LNAs), have been shown to enhance the specificity of RNA-DNA hybridization by locking the modified oligonucleotide into a more favorable (stable) three-dimensional structure. Therefore, the gRNAs disclosed herein may comprise one or more modified RNA oligonucleotides. For example, the truncated guide RNA molecules described herein have one, some, or all of the regions of the guide RNA complementary to the target sequence modified, e.g., locked (2'-O-4'-C methylene bridge), 5'-methylcytidine, 2'-O-methyl-pseudridine, or a ribose phosphate backbone substituted with a polyamide chain (peptide nucleic acid), e.g., synthetic ribonucleic acid.

[0038] gRNA may be provided as is or in an expression vector. Vectors for expressing gRNA may include an RNA Pol III promoter that drives gRNA expression, such as the H1, U6, or 7SK promoter. These human promoters can express gRNA in mammalian cells after plasmid transfection. Alternatively, a T7 promoter may be used for in vitro transcription, for example, allowing the RNA to be transcribed and purified in vitro. Vectors suitable for expressing short RNAs, such as siRNA, shRNA, or other small RNAs, can be used.

[0039] The sequence (promoter and gene, and optionally additional sequences) can be delivered by any delivery system suitable for delivery to the CNS, either by direct delivery to the CNS or systemic delivery. In various embodiments, the WWOX wild-type gene or its functional derivatives are delivered using viral vectors, polymer nanoparticles, inorganic nanoparticles, lipid nanoparticles, or exosomes. In some embodiments, the WWOX wild-type gene or its functional derivatives are delivered by viral vectors. The viral vector may be an adeno-associated virus (AAV) delivery system. In some embodiments, the AAV delivery system is AAV9, which crosses the blood-brain barrier better than other AAV serotypes. Further viral delivery systems that may be used include lentivirus and herpes simplex virus delivery systems.

[0040] Another suitable delivery vehicle for the CNS typically involves nanoparticles having a size of less than 200 nm, or about less than 150 nm, or about less than 100 nm. These may include lipid-based nanoparticles, polymer nanoparticles, dendrimers, and inorganic nanoparticles, some of which can be modified to cross the blood-brain barrier (BBB). In some embodiments, the delivery system enhances the uptake of nanoparticles via the BBB by actively targeting delivery using a transporter or receptor ligand. A preferred pathway in this approach is receptor (or transporter)-mediated transcytosis, thereby transporting cargo (e.g., nanoparticles) between the apical and basal surfaces of the brain's EC. For example, low-density lipoproteins undergo transcytosis via the EC through a receptor-mediated process, bypassing the lysosomal compartment and being released at the basal surface on the brain side. Furthermore, since the BBB contains transporters to amino acids, using a natural arginine transporter for delivery is one approach for delivery to the brain.

[0041] Another medium for brain delivery is exosomes, which are small extracellular vesicles secreted by cells. The main advantage of exosomes over other synthetic nanoparticles is their non-immunogenicity, which leads to a long and stable circulation.

[0042] In some embodiments, compounds or electrical stimulation can be used for delivery to the brain to temporarily open the blood-brain barrier (BBB), allowing systemically administered high concentrations of polynucleotides to reach the brain. Examples of such compounds include cerepot (a bradykinin analog) or regadenoson (an adenosine receptor agonist). Another method to enhance penetration is by ultrasound, which is an attractive technique for facilitating drug crossing the BBB. Microbubble-enhanced ultrasound (MEUS), a non-invasive technique, effectively assists drug crossing the BBB. Another approach is transcranial magnetic stimulation (TMS), which stimulates neuronal activity, increasing glutamate release and facilitating delivery across the BBB. (Review by Xiaowei Don. 2018;8(6):1481-149 – the entire work is incorporated herein by reference).

[0043] The route of administration of the desired delivery vehicle may be systemic delivery without further manipulation (using particles or viral vectors that essentially enter the BBB), or it may be systemic in connection with various procedures to temporarily open the BBB (such as microbubble-enhanced ultrasound (MEUS) or transcranial magnetic stimulation (TMS). In other embodiments, delivery is by nasal administration.

[0044] In yet another embodiment, delivery to the brain is by delivery to the cerebrospinal fluid via an intraventricular pathway. Another option is delivery via the cisterna magna pathway by injection, which is an alternative method to delivery to the cerebrospinal fluid (CSF) and delivers the gene extensively throughout the CNS. In some embodiments, administration is by direct injection into the parenchyma or injection into the cerebrospinal fluid via an intraventricular pathway, and by an intrathecal (cisterna magna or lumbar) pathway.

[0045] In some embodiments, the individual being treated is a pediatric or neonatal patient (e.g., a patient with WOREE or SCAR12). In some embodiments, early treatment prevents the manifestation of several clinical parameters of the disease, such as growth retardation, epileptic seizures, cognitive impairment, and intellectual disability. In some embodiments, the individual is an adult patient (e.g., with WOREE or SCAR12), and treatment can improve one or more clinical parameters, such as epileptic seizures. In various embodiments, the individual or patient exhibits one or more symptoms selected from growth retardation, epileptic seizures, cognitive impairment, social dysfunction, fertility impairment, ataxia, retinopathy, intellectual disability, and microcephaly. In various embodiments, treatment substantially reduces the frequency and / or severity of epileptic seizures in patients with WOREE or SCAR12.

[0046] In various embodiments, the number of administration episodes to an individual does not exceed 10, 9, 8, 7, 6, 5, or 4. In various embodiments, the number of administration episodes is 3 or less. In some embodiments, the number of administration episodes is 2 or less. For example, in various embodiments, the number of administration episodes is 1.

[0047] For example, in some embodiments, the present invention provides a method for treating WOREE syndrome or SCAR12, the method comprising administering an AAV9 gene delivery system containing the WWOX wild-type gene under the control of the synapsin I promoter (i.e., encoding the polypeptide of SEQ ID NO: 2) to the brain of a patient requiring such treatment. The AAV9 delivery system substantially comprises the nucleotide sequences described in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 4, and / or SEQ ID NO: 5.

[0048] In other embodiments, the disclosure provides expression constructs comprising the WWOX wild-type gene or a functional derivative thereof under the expression control of a neuron-specific promoter. Exemplary neuron-specific promoters include the synapsin I promoter, the CamKII promoter, the MeCP2 promoter, the NSE promoter, and the Hb9 promoter, or derivatives thereof. In various embodiments, the promoter is synapsin I or a derivative thereof, as previously described. In some embodiments, the nucleotide sequence comprises the sequence substantially described in SEQ ID NOs: 1, 3, 5, and / or 5.

[0049] In various embodiments, the expression construct is a viral vector, such as an adeno-associated virus (AAV) delivery system. In some embodiments, the expression construct is an AAV9 delivery system. Specific examples of such vectors are shown in Figure 7A.

[0050] In some embodiments, the expression construct is incorporated into a pharmaceutical composition for administration to the brain. The composition further comprises a pharmaceutically acceptable carrier suitable for injection, including direct injection into the brain or CNS, or systemic administration.

[0051] In yet another embodiment, the present invention provides a method for treating WOREE syndrome or SCAR12, comprising administering a pharmaceutical composition to a patient in need of treatment. Thus, the present invention provides the use of a pharmaceutical composition in the treatment of WOREE or SCAR12. The present invention encompasses embodiments described in the following sections. [Section 1] A method for treating a WW domain-containing oxidodriductase (WWOX)-related CNS disease, the method comprising administering to the brain of a patient requiring such treatment a WWOX wild-type gene under the control of a regulatory element that expresses WWOX in the brain, or a functional derivative thereof. [Section 2] The method according to item 1, wherein the WWOX-associated CNS disorder is selected from WWOX-associated epileptic encephalopathy (WOREE) syndrome, spinocerebellar ataxia, autosomal recessive 12 (SCAR12), Alzheimer's disease, West syndrome, autism, multiple sclerosis, and disorders of sex development (DSD). [Section 3] The method according to item 2, wherein the WWOX-related CNS disorder is WOREE syndrome or SCAR12. [Section 4] The method according to any one of items 1 to 3, wherein the patient has a compound heterozygous mutation in WWOX. [Section 5] The method according to any one of claims 1 to 4, wherein the regulatory element is a promoter that directs the expression of the WWOX gene in a neuron. [Section 6] The method according to item 5, wherein the promoter is a universal promoter. [Section 7] The method according to claim 6, wherein the promoter is the CMV promoter, the E2F1 promoter, and the U1snRNA promoter, or derivatives thereof. [Section 8] The method according to item 5, wherein the regulatory element is a promoter that is specifically expressed in neurons. [Section 9] The method according to claim 8, wherein the promoter is selected from the synapsin I promoter, the CamKII promoter, the MeCP2 promoter, the NSE promoter, and the Hb9 promoter, or derivatives thereof. [Section 10] The method according to item 8 or 9, wherein the promoter is not expressed in glial cells or is expressed at a lower level. [Section 11] The method according to item 10, wherein the promoter is not expressed or is expressed at a lower level in oligodendrocytes and / or astrocytes. [Section 12] The method according to any one of claims 8 to 11, wherein the regulatory element is a synapsin I promoter or a derivative thereof. [Section 13] The method according to any one of claims 1 to 4, wherein the regulatory element is a promoter that directs the expression of the WWOX gene in an oligodendrocyte. [Section 14] The method according to claim 13, wherein the promoter is selected from the MBP promoter, the PLP1 promoter, and the CNP promoter, or derivatives thereof. [Section 15] The method according to any one of claims 1 to 4, wherein the regulatory element is a promoter that directs the expression of the WWOX gene in astrocytes. [Section 16] The method according to claim 15, wherein the promoter is a GFAP promoter or an S100b promoter, or a derivative thereof. [Section 17] The method according to any one of claims 5 to 16, wherein the promoter further comprises one or more enhancer sequences. [Section 18] The method according to any one of items 1 to 17, wherein the WWOX gene includes an untranslated sequence that enhances mRNA stability. [Section 19] The method according to any one of items 1 to 17, wherein the WWOX wild-type gene is delivered together with one or more detectable labels. [Section 20] The method according to item 19, wherein the detectable label is an encoded fluorescent protein. [Section 21] The method according to any one of claims 1 to 20, wherein the WWOX wild-type gene or a functional derivative thereof is delivered using polymer nanoparticles, inorganic nanoparticles, liponoparticles, or exosomes. [Section 22] The method according to any one of claims 1 to 21, comprising delivering the WWOX wild-type gene or a functional derivative thereof together with a Cas enzyme or a polynucleotide encoding the Cas enzyme, and a gRNA or a polynucleotide encoding the gRNA to instruct the insertion of the WWOX wild-type gene or a portion thereof. [Section 23] The method according to any one of claims 1 to 22, wherein the WWOX wild-type gene or a functional derivative thereof is delivered by a viral vector. [Section 24] The method according to item 23, wherein the viral vector is an adeno-associated virus (AAV) delivery system. [Section 25] The method according to item 24, wherein the AAV delivery system is AAV9. [Section 26] The method according to any one of items 1 to 25, wherein the WWOX wild-type gene encodes the amino acid sequence of SEQ ID NO: 2. [Section 27] The method according to item 26, wherein the WWOX wild-type gene comprises one or more introns. [Section 28] The method according to item 26, wherein the WWOX wild-type gene is cDNA. [Section 29] The method according to claim 28, wherein the WWOX wild-type gene or a functional derivative thereof, under the control of a regulatory element, comprises a nucleotide sequence substantially described in SEQ ID NO: 1 or 3. [Section 30] The method according to any one of claims 1 to 29, wherein the administration is by a route selected from direct injection into the parenchyma, injection into the cerebrospinal fluid via the ventricles, and an intrathecal (cisternus or lumbar) route. [Section 31] The method according to any one of items 1 to 30, wherein the individual is a pediatric patient or a neonatal patient. [Section 32] The method according to any one of items 1 to 31, wherein the individual is an adult patient. [Section 33] The method according to paragraph 31 or 32, wherein the patient exhibits one or more symptoms selected from growth retardation, epileptic seizures, cognitive impairment, social dysfunction, reproductive disorders, ataxia, retinopathy, intellectual disability, and microcephaly. [Section 34] The method described in any one of items 1 to 33, wherein the number of administration episodes is three or less. [Section 35] The method according to item 34, wherein the number of administration episodes is two or less. [Section 36] The method according to item 34, wherein the administration episode is a single episode. [Section 37] A method for treating WOREE syndrome or SCAR12, the method comprising administering an AAV9 gene delivery system containing a WWOX wild-type gene under the control of the synapsin-1 promoter to the brain of a patient requiring such treatment. [Section 38] The method according to claim 37, wherein the AAV9 delivery system comprises a nucleotide sequence substantially described in SEQ ID NO: 1 or SEQ ID NO: 3. [Section 39] An expression construct comprising the WWOX wild-type gene, or a functional derivative thereof, under the expression control of a neuron-specific promoter. [Section 40] The expression construct according to claim 39, wherein the promoter is selected from the synapsin 1 promoter, the CamKII promoter, the MeCP2 promoter, the NSE promoter, and the Hb9 promoter, or derivatives thereof. [Section 41] The expression construct according to claim 40, wherein the promoter is synapsin 1 or a derivative thereof. [Section 42] An expression construct according to item 41, comprising a nucleotide sequence substantially described in SEQ ID NO: 1, 3, 4, or 5. [Section 43] The expression construct according to any one of items 39 to 42, wherein the expression construct is a viral vector. [Section 44] The expression construct according to item 43, wherein the viral vector is adeno-associated virus (AAV). [Section 45] The expression construct according to item 44, wherein the AAV is AAV9. [Section 46] A pharmaceutical composition for direct administration to the brain, comprising an expression construct described in any one of items 39 to 45, and a pharmaceutically acceptable carrier suitable for direct injection into the brain. [Section 47] A method for treating WOREE syndrome or SCAR12, comprising administering the pharmaceutical composition described in item 46 to a patient in need. [Section 48] Use of the pharmaceutical composition described in item 46 in the treatment of the WOREE or SCAR12.

[0052] definition To better understand this application, several definitions are provided below. Such definitions are intended to encompass grammatical equivalents.

[0053] The terms "a" or "an" refer to one or more entities, that is, to multiple referents. Therefore, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably in this specification. Furthermore, a reference to an "element" using the indefinite article "a" or "an" does not preclude the possibility of two or more elements existing unless the context clearly requires that only one element exists.

[0054] The term "identity" relating to two or more nucleic acid sequences or polypeptide sequences means that two or more sequences or subsequences have a certain proportion of nucleotide or amino acid residues that are identical when compared and aligned for the greatest correspondence, as measured by one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to those skilled in the art) or by visual inspection.

[0055] The term "approximately" means ±10% of the relevant value, unless the context suggests otherwise. [Examples]

[0056] Example 1: Neuron loss of Wwox related to WOREE syndrome causes epilepsy and myelin deficiency The oxidoreductase containing WW domain (WWOX) gene is mapped to chromosome 16q23.1-q23.2, which includes FRA16D, one of the most active chromosomal fragile sites. 1,2 WWOX encodes a 46 kDa protein, and its ability of protein - protein interaction shows tumor suppressor function in several types of cancers. 3,4 In fact, WWOX acts as a scaffold protein and regulates the localization, stability and function of its partners. 5 Multiple evidences show that WWOX function is associated with genomic stability, cell metabolism, and maintenance of cytoskeletal organization. 1,3,6,7 Surprisingly, high expression levels of WWOX have been observed in cells of the central nervous system (CNS) 8,9 , suggesting an important role of WWOX in CNS biology. However, the exact role of WWOX in CNS development and diseases is unclear.

[0057] In recent years, germline deleterious mutations (missense, nonsense, partial deletion / complete deletion) in the WWOX gene have been associated with SCAR12 (spinocerebellar ataxia, autosomal recessive - 12, OMIM614322) and WOREE syndrome (WWOX - related epileptic encephalopathy), and the latter is also known as early infantile epileptic encephalopathy - 28 (EIEE28, OMIM616211). 10,11 It was inferred that the severity of this disease largely depends on the type of mutation and its impact on WWOX expression. For example, the most severe phenotypes were observed in children with WOREE syndrome, in whom WWOX was completely lost, resulting in refractory seizures and prenatal or postnatal lethality. 10 Patients with SCAR12 mainly caused by missense mutations of WWOX showed mild phenotypes such as ataxia and epilepsy. 10,11 Recently, WWOX mutations have also been discovered in patients with West syndrome characterized by epileptic spasms accompanied by arrhythmia 12,13In most cases, magnetic resonance imaging (MRI) of children with mutations in WWOX revealed abnormalities such as corpus callosum malformation, progressive brain atrophy, delayed myelin formation, and optic nerve atrophy. 10,14-18 Little is known about how the deficiency in WWOX causes these neurological abnormalities.

[0058] Target deletions in mouse Wwox and spontaneous Wwox mutations in Lde rats phenotypicly mimicked a complex human neurological phenotype, including epileptic seizures, growth retardation, ataxia, and postnatal lethality. 11,19-21 To further shed light on the key cellular and molecular players in Wwox models and human diseases, we conditionally generated mouse models with Wwox deficiency in either neural stem cells and progenitor cells (using nestin-Cre;N-KO), mature neurons (synapsin I-Cre;S-KO), oligodendrocytes (Olig2-Cre;O-KO), or astrocytes (GFAP-Cre;G-KO), and studied their phenotypes and disease mechanisms alongside the previously described Wwox-null mice. 19,20 Surprisingly, WWOX deficiency in either neural stem cells and progenitor cells (N-KO) or neuronal cells (S-KO mice) resulted in severe epilepsy, ataxia, and premature death within 3–4 weeks, reproducing the phenotype observed in WWOX-null mice. Characterization of molecular and cellular changes in S-KO mice revealed marked myelin dysplasia and a decrease in the number of mature oligodendrocytes, attributed to WWOX's non-cellular autonomous function. Furthermore, modeling the complete loss of WWOX and the generation of human oligocortical spheroids (OS) in human embryonic stem cells (hESCs) further confirmed the role of WWOX in epilepsy and myelin dysplasia. These findings highlight the central role of WWOX in CNS biology and its therapeutic potential in the treatment of WWOX-related neurological disorders.

[0059] Materials and methods Cell culture and plasmids WiBR3 hES cells were maintained under 5% CO2 conditions on irradiated DR4 mouse embryonic fibroblast (MEF) feeder plates under FGF / KOSR conditions: DMEM-F12 (Gibco; 21331-020 or Biological Industries; 01-170-1A) supplemented with 15% Knockout Serum Replacement (KOSR, Gibco; 10828-028), 1% GlutaMax (Gibco; 35050-038), 1% MEM non-essential amino acids (NEAA, Biological Industries; 01-340-1B), 1% sodium pyruvate (Biological Industries; 03-042-1B), 1% penicillin-streptomycin (P / S, Biological Industries; 03-031-113), and 8 ng / mL bFGF (Peprotech; 100-18B). The culture medium was changed daily, and the cultures were subculturified every 5-7 days by trypsin treatment with type C trypsin (Biological Industries; 03-053-1B). For 24-48 hours after trypsin treatment, hESCs were treated with a 10 μM concentration of Rho-related kinase inhibitor (ROCKi, also known as Y27632) (Cayman; 10005583). For hESC transfection, cells were cultured in 10 μM ROCKi 24 hours before electroporation. Cells were detached using trypsin C solution and mixed with a total of 100 μg of DNA construct (px330 plasmid containing sgRNA targeting exon 1 mixed with pNTK-GFP in a 1:5 ratio) in PBS (Ca 2+ and Mg 2+ The cells were resuspended in (containing) and electroporated using a Gene Pulser Xcell System (Bio-Rad; 250V, 500μF, 0.4cm cuvette). Subsequently, the cells were plated onto an MEF feeder layer under the above conditions. After 48 hours, GFP-positive cells were selected and then, approximately 10 days later, sparsely seeded (2,000 cells per 10cm plate) onto MEF feeder plates for colony isolation.

[0060] mouse The generation of Wwox null mice has been reported previously. 19 These mice were maintained in an FVB background. Mice possessing two loxp sites adjacent to exon 1 of the Wwox genome locus (Wwox flox / flox ) is previously described 22 In the current study, these mice were used to conditionally eliminate WWOX expression in CNS cells. Mice possessing transgenic Cre recombinase under the promoter of Nestin (origin Jax strain: 003771, B6.Cg-Tg(Nes-cre)1Kln / J) were a generous gift from Dr. Tal Burstyn-Cohen of the Faculty of Medicine, Hadassah Dental College, Hebrew University. Synapsin I-Cre (stock: 003966, B6.Cg-Tg(Syn1-cre)671Jxm / J), Olig2-Cre (stock: 025567, B6.129-Olig2 tm1.1(cre)Wdr The mouse strains / J) and GFAP-Cre(B6.Cg-Tg(GFAP-cre)77.6Mvs / 2J) were purchased from Jackson Laboratory in the United States. The Wwox mice possess transgenic Cre recombinase. flox / flox The mouse is considered to have a homozygous conditional deletion of Wwox, and N-KO(Wwox flox / flox Nestine-Cre + ), S-KO (Wwox flox / flox Synapsin-Cre + ), O-KO (Wwox flox / flox ;Olig2 cre / + ) and G-KO (Wwox flox / flox ;Gfap-Cre +It is expressed as ). All mice were PCR-typed by extracting tail / ear DNA using specific primers. All conditional models were maintained in a C57BL6 / J;129sv mixed genetic background. All conditional models contained the Rosa26-loxp-STOP-tdTomato reporter allele. Animals were maintained in SPF units with free access to food and water in a 12-hour light / dark cycle. All animal-related experiments were conducted in accordance with the prior approval of the Institutional Animal Care Use Committee (HU-IACUC) of Hebrew University.

[0061] DRG-OPC co-culture Co-culture experiments of DRG neurons and OPCs are performed using previously published protocols. 23The procedure was carried out according to the following guidelines. Briefly, DRG neurons were isolated from mouse embryos at E13.5. The embryo genotype was determined, and DRGs were recovered in cold L-15 medium. The tissue was dissociated with 0.25% trypsin, pulverized, centrifuged, and resuspended in NB medium (Neurobasal, B27 supplement, 0.5 mM L-glutamine, and penicillin-streptomycin). Pre-washed 13 mm diameter glass coverslips were placed in a 4-well dish, coated with Matrigel (at room temperature for 1 hour), and then coated with poly-D-lysine (at room temperature for 30 minutes). Cells were seeded in NB medium at a density of 40,000 cells / 13 mm coverslip and maintained in a humidified incubator at 37°C and 5% CO2. Non-neuronal cells were removed by treating the cultures with fluorodeoxyuridine in divs 2, 4, and 6. 50% of the cell medium was replaced every 3 days, and OPCs were added in DIV15. OPCs were isolated from P0-P2 year pup mice. The cortex was isolated in ice-cold L-15 medium, dissociated using a syringe (19G followed by 21G for mouse tissue), ground, centrifuged, and resuspended in glial seeding medium (DMEM containing 10% fetal bovine serum and penicillin-streptomycin) on a PDL-coated flask. Glial cells were maintained in a humidified incubator at 37°C and 5% CO2, and 50% of the cell medium was replaced every 3 days. In DIV10, OPCs were isolated by rapidly adhering the flask to a culture dish (3 times for 10 minutes at 37°C) after vigorously shaking it to deplete the astrocytes, or by seeding purified OPCs (200,000 / coverslip) onto DRG neuron cultures and maintaining them in co-culture medium (DMEM containing B27 and N2 supplements, 5 mg / ml N-acetylcysteine, 5 mM forskolin, and penicillin-streptomycin). After changing the medium every other day for 9-11 days, the cultures were fixed and stained for analysis.

[0062] Generation and culture of oligocortical spheroids Brain organoids were generated from hESCs, as previously described. 24In short, human WiBR3 cells were maintained in mitotically inactivated MEFs. Four to seven days before the start of the protocol, the cells were passaged onto 60 mm plates coated with MEFs and grown until a concentration of 70-80% was reached. On day 0, hESC colonies were detached from the MEFs using 0.7 mg / ml collagenase D solution (Sigma; 11088858001), and dissociated into single-cell suspensions using type C trypsin for 2 minutes. After dissociation, the cells were counted and supplemented with 20% KOSR, 1% GlutaMax, 1% NEAA, 1% P / S, and 100 μM 2-mercaptoethanol (Sigma; M3148). The cells were then supplemented with 10 μM dorsomorphine (Sigma; P5499) or 100 nm LDN-193189 (Axon medchem; Axon1509), as well as 10 μM SB-431542 (Sigma; S4317) and 10 μM Rocki, and suspended in hESC medium consisting of DMEM / F12 sterilized through a 0.22 μm filter. For embryoid body (EB) formation, 10,000 cells were seeded in each well of an ultra-low adhesion (ULA) 96v well plate (S-Bio Prime; MS-9096VZ). EB was supplied daily until day 6 (approximately 100 μl per well) by aspirating and replacing half of the culture medium, along with the addition of fresh dorsomorphine / LDN-193189 and SB-431542.

[0063] From days 7 to 50, all media used were based on Neural Medium (NM) consisting of Neurobasal medium (Gibco; 21103049 or Biological Industries; 06-1055-110-1A), 2% B27 supplement (Gibco; 17504044), 1% GlutaMax, 1% P / S, and from day 27 onwards, 1% Matrigel (Corning; 356231), all sterile filtered through a 0.22 μm filter. On day 7, approximately 75% of the medium was replaced with EBX (Evolved Biopharmaceutical X-ray) medium consisting of NM supplemented with 20 ng / ml FGF-2 and 20 ng / ml EGF (Peprotech; AF-100-15). Half of the EBX medium was replaced daily until day 15, and thereafter, half of the medium was replaced every other day. Around day 20, the spheroids proliferated beyond the 96-well plate, so they were transferred to a 24-well ULA (Corning; 3473), and half of the culture medium was changed every other day until day 26. On day 27, the spheroids were transferred to a sterile 90mm untreated culture dish (Miniplast; 825-090-15-017), and the medium was changed to neuronal differentiation medium (NDM) consisting of NM supplemented with 20 ng / ml BDNF (Peprotech; 450-02) and 20 ng / ml NT-3 (Peprotech; 450-03). On day 41, the medium was changed back to NM without supplementation.

[0064] From day 51 onward, all culture media were based on oligo-maturation medium (OMM) containing Neurobasal medium supplemented with 1% B27 supplement, 1% GlutaMax, 1% P / S, and 1% Matrigel, with half of the medium replaced every two days. For OPC proliferation, on day 51, the medium was changed to OPC proliferation medium (OEM) consisting of OMM supplemented with 10 ng / ml PDGF-AA (R&D systems; 221-AA) and 10 ng / ml IGF-1 (R&D systems; 291-G1), with half of the medium replaced every two days. For oligodendrocyte differentiation, 40 ng / ml T3 (Sigma; T2877) was added to OMM to form oligo-differentiation medium (ODM). Finally, from day 71 onward, spheroids were cultured in OMM, with complete medium replacement every two days.

[0065] Throughout the protocol, spheroids were cultured under static conditions of 37°C and 5% CO2, with growth factors and cytokines added fresh before each medium change, and the spheroids were transferred to fresh plates at least once every 30 days. Unless otherwise specified, the same batch of organoids was used for all analyses.

[0066] Immunofluorescence Mice of different genotypes (P17-P18) were euthanized with CO2 and perfused transcardiacally with 2% PFA / PBS. Dissected brains were fixed on ice for 30 minutes. For immunofluorescence testing, brains were incubated overnight at 4°C in 30% sucrose, then embedded in OCT and sectioned (12–14 μm) using a cryostat. Sagittal sections were washed with PBS, blocked with 5% goat serum containing 0.5% TritonX-100, incubated at room temperature for 1 hour, and then incubated overnight at 4°C with the primary antibody. Sections were then washed with PBS and incubated at room temperature for 1 hour with the secondary antibody tagged with the corresponding Alexa Fluorophore, washed with PBS, and mounted with mounting medium.

[0067] Fixation and immunohistochemical staining of oligocortical spheroids were performed as previously described. 25In short, the organoids were washed three times with PBS, then transferred to 4% ice-cold paraformaldehyde for 45 minutes for fixation, washed three times with cold PBS, and equilibrated overnight in 30% sucrose solution for cryoprotection. The following day, the spheroids were embedded in OCT, snap-frozen with dry ice, and sectioned to 10 μm using a Leica CM1950 cryostat. For immunofluorescence staining, the sections were warmed to room temperature, washed with PBS for rehydration, permeabilized with 0.1% TritonX (PBT) in PBS, and then blocked for 1 hour in blocking buffer containing 5% normal goat serum (NGS) and 0.5% BSA in PBT. The sections were then incubated overnight at 4°C with the primary antibody diluted in the blocking solution. The following day, the sections were subsequently washed three times with shaking in PBS (PBST) containing 0.05% Tween-20 and incubated with the secondary antibody for 1.5 hours. The slides were washed four times with PBST while shaking, and then mounted using Immunofluorescence Mounting Medium (DAKO; s3023).

[0068] Luxol Fast Blue staining Luxol Fast Blue (LFB) staining was performed using the Nova Ultra Luxol Fast Blue staining kit, following a previously published protocol. 23 The procedure was carried out according to the following guidelines. Briefly, paraffin-embedded brain sections (6 μm) from at least three mice of each genotype were dewaxed, then rehydrated with 95% ethanol, and incubated overnight at 56°C in LFB solution (0.1% LFB / 0.5% acetic acid in 95% ethanol). The sections were then washed with 95% ethanol and ddH2O, followed by 0.05% lithium carbonate for 30 seconds, and then washed with 70% ethanol until the gray matter was colorless and the white matter appeared blue. The sections were then rinsed with ddH2O and counterstained with preheated 0.1% Cresyl Violet acetate solution for 30-40 seconds. Finally, the sections were rinsed with ddH2O, dehydrated with 100% ethanol and xylene, and mounted in resin medium.

[0069] Electron microscopy Mice were anesthetized and perfused with a fixative containing 4% PFA, 2.5% glutaraldehyde, and 0.1 M cacodylate buffer. Brains were isolated, incubated overnight in the fixative at room temperature, and treated as previously described. 26 Samples were examined using either an XF416 TVIP camera or a US4000 Gatan camera equipped with an FEI Tecnai T12 transmission electron microscope or a Tecnai F20 S / TEM. EM micrographs were analyzed using computer-aided ImageJ analysis software. To calculate the g ratio, myelinated axons (approximately 600, 100 axons per mouse, n=3 per genotype) from either the corpus callosum or optic nerve in the EM images were analyzed by dividing the inner diameter of the axon by the total diameter of the axons.

[0070] Image acquisition and analysis LFB-stained sections were imaged using a panoramic digital slide scanner (3DHISTECH). Immunostained sections were imaged using a panoramic digital slide scanner or an Olympus FV1000 confocal laser scanning microscope or a Nikon A1R+ confocal microscope. The total fluorescence intensity of CNP and MBP staining in the cortex and cerebellum was calculated using NIS elements software. Acquired images were processed using the relevant microscopy software programs, namely CaseViewer, F-10-ASW viewer, and NIS elements. Images were analyzed using ImageJ software. Images were analyzed with genotype blinding, and processing included overall changes in brightness and contrast.

[0071] Record of spontaneous seizures During monitoring of mice in an animal facility, mice experiencing spontaneous seizures were recorded using a mobile camera. Spontaneous seizures or abnormal activity (runaway behavior) were observed in Wwox mutant mice. The duration of spontaneous seizures (in seconds) was calculated and shown. n=6.

[0072] Electrophysiology Electrophysiological recordings were performed in mice in which neuronal Wwox was conditionally deleted using synapsin-Cre recombinase. For these experiments, S-control (Wwox) + / + ;Synapsin-Cre + ), S-HT (Wwox + / flox ;Synapsin-Cre + ) and S-KO (Wwox flox / flox ;Synapsin-Cre + Male or female mice from P13–P17 of the ) were humanely euthanized in accordance with guidelines outlined by the Canadian Council of Animal Care (CCAC). All surgical procedures were approved and performed in accordance with the guidelines of the Animal Care Committee of the University Health Network.

[0073] In vivo preparation. Ketamine-xylazine (100 mg / kg ketamine and 10 mg / kg xylazine) dissolved in phosphate-buffered saline (PBS) was intraperitoneally injected into mice. The pedal reflex was used to determine the depth of anesthesia. Once the mice were deeply anesthetized, they were placed in a stereotactic frame. A local anesthetic (lidocan) was injected just above the incision site in the skull, and then, after a short time (approximately 5 minutes), the skin was removed to expose the skull. An incision was made in the skull using a drill, and then the skull was peeled back using forceps to expose the cortical tissue. Thin-walled glass electrodes (1.5 mm in diameter, World Precision Instruments) were pulled using a vertical puller. These were filled with PBS. The electrodes were positioned 1.6–2 mm posterior to the bregma and 4 mm lateral to the midline. Electrodes were placed at multiple depths, and activity in the subcortical brain and hippocampus of the neocortex was recorded for 3 minutes at each depth.

[0074] In vitro preparation. Mice were anesthetized with pentobarbital (50 mg / kg). The depth of anesthesia was tested using the pedal reflex. Once the mice were deeply anesthetized, they were rapidly decapitated and the brains were removed. The cerebellum and olfactory bulb were removed, and the remaining tissue was placed caudally down on a platform in an ice-cold sucrose solution containing (mM): 248 sucrose, 26 NaHCO3, 10 glucose, 2 KCl, 3 MgSO4-7H2O, 1.25 NaH2PO4, 1 CaCl2-2H2O. The neocortex was coronally cut to a thickness of 400-500 μm (velocity 0.6 mm / sec, amplitude 1 mm) using a Leica 1200 vibratome. Subsequently, the sections were incubated in artificial cerebrospinal fluid (ACSF) containing (mM) 123NaCl, 25NaHCO3, 10glucose, 3.5KCl, 1.3MgSO4-7H2O, 1.2NaH2PO4, 1.5CaCl2-2H2O, pH 7.3-7.4. After holding the sections at 34°C for 30 minutes, they were transferred to room temperature for at least 60 minutes before the experiment. Local collective potential (LFP) glass electrodes (1.5 mm, World Precision Instruments) containing ACSF were drawn using a vertical puller (Narishige, Japan PP-83) and placed in layers II and III of the neocortex or the CA3 region of the hippocampus. An Olympus BX51 microscope (OLY-150IR camera-video monitor unit) was used as guidance for appropriate electrode placement. To assess the excitability of the network, layer V cortical or dentate gyrus stimulation was performed using bipolar concentric tungsten electrodes positioned along the same vertical column as the recordings from layers II / III. A GRASS S88 stimulator connected to a photoelectric stimulation isolation unit was used to apply current pulses of varying intensities with durations of 0.1 milliseconds every 30 seconds. The amplitude of the maximum steady-state response was compared between S-control, S-HT, and S-KO mice (100 μA).

[0075] Power Spectrum Analysis First, the data was decimated so that the final sampling frequency was 1000 Hz. Next, the data was notch-filtered at 60 Hz and its harmonics. Spectral power was analyzed using the Fast Fourier Transform and a bin size of 1 Hz in MATLAB. For each animal, the power spectrum was averaged over 2.5 minutes at a depth of 300 μm using a 10-second window with a 5-second overlap. The power spectrum was plotted as the average for all subjects.

[0076] Electrophysiological recordings from oligocortical spheroids The organoids at the specified time point were embedded in 3% cold-gelled agarose (at approximately 36°C), incubated on ice for 5 minutes, and then sectioned to 400 μm in sucrose solution (mM: 87 NaCl, 25 NaHCO3, 2.5 KCl, 25 glucose, 0.5 CaCl2, 7 MgCl2, 1.25 NaHPO4, 75 sucrose) at 4°C using a Leica 1200S Vibratome. The sections were incubated in artificial cerebrospinal fluid (ACSF, mM: 125 NaCl, 25 NaHCO3, 2.5 KCl, 10 glucose, 2.5 CaCl2, 1.5 MgCl2; pH 7.38, 300 mOsm) at 37°C for 30 minutes, followed by 1 hour at room temperature. During recording, sections were incubated in ACSF at 37°C with perfusion carbogen (95% O2, 5% CO2) under baseline conditions. Local set potential (LFP) and whole-cell patch-clamp recordings were performed using electrodes withdrawn from borosilicate capillary glass and placed 150 μm from the outer edge of each section (see Figure 6A). The LFP electrodes were filled with ACSF, while the patch electrodes were filled with internal solution. Data were recorded using MultiClamp software at a sampling rate of 25,000 Hz. Data were analyzed using MATLAB software. Traces were filtered by (1) removing noise using a 60-notch filter (5th harmonic) and (2) removing variations from the recording setup using a 0.1 Hz high-pass IIR filter.

[0077] statistical analysis All graphs and statistical analyses were performed using either Excel or GraphPad Prism5. Experimental results are shown as mean ± SEM. Statistical significance was tested using a two-tailed independent Student t-test. Results were considered significant if P < 0.05, otherwise expressed as ns (not significant). Data analysis was performed openly against genotypes.

[0078] result Conditional deletion of Wwox in neural stem cells / progenitor cells or neuronal cells reproduces the Wwox null phenotype. Wwox null mice are born according to Mendelian ratios and are indistinguishable from wild-type littermates. 19,20 Within the first few days of life, the mice begin to show signs of developmental delay and seizures, which persist until they die by 3-4 weeks of age (Figure 1A-C). These phenotypes are very similar to those observed in WOREE / EIEE28 patients, most of whom die between 2 and 4 years of age. 10 To better understand the precise function of WWOX in the central nervous system, we created a conditional mouse model of the brain, taking into account the high expression of WWOX during embryonic brain development in various regions. 8 To achieve this, Wwox fl / fl Mouse (possessing the loxP region) 22 Rat nestin (Nes), which is expressed at embryonic stage E10.5, promotes WWOX excision in neural stem cells / progenitor cells of the mouse brain, generating N-KO mice. 27Transgenic mice possessing Cre recombinase under the promoter and enhancer of WWOX were crossed. WWOX excision was validated in the brains of N-KO mice using immunofluorescence. Monitoring of N-KO mice revealed similarities to the phenotype of Wwox null mice. N-KO mice were born at Mendelian ratios and showed no macroscopic abnormalities until postnatal 2-3 days, when they began to show clear overall developmental delay at P7 (Figure 1D,E). Furthermore, N-KO mice exhibited tremors / seizures and ataxia (lack of coordination in the hindlimb clasping test) similar to those observed in Wwox null mice. All (100%) N-KO mice died by postnatal 4 weeks (Figure 1F). Mice possessing one intact Wwox allele (Wwox + / fl The nestin-Cre+ group did not exhibit any visible abnormal phenotypes, which is consistent with the absence of neurological symptoms in heterozygous carriers in human individuals. These phenotypes indicate a crucial role of WWOX in the CNS, suggesting that its removal in nestin-positive cells and their offspring is responsible for the complex phenotypes observed in WWOX rodent models and human patients.

[0079] Since the nestin promoter is expressed in precursor neurons and glial cells, the effect of WWOX removal was analyzed separately in neurons and glial cells. First, the synapsin I gene, which is expressed at E12.5 and leads to WWOX deletion, especially in most differentiated neurons. 28We conditionally deleted Wwox in neuronal cells using a transgenic mouse line possessing Cre recombinase under the promoter of . We validated the excision of WWOX, which is specific to neurons but not to other cells, by observing that intact WWOX levels in oligodendrocytes (OLs) (stained with CC1) in S-KO brain tissue were comparable to those of control mice. Phenotypic analysis of conditional excision of WWOX in neuronal cells revealed growth retardation up to 3–4 weeks of age (Figure 1G, H) and premature death (Figure 1I), which phenotypicly mimicked N-KO and Wwox null mice. S-KO mice also exhibited disorganized spontaneous tonic-clonic seizures starting from P9 and ranging from a few seconds to a few minutes (68.5 ± 13.4 seconds; n=6 for P14–P18). Furthermore, these mice exhibited phenotypes of lack of coordination and ataxia. Heterozygotes possessing one intact Wwox allele (Wwox + / fl The (synapsin Cre+) mice did not exhibit abnormal phenotypes and were behaviorally indistinguishable from control mice. The phenotype of the S-KO model strongly suggests that WWOX plays a crucial role in neurons, and its deficiency leads to dramatic neurological phenotypes.

[0080] To confirm the WWOX function of specific neuronal cells, oligodendrocytes (Olig2-Cre + / - (using) 29 and astrocytes (using GFAP-Cre) 30 We examined the results of removing WWOX expression in these cells, but no phenotypic abnormalities were observed (Figure 1J-O). The results of conditional deletion of WWOX in oligodendrocytes and astrocytes are shown by immunofluorescence staining of CC1 and GFAP, respectively. In summary, these findings indicate that WWOX is expressed in astrocytes, oligodendrocytes, and neurons, but its function in neurons is important for animal survival and CNS homeostasis.

[0081] Wwox neuronal loss causes epileptic seizures. To characterize epileptic activity in S-KO brains, electrophysiological recordings were performed from the brains of S-KO mice, as well as controls (S-controls) and heterozygotes (S-HTs) (Figures 2A-F). For this purpose, mice were anesthetized, craniotomy was performed, and brain regions above the sensory cortex were exposed. Local collective potential (LFP) electrodes containing saline were used to passively record at various depths of the cortex. Specifically, in the superficial neocortex, S-KO mice showed large-amplitude burst activity that was not present in S-controls (Figure 2A). These bursts were not observed in subcortical structures, suggesting a neocortical origin for burst generation. Further characterization of activity at an in vivo recording depth of 300 μm revealed a peak-to-trough amplitude of 2.06 mV (2.01–3.09 mV, 25th and 75th percentiles), a burst interval of 8.62 seconds (6.25–21.27 seconds, 25th–75th percentiles), and a median epileptic burst duration of 350 milliseconds (206–472 milliseconds, 25th and 75th percentiles).

[0082] These bursts were also observed in LFP recordings obtained from acute neocortical brain sections (Figure 2B), indicating that the neocortex is the source of these abnormal burst discharges. In total, 0% of S-control sections showed bursts (0 out of n=11 sections across 7 animals), 17% of sections from S-HT animals showed bursts (4 out of n=23 sections across 14 animals), and 86% of sections from S-KO animals showed bursts (36 out of n=42 sections across 24 animals).

[0083] S-KO mice exhibited large-amplitude activity, which is thought to be due to increased excitability of the neocortical circuits. To assess this, power spectral analysis was performed on spontaneous electric field activity (Figure 2C, D) and the neocortical layer II / III electric field response to electrically evoked stimuli in layer V was recorded (Figure 2E, F). Traces with spontaneous activity showed increased spectral power in a broad frequency band containing δ (<5 Hz), θ (5–9 Hz), α (10–15 Hz), and β (15–30 Hz) rhythms, both in vivo and in vitro (Figure 2C, D). Furthermore, the electrophysiologically evoked electric field response in vitro was greater in S-KO compared to S-control and S-HT (Figure 2E, F). Since larger evoked responses and increased spectral power are biomarkers of hyperexcitability, these results indicate enhanced neocortical excitability in S-KO mice, and the in vitro data suggest increased innate cortical hyperexcitability.

[0084] RNA-seq and mononuclear RNA-seq (snRNA-seq) have revealed transcriptome changes in myelination and cell turnover in the Wwox mutation model. To analyze the molecular changes underlying the phenotypes observed during neuronal deletion in WWOX, bulk RNA sequencing (RNA-seq) was performed on the whole cortex and hippocampus of S-KO and S-control mice in P17. The analysis revealed a total of 730 upregulated genes and 579 downregulated genes between the two genotypes, highlighting differential expression of several known oligodendrocyte-specific genes and genes induced during epileptic seizures, including astrocyte activation (P-value < 0.01, magnification > 1.5). 31,32Importantly, gene ontology (GO) term analysis revealed significant downregulation of genes related to neuronal myelination and covering. Gene set enrichment analysis (GSEA) also revealed downregulation in S-KO mice by enriching genes related to myelination compared to S-controls. Detailed analysis of RNA-seq data from the cortex and hippocampus of S-KO mice showed significant downregulation of genes involved in oligodendrocyte (OL) maturation (Gjb1, Gjc2, and Olig1), myelin development, maintenance, and function compared to S-controls. 31,33-36 (Ermn, Ugt8a, Plp1, Otud7b, Mal, Eml1, Mobp, Hist1h2be, Cldn11, Mbp, Gal3st1, Fa2h, Gsn, Adamts4, Cnp, Mog, Oplalin, Enpp, Mag, and Myrf. These findings suggest that intracellular resection of neurons in WWOX may affect the myelination process.

[0085] To test whether the observed molecular changes were directly related to WWOX function and to further validate the results, bulk RNA-seq was performed on entire hippocampal tissues of WWOX-null and N-KO models and compared to the S-KO model. Unsupervised clustering analysis of the three models revealed significant downregulation of myelin-related genes, as well as genes involved in OL development, maturation, and myelination processes, while samples were grouped according to WWOX status rather than Cre recombinase promoters. The top 25 DEGs indeed showed downregulation of myelination-related transcripts, including Cnp, Ugt8a, Plp1, Ermn, Otud7b, Mettl7a1, Prr18, Adamts4, Klhdc7a, Mobp, Cldn11, and Mbp. GSEA and GO term analysis revealed significant negative FDR values ​​associated with myelination, neuronal sheathing, and axon sheathing.

[0086] To further analyze the molecular effects of WWOX-induced neuronal loss, mononuclear RNA-seq (snRNA-seq) analysis was performed in the hippocampus of S-KO and S-control mice. Different cell population clusters were identified based on the expression levels of gene sets specific to each cell type or subtype. 37-41 Uniform Manifold Approximation and Projection (UMAP) analysis revealed a decrease in the number of mature myelinated oligodendrocyte cells (15%), COPs (fate-determined oligodendrocyte progenitor cells) (68%), and a greater number (150%) of oligodendrocyte progenitor cells (OPCs) in S-KO compared to S-control. Overall, both bulk RNA-seq and snRNA-seq analyses revealed impaired oligodendrocyte maturation and demonstrated the potential non-cellular autonomy effects of neuronal cell deletion in WWOX.

[0087] WWOX excision of neurons results in myelin sheathing failure, reduced oligodendrocyte maturation, and axonal conduction disorders.

[0088] Next, we investigated whether transcriptome changes in OL-specific genes affect myelination in the brains of S-KO mice and other Wwox mutation models. To test this, sagittal sections of brain tissue obtained at P18 were immunostained for myelin markers such as CNPase (CNP) and myelin basic protein (MBP). Immunofluorescence analysis showed a significant decrease in staining of both myelin markers in S-KO brain tissue compared to age-matched S-controls, indicating myelination defects. Compared to S-controls, decreased myelin staining was observed in various regions of the brain of S-KO mice, including the cortex, cerebellum, caudate nucleus putamen, fimbriae, and fornix. Quantification of fluorescence intensity for CNP and MBP staining revealed significant decreases in the cortex and cerebellum of S-KO mice (Figure 3A, C). Furthermore, Luxol fast blue (LFB) staining of S-KO brain tissue at age P18 showed myelination defects in the white matter tracks compared to age-matched controls. Furthermore, compared to the corresponding control tissue, the expression levels of OL maturation and myelination genes were reduced in S-KO, as assessed by quantitative real-time PCR (qRT-PCR) in the cortex of P18. In particular, early OPC marker genes such as Pdgfra and Cspg4 were slightly elevated, suggesting a specific deficiency in OL maturation.

[0089] Next, we evaluated cellular changes associated with myelin sheathing in the brain tissue of S-KOs. Immunostaining with CC1 (a marker of mature OL) and anti-PDGFRα (OPC) showed a twofold decrease in CC1-positive cells and significantly more OPCs in the corpus callosum of S-KO brains (Figure 3E) compared to age-matched controls. Furthermore, immunostaining of brain sections for NG2 and CC1 was performed to test the transition from OPCs to mature OLs, and a decrease in the number of double-positive cells was found in the corpus callosum and cerebellum of S-KO tissue, further confirming the deficiency of OPC differentiation into mature OLs. Notably, no significant OL cell death was observed in S-KO tissue when stained for CC1 and cleaved caspase-3. Similar results of myelin sheathing and OL maturation deficiencies were observed in brain tissue of N-KOs and Wwox null brain tissue.

[0090] To further evaluate the hypomyelination phenotype observed in S-KO mice, electron microscopy of the corpus callosum and optic nerve was performed at P17. Electron microscopy images showed a substantial decrease in the number of myelinated axons in the corpus callosum of S-KO mice (approximately 3.5–4 times) and a significantly higher number (approximately 6 times) of unmyelinated axons in the optic nerve compared to S-control mice of the same age (Figure 4A). The number of myelinated axons in the corpus callosum (S-control, mean = 180±40, S-KO, mean = 55±35) and unmyelinated axons in the optic nerve (S-control, mean = 55±20, S-KO, mean = 270±60) was counted per field of view (FOV) and is shown in Figure 4B. Furthermore, the g ratio of myelinated axons was calculated, and a significantly higher ratio was observed in the corpus callosum and optic nerve of S-KO mice compared to controls of the same age, indicating a decrease in myelin thickness (Figure 4C).

[0091] To test whether observed myelin sheathing defects delay axonal conduction and lead to dysfunction 42,43 The corpus callosum was stimulated and recorded from neocortical layer V in in vitro section specimens. Compared to controls, a significant latency of nerve conduction was observed in S-KO (Figure 4D-i), which is consistent with myelin formation impairment. As seen in Figure 4D, the amplitude ratio of N1 to N2 indicated a longer response latency of axonal propagation (Figure 4D-ii, iii) and significantly lower peak amplitude of evoked potentials.

[0092] WWOX in neurons promotes differentiation from OPCs to mature oligodendrocytes. To date, our results suggest that excision of WWOX in mature neurons leads to myelin formation defects due to impaired differentiation of OPCs. To further investigate whether the non-cellular autonomy of WWOX in neurons modulates differentiation from OPCs to mature OLs in vitro, we performed co-culture assays with wild-type OPCs and dorsal root ganglion (DRG) neurons isolated from either WT or Wwox null mice. Surprisingly, OPCs cultured in Wwox null DRGs showed significantly reduced differentiation into myelinated OLs compared to OPCs seeded in WT-DRGs (Figure 5). Interestingly, an increase in the number of pre-myelinated OLs was observed under these conditions, which are likely to suggest a compensatory effect (Figure 5).

[0093] These results facilitated the determination of whether WWOX-specific deletion in oligodendrocytes (O-KO) leads to altered myelination in the early postnatal period. For this purpose, MBP staining was examined in P17 of O-KO and control littermates, and no significant changes were observed. Overall, these findings suggest that WWOX excision of neuronal cells causes myelination defects.

[0094] Modeling WWOX deletion in human oligocortical spheroids reveals hyperexcitability and myelin sheathing. Next, we modeled WWOX loss in embryonic stem cells (hESCs) and generated human brain organoids known as oligocortical spheroids (OS) to assess the relevance of our findings to humans. These spheroids consist of functional neurons and glial cells that model brain cell architecture and interpopulation interactions, mimicking the natural myelination process observed in humans. To study the effects of WWOX loss on human neuronal activity and myelination, we utilized a CRISPR / Cas9 system that knocks out WWOX in WiBR3 hESC lines. OS derived from both WT (OS-WT) and WWOX-KO hESCs (OS-WWOX-KO) were generated using a recently published protocol. 24 It was generated according to the following.

[0095] To characterize the functional properties of OS, whole-cell patch and local aggregate potential (LFP) recordings were performed on 15-week-old organoid sections (as described in the Methods). To compare electric field and single-cell recordings, LFPs and patch electrodes were placed 10–15 μm apart (Figure 6A). The resting membrane potential (RMP) of patched cells, derived from both OS-WT and OS-WWOX-KO lines, was found to be non-negative compared to typical mature neurons, suggesting that these cells are still developing (Figure 6B). Pre et al. 44 As previously noted, the RMP of iPSC-derived neurons becomes even more negative during maturation, reaching -50mV in healthy cells at 48-55 days when grown in 2D culture. In comparison, our spheroids showed a similar RMP around 105 days (15 weeks). The difference in the time it takes to reach -50mV may be due to the difference in development between 3D spheroids and 2D cultures. OS-WWOX-KO cells showed significantly more depolarization RMP than the corresponding WT cells, suggesting developmental delay in OS-WWOX-KO. From LFP recordings, a prominent peak was observed in the low frequency range of 0.5–7.9Hz. Oscillatory activity was quantified by the area under the curve (Figure 6C, D), which was significantly higher in OS-WWOX-KO than at the frequency of interest. Differences in signal characteristics were also observed, as shown in the sample recordings and their corresponding time-frequency spectrograms. Overall, low-frequency activity is increased, particularly in the δ and θ ranges of OS-WWOX-KO, which is absent in OS-WT. Increased low-frequency activity generally corresponds to seizure-like, epileptic-like activity. 45 This is due to the fact that it reveals the early signs of the OS-WWOX-KO phenotype in developing spheroids and is consistent with data from human patients and Wwox-deficient mouse phenotypes.

[0096] To assess the state of oligodendrocytes, we followed the timeline of oligodendrocyte and myelin development and maturation, as previously described. First, at 14 weeks of OS, the first point in time when mature OLs are observed, we performed immunostaining for CC1 and anti-PDGFRα. Staining revealed that OPCs were present in similar proportions, but CC1 + The number of cells was reduced in OS-WWOX-KO compared to OS-WT. Next, OL and OPC were tested at week 20, the first time point when myelin was expected to be present, by staining for myelin protein CNP and OPC marker NG2. + A significant decrease in cells was observed, which is NG2 + In cells, the effects were not clear, but both a decrease in the number of OLs and a reduction in myelin formation were suggested. Finally, we tested overall survival (OS) at 30 weeks, the point at which compact and mature myelin was described. 24 In OS-WT, Tuj + WWOX expression was prominent in the cells. At this stage, OS-WWOX-KO showed a significant decrease in staining of both MBP and CNP (Figure 6E), which was also supported by transcriptional levels. Interestingly, OS-WWOX-KO was found to have slightly increased RNA levels of early OPC markers such as SOX10 and PDGFRα. Furthermore, electron microscopy images showed that OS-WT had more myelinated axons compared to OS-WWOX-KO when tested at 37 weeks (Figure 6F,G). Overall, these findings in our human model are consistent with mouse data regarding the effects of WWOX loss in the brain and support its important role in the physiological development of myelin and the pathological development of epilepsy.

[0097] Consideration While some evidence suggests that WWOX plays a crucial role in maintaining brain homeostasis, the precise cellular role of WWOX in the CNS remains unclear. This current study reveals previously unknown cellular roles of WWOX resulting from its complex phenotype.

[0098] WWOX expression is observed in neurons, oligodendrocytes, and astrocytes of the central nervous system (CNS). 46 To identify the cell type contributing to the deletion of Wwox null mice, we systematically mutated the gene in different neuronal populations. Conditional deletion of WWOX in either neural stem cells and progenitor cells or mature neurons reproduced the Wwox null phenotype, including growth retardation, epileptic seizures, ataxia, and premature death, consistent with EEG recordings from WWOX patients. 10,13-18 It was revealed that neuronal deletion in WWOX is associated with neocortical hyperexcitability and spontaneous epileptic activity.

[0099] One of the most significant findings in the analysis of neuronal deletions in WWOX is the phenotype of marked myelin deficiency. This remarkable phenotype was initially observed using large amounts of RNA-seq and snRNA-seq, but later became limited to abundant nuclear transcripts. These observations were further confirmed using immunofluorescence and electron microscopy of various tissue compartments. Overall, these results are consistent with delayed myelin formation and thin corpus callosum described in the white matter tracks of most WOREE children observed on MRI images. 10,13-18 At the cellular level, this myelination defect was found to be a result of a reduced number of mature OLs. This reduction likely represents a differentiation defect, as an increase in the number of OPCs was also detected in neuron-specific WWOX knockout brains. Interestingly, both in vitro and in vivo, evidence of WWOX's non-cellular autonomous function in positively regulating the differentiation of OPCs into mature myelinated OLs has been demonstrated.

[0100] To further investigate the critical importance of WWOX function for neurons, we specifically excised Wwox in oligodendrocytes or astrocytes. Interestingly, deletion of Wwox in either cell type did not result in these phenotypes. In summary, these findings do not rule out the cell-autonomous function of WWOX in oligodendrocytes or astrocytes in other neurological diseases, but they do suggest that a non-cell-autonomous role of WWOX in neurons has some influence on the differentiation of OPCs into oligodendrocytes. Overall, these findings highlight the important and novel role of neuronal WWOX in CNS biology.

[0101] Our results clearly demonstrate that neuronal hyperexcitability and myelin sheathing are major defects in Wwox mutant mice. These two phenotypes are not necessarily mutually exclusive. Recent reviews of white matter imaging in epilepsy have suggested that neurological disorders associated with abnormal myelin content are associated with increased susceptibility to epileptic seizures. 47 Demyelination or hypomyelination is actually a common finding in refractory childhood epilepsy and in animal models of epilepsy. 48 The reason why myelin deficiency or demyelination causes focal hyperexcitability remains unclear. WWOX deficiency may affect several CNS functions, including myelin formation, which leads to imbalances in neuronal activity and the complex phenotype observed in WOREE syndrome.

[0102] The differentiation of OPCs into oligodendrocytes is regulated by numerous endogenous and exogenous factors in the central nervous system (CNS). 49-51 More evidence indicates that neuronal activity and glutamate signaling can promote the migration, proliferation, differentiation, and myelination of developing OPCs. 52,53It remains unclear whether neuronal WWOX influences oligodendrocyte differentiation through neuronal activity or other mechanisms. WWOX influences Wnt / β-catenin through physical interactions with major proteins. 54-56 TGFβ / SMAD 57,58 and DNA damage response 6,59 Since WWOX is known to modulate many signaling pathways, including those mentioned above, it remains to be investigated whether loss of function in WWOX would demodulate these important pathways and affect CNS homeostasis.

[0103] In recent years, brain organoids have attracted considerable attention from the scientific community due to their ability to model human diseases. 60-64 Modeling of WWOX deficiency in brain organoids reproduced several phenotypes observed in Wwox mutant mice. This made it possible to model epilepsy-like activity, which was shown as an increase in power in the low-frequency range. This range corresponds to the delta and theta waves involved in epilepsy. 45 Cell patch recording revealed depolarization RMP in OS-WWOX-KO neurons, which indicates developmental delay and may explain increased excitation. Interestingly, neuronal changes were observed as early as 15 weeks, a time when the literature typically describes myelin deficiency. These data suggest a role for WWOX in regulating hyperexcitability and support the concept that intraneuronal WWOX expression plays a crucial role in disease development.

[0104] WWOX-deficient brain organoids also replicated the myelin sheathing observed in WWOX mutant mice. The phenotype proved progressive, eventually leading to a clear decrease in myelin staining and an increase in unmyelinated axons, suggestive of myelin sheathing. Overall, the results in this system further support the function of WWOX in OLs and myelin sheathing in humans.

[0105] In summary, our findings indicate that WWOX deficiency in neuronal cells leads to hyperexcitability and myelin formation defects.

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WWOX-related encephalopathies: delineation of the phenotypical spectrum and emerging genotype-phenotype correlation. J Med Genet. Jan 2015;52(1):61-70. doi:10.1136 / jmedgenet-2014-102748 jmedgenet-2014-102748 [pii] 19. Aqeilan RI, Trapasso F, Hussain S, et al. Targeted deletion of Wwox reveals a tumor suppressor function.Proc Natl Acad Sci U S A. March 6, 2007 2007;104(10):3949-3954. 20. Aqeilan RI, Hassan MQ, de Bruin A, et al. The WWOX tumor suppressor is essential for post-natal survival and normal bone metabolism. J Biol Chem. Aug 1 2008;283(31):21629-39. 21. Suzuki H, Katayama K, Takenaka M, Amakasu K, Saito K, Suzuki K. A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy. Genes, brain, and behavior. May 20 2009;doi:GBB502 [pii] 10.1111 / j.1601-183X.2009.00502.x 22. Abdeen SK, Del Mare S, Hussain S, et al. Conditional inactivation of the mouse Wwox tumor suppressor gene recapitulates the null phenotype. Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov’t.Journal of cellular physiology. Jul 2013;228(7):1377-82. doi:10.1002 / jcp.24308 23. Elazar N, Vainshtein A, Golan N, et al. Axoglial Adhesion by Cadm4 Regulates CNS Myelination. Neuron. Jan 16 2019;101(2):224-231 e5. doi:10.1016 / j.neuron.2018.11.032 24. Madhavan M, Nevin ZS, Shick HE, et al. Induction of myelinating oligodendrocytes in human cortical spheroids. Nat Methods. Sep 2018;15(9):700-706. doi:10.1038 / s41592-018-0081-4 25. Mansour AA, Goncalves JT, Bloyd CW, et al. An in vivo model of functional and vascularized human brain organoids. Nat Biotechnol. Jun 2018;36(5):432-441. doi:10.1038 / nbt.4127 26. Yang HJ, Vainshtein A, Maik-Rachline G, Peles E. G protein-coupled receptor 37 is a negative regulator of oligodendrocyte differentiation and myelination. Nat Commun. Mar 10 2016;7:10884. doi:10.1038 / ncomms10884 27. Tronche F, Kellendonk C, Kretz O, et al. Disruption of the glucocorticoid receptor gene in the nervous system results in reduced anxiety. Nat Genet. Sep 1999;23(1):99-103. doi:10.1038 / 12703 28. Zhu Y, Romero MI, Ghosh P, et al. Ablation of NF1 function in neurons induces abnormal development of cerebral cortex and reactive gliosis in the brain. Genes & development. Apr 1 2001;15(7):859-76. doi:10.1101 / gad.862101 29. Lu QR, Sun T, Zhu Z, et al. Common developmental requirement for Olig function indicates a motor neuron / oligodendrocyte connection. Cell. Apr 5 2002;109(1):75-86. doi:10.1016 / s0092-8674(02)00678-5 30. Gregorian C, Nakashima J, Le Belle J, et al. Pten deletion in adult neural stem / progenitor cells enhances constitutive neurogenesis. J Neurosci. Feb 11 2009;29(6):1874-86. doi:10.1523 / JNEUROSCI.3095-08.2009 31. Cahoy JD, Emery B, Kaushal A, et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci. Jan 2 2008;28(1):264-78. doi:10.1523 / JNEUROSCI.4178-07.2008 32. Hansen KF, Sakamoto K, Pelz C, Impey S, Obrietan K. Profiling status epilepticus-induced changes in hippocampal RNA expression using high-throughput RNA sequencing. Sci Rep. Nov 6 2014;4:6930. doi:10.1038 / srep06930 33. Vejar S, Oyarzun JE, Retamal MA, Ortiz FC, Orellana JA. Connexin and Pannexin-Based Channels in Oligodendrocytes: Implications in Brain Health and Disease.Front Cell Neurosci. 2019;13:3. doi:10.3389 / fncel.2019.00003 34. Dugas JC, Tai YC, Speed TP, Ngai J, Barres BA. Functional genomic analysis of oligodendrocyte differentiation. J Neurosci. Oct 25 2006;26(43):10967-83. doi:10.1523 / JNEUROSCI.2572-06.2006 35. Rosenberg SS, Powell BL, Chan JR. Receiving mixed signals: uncoupling oligodendrocyte differentiation and myelination.Cell Mol Life Sci. Dec 2007;64(23):3059-68. doi:10.1007 / s00018-007-7265-x 36. Emery B, Agalliu D, Cahoy JD, et al. Myelin gene regulatory factor is a critical transcriptional regulator required for CNS myelination. Cell. Jul 10 2009;138(1):172-85. doi:10.1016 / j.cell.2009.04.031 37. Habib N, Li Y, Heidenreich M, et al. Div-Seq: Single-nucleus RNA-Seq reveals dynamics of rare adult newborn neurons. Science. Aug 26 2016;353(6302):925-8. doi:10.1126 / science.aad7038 38. Habib N, Avraham-Davidi I, Basu A, et al. Massively parallel single-nucleus RNA-seq with DroNc-seq. Nat Methods. Oct 2017;14(10):955-958. doi:10.1038 / nmeth.4407 39. Zeisel A, Munoz-Manchado AB, Codeluppi S, et al. Brain structure. Cell types in the mouse cortex and hippocampus revealed by single-cell RNA-seq.Science. Mar 6 2015;347(6226):1138-42. doi:10.1126 / science.aaa1934 40. Marques S, Zeisel A, Codeluppi S, et al. Oligodendrocyte heterogeneity in the mouse juvenile and adult central nervous system. Science. Jun 10 2016;352(6291):1326-1329. doi:10.1126 / science.aaf6463 41. Artegiani B, Lyubimova A, Muraro M, van Es JH, van Oudenaarden A, Clevers H. A Single-Cell RNA Sequencing Study Reveals Cellular and Molecular Dynamics of the Hippocampal Neurogenic Niche. Cell Rep. Dec 12 2017;21(11):3271-3284. doi:10.1016 / j.celrep.2017.11.050 42. Barak B, Zhang Z, Liu Y, et al. Neuronal deletion of Gtf2i, associated with Williams syndrome, causes behavioral and myelin alterations rescuable by a remyelinating drug. Nat Neurosci. May 2019;22(5):700-708. doi:10.1038 / s41593-019-0380-9 43. Cicciarelli J, Terasaki PI. DR phenotype matching and transfusion in patients treated with cyclosporine.Transplant Proc. Dec 1988;20(6):1082-3. 44. Pre D, Nestor MW, Sproul AA, et al. A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs). PLoS One. 2014;9(7):e103418. doi:10.1371 / journal.pone.0103418 45. Clemens B. Pathological theta oscillations in idiopathic generalised epilepsy. Clin Neurophysiol. Jun 2004;115(6):1436-41. doi:10.1016 / j.clinph.2004.01.018 46. Tochigi Y, Takamatsu Y, Nakane J, Nakai R, Katayama K, Suzuki H. Loss of Wwox Causes Defective Development of Cerebral Cortex with Hypomyelination in a Rat Model of Lethal Dwarfism with Epilepsy. Int J Mol Sci. Jul 23 2019;20(14)doi:10.3390 / ijms20143596 47. Drenthen GS, Backes WH, Aldenkamp AP, Vermeulen RJ, Klinkenberg S, Jansen JFA. On the merits of non-invasive myelin imaging in epilepsy, a literature review.J Neurosci Methods. May 15 2020;338:108687. doi:10.1016 / j.jneumeth.2020.108687 48. Gibson EM, Geraghty AC, Monje M. Bad wrap: Myelin and myelin plasticity in health and disease. Dev Neurobiol. Feb 2018;78(2):123-135. doi:10.1002 / dneu.22541 49. Zuchero JB, Barres BA. Intrinsic and extrinsic control of oligodendrocyte development. Curr Opin Neurobiol. Dec 2013;23(6):914-20. doi:10.1016 / j.conb.2013.06.005 50. Simons M, Trajkovic K. Neuron-glia communication in the control of oligodendrocyte function and myelin biogenesis. J Cell Sci. Nov 1 2006;119(Pt 21):4381-9. doi:10.1242 / jcs.03242 51. Emery B. Regulation of oligodendrocyte differentiation and myelination. Science. Nov 5 2010;330(6005):779-82. doi:10.1126 / science.1190927 52. Barres BA, Raff MC. Proliferation of oligodendrocyte precursor cells depends on electrical activity in axons.Nature. Jan 21 1993;361(6409):258-60. doi:10.1038 / 361258a0 53. Bergles DE, Roberts JD, Somogyi P, Jahr CE. Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus.Nature. May 11 2000;405(6783):187-91. doi:10.1038 / 35012083 54. Bouteille N, Driouch K, Hage PE, et al. Inhibition of the Wnt / beta-catenin pathway by the WWOX tumor suppressor protein. Oncogene. May 25 2009;doi:onc2009120 [pii] 10.1038 / onc.2009.120 55. Abu-Odeh M, Bar-Mag T, Huang H, et al. Characterizing WW Domain Interactions of Tumor Suppressor WWOX Reveals Its Association with Multiprotein Networks. The Journal of biological chemistry. Mar 28 2014;289(13):8865-80. doi:10.1074 / jbc.M113.506790 56. Wang HY, Juo LI, Lin YT, et al. WW domain-containing oxidoreductase promotes neuronal differentiation via negative regulation of glycogen synthase kinase 3beta. Cell death and differentiation. Jun 2012;19(6):1049-59. doi:10.1038 / cdd.2011.188 57. Khawaled S, Nigita G, Distefano R, et al. Pleiotropic tumor suppressor functions of WWOX antagonize metastasis. Signal Transduct Target Ther. Apr 17 2020;5(1):43. doi:10.1038 / s41392-020-0136-8 58. Ferguson BW, Gao X, Zelazowski MJ, et al. The cancer gene WWOX behaves as an inhibitor of SMAD3 transcriptional activity via direct binding. Meta-Analysis Research Support, N.I.H., Extramural. BMC cancer. 2013;13:593. doi:10.1186 / 1471-2407-13-593 59. Abu-Odeh M, Salah Z, Herbel C, Hofmann TG, Aqeilan RI. WWOX, the common fragile site FRA16D gene product, regulates ATM activation and the DNA damage response.Proc Natl Acad Sci U S A. Nov 4 2014;111(44):E4716-25. doi:10.1073 / pnas.1409252111 60. Lancaster MA, Knoblich JA. Organogenesis in a dish: modeling development and disease using organoid technologies.Science. Jul 18 2014;345(6194):1247125. doi:10.1126 / science.1247125 61. Lancaster MA, Renner M, Martin CA, et al. Cerebral organoids model human brain development and microcephaly. Nature. Sep 19 2013;501(7467):373-9. doi:10.1038 / nature12517 62. Sidhaye J, Knoblich JA. Brain organoids: an ensemble of bioassays to investigate human neurodevelopment and disease.Cell Death Differ. Jun 1 2020;doi:10.1038 / s41418-020-0566-4 63. Aqeilan RI. Engineering organoids: a promising platform to understand biology and treat diseases.Cell Death Differ. Jan 2021;28(1):1-4. doi:10.1038 / s41418-020-00680-0 64. Steinberg DJ, Saleem A, Repudi SR, et al. 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[0107] Example 2: Neonatal neuron WWOX gene therapy rescues WWOX null phenotype. In recent years, evidence has been proposed linking WWOX function to the regulation of central nervous system (CNS) homeostasis. 9,10 Germline recessive mutations (missense, nonsense, and partial / complete deletions of the WWOX gene) have been found to be associated with two major phenotypes: SCAR12 (spinocerebellar ataxia, autosomal recessive-12, OMIM614322) and WOREE syndrome (WWOX-associated epileptic encephalopathy). The latter is also known as developmental and epileptic encephalopathy-28 (DEE28, OMIM616211). 9 WOREE is a complex and devastating neurological disorder found in children with early immature arrest codons or complete loss of WWOX. 11 The clinical spectrum of WOREE includes severe developmental delay and early onset of severe epilepsy with various seizure manifestations (tonic, clonic, tonic-clonic, myoclonic, infantile spasms, and absence seizures). Most affected patients do not make eye contact and are unable to sit, speak, or walk. 9 WOREE syndrome is refractory to current anticonvulsant medications, so there is an urgent need to develop alternative therapies to help children with WOREE syndrome. Children with SCAR12, mainly caused by missense mutations in WWOX, exhibit mild phenotypes such as ataxia and epilepsy. 12 SCAR12 epilepsy can be treated with anticonvulsant drugs, but children still exhibit ataxia and intellectual disability. Furthermore, WWOX mutations have been reported in patients with West syndrome, which is characterized by epileptic seizures accompanied by arrhythmias. 13 Brains of children with WWOX gene mutations have been found to be abnormal upon evaluation by magnetic resonance imaging (MRI). Brain abnormalities such as corpus callosum malformation, progressive brain atrophy, delayed myelination, and optic nerve atrophy have been reported in most cases. How WWOX mutations or loss of WWOX function lead to these CNS-related abnormalities is not well understood.

[0108] There are significant similarities between human WWOX (hWWOX) and mouse WWOX (mWwox). In fact, the human WWOX protein sequence is 93% identical and 95% homologous to the mouse WWOX protein sequence. Surprisingly, targeted loss of Wwox function in rodent models (mice and rats) phenotypicly mimics a complex human neurological phenotype, including severe epileptic seizures, growth retardation, ataxia, and premature death. 12,14,15 Wwox null mice also exhibit phenotypes associated with bone metabolism disorders and steroid production. 16,17 Example 1 of this disclosure shows that conditional excision of mouse Wwox in either neural stem cells and progenitor cells (N-KO) or neuronal cells (S-KO mice) resulted in severe epilepsy, ataxia, and premature death within 3–4 weeks, replicating the phenotype observed in Wwox null mice. These results highlight the crucial role of WWOX in neuronal function and prompt us to investigate whether the observed phenotype could be reversed by restoring WWOX expression in the neuronal compartment of Wwox null mice. For this purpose, WWOX expression was restored using an adeno-associated virus (AAV) vector. It was shown that an AAV vector having an open reading frame of mWwox or hWWOX and driven by a human neuronal synapsin I promoter could reverse the Wwox null phenotype. A single intraventricular (ICV) injection of AAV9-synapsin I-WWOX rescued growth retardation, epileptic seizures, ataxia, and premature death in Wwox null mice. Furthermore, WWOX repair improved myelin formation and reversed the abnormal behavioral changes in WWOX null mice. Overall, these remarkable results suggest that WWOX gene therapy may be a promising therapeutic approach for children with WOREE and SCAR12.

[0109] result Neuronal WWOX repair rescues growth retardation and postnatal lethality in Wwox null mutant mice. In Example 1, conditional excision of WWOX in neurons was shown to mimic the phenotype of Wwox null mice, including growth retardation, spontaneous epileptic seizures, ataxia, and premature death at 3–4 weeks. These results suggest that WWOX is a key neuronal gene that regulates CNS homeostasis. Prompted by these remarkable findings, we wanted to investigate whether neuron-specific expression of WWOX in Wwox null mice could rescue the lethality and associated phenotypes of these mice. We designed adeno-associated virus (AAV) vectors expressing mouse Wwox (mWwox) or human WWOX (hWWOX) cDNA driven by the human synapsin-I (hSynI) promoter (Figure 8A), and packaged these with the AAV9 serotype, which is highly CNS-tropic and used in CNS-based gene therapy studies. 22,24 The IRES-EGFP sequence was cloned downstream of the Wwox / WWOX sequence to allow for expression tracking. Successful delivery of AAV9-hSynI-mWwox-IRES-EGFP (AAV9-hSynI-mWwox) should result in the expression of intact WWOX protein in synapsin I-positive non-dividing / mature neurons. AAV9-hSynI-EGFP was used as a control. WWOX and GFP expression were first verified by in vitro infection of primary Wwox null dorsal root ganglion (DRG) neurons with viral particles.

[0110] Next, we evaluated the expression and function of AAV in vivo. Viral particles of AAV9-hSynI-mWwox or AAV9-hSynI-EGFP (2 × 10⁻¹⁰) were evaluated. 10 We achieved widespread neuronal transduction throughout the brain by injecting (in the hemisphere) into the ventricular region of Wwox null mice at birth (P0). 25,26 Immunofluorescence using anti-NeuN and anti-WWOX antibodies confirmed that transgene expression was successful in neurons but not in oligodendrocytes (CC1-positive cells).

[0111] Monitoring of treated mice revealed that mice injected with AAV9-hSynI-mWwox grew normally (Figure 7B), gradually increased in weight (Figure 7C), and became indistinguishable from wild-type mice by 6-8 weeks of age. Mice injected with AAV9-hSynI-EGFP showed a similar phenotype to Wwox null mice (Figure 7C). Notably, compared to wild-type mice, Wwox null and AAV9-hSynI-EGFP injected mice were hypoglycemic from week 2 until death, while AAV9-hSynI-mWwox injected mice had normal blood glucose levels (Figure 7D). Surprisingly, all rescued mice had a longer lifespan compared to Wwox null or Wwox null injected with AAV9-hSynI-EGFP, with a median survival time of 240 days (p < 0.0001) (Figure 7E). Replacing mWwox with hWWOX cDNA yielded similar results and outcomes, although these mice have only been followed for a maximum of 100 days so far (Figure 7F). Notably, the rescued mice were active and highly fertile in both males and females. Wwox null mice were previously shown to lack testicular Leydig cells. 16 Next, we determined whether the repair of WWOX neuronal cells rescued this phenotype, and indeed, intact Leydig cells were found in AAV9-hSynI-mWwox-treated mice at P17. Since bone growth disorders had also been previously reported in Wwox mutant mice, 17,27-30 Upon examining the bones of the rescued mice, it was observed that the cortical bone was of comparable size and thickness to that of WT mice. These results suggest that WWOX repair in neurons may be sufficient to rescue the abnormal phenotype of Wwox null mice.

[0112] Repairing WWOX in neurons reduces hyperexcitability in Wwox null mice. Wwox null mutants exhibit spontaneously recurrent seizures. 12,14,18,31Since spontaneous seizures were not observed in the rescued mice, epileptic activity in the brains of wild-type (WT), Wwox null (KO), and Wwox null mice injected with AAV9-hSynI-mWwox (KO+AAV9-Wwox) at P21–22 was then measured by performing electrophysiological recordings connected to cells. As expected, KO offspring showed severe hyperactivity. Representative traces with spontaneous firing of action potentials are shown in Figure 8A. Clear hyperexcitability is observed from representative traces (WT, KO+AAV9-Wwox, and KO). Brain activity in KOs typically resulted in bursts of action potentials and a dramatic increase in overall firing rate. The average firing rate of neurons recorded for 20WT, 20KO+AAV-Wwox, and 30+KOs was approximately 6 times higher in KO offspring compared to WT offspring (p=2.6e-7). No significant difference was observed in the average firing rate between KO+AAV-Wwox and WT offspring mice.

[0113] Since the KO mice died within four weeks, in vivo recordings in adult KO mice could not be performed. Therefore, in vivo recordings of cell adhesion were performed only in adult WT and KO+AAV9-Wwox mice (Figure 8B). Representative traces for WT and KO+AAV9-mWwox are shown, along with the mean firing rates of 60WT and 60KO+AAV9-mWwox neurons (Figure 8B). There was no significant difference in firing rates between adult WT and KO+AAV9-mWwox cortical neurons. These findings suggest that AAV9-hSynI-mWwox can prevent epileptic seizures caused by WWOX loss.

[0114] Repair of WWOX in neurons promotes myelination in Wwox null mice, possibly by facilitating OPC differentiation. Example 1 linked WWOX loss with myelination failure. Indeed, it was shown that WWOX excision in neurons resulted in non-cellular autonomy and impaired oligodendrocyte precursor cell (OPC) differentiation. Therefore, we next investigated whether the myelination failure phenotype in Wwox null mice could be rescued by repairing WWOX in neurons using AAV. Immunofluorescence analysis of P17 sagittal brain tissue using an anti-MBP antibody revealed improved myelination in all parts of the brain (cortex, hippocampus, and cerebellum) of rescued AAV9-hSynI-mWwox-treated mice compared to Wwox null mice injected with a control virus (Figure 9A). Furthermore, we investigated whether this improved myelination was related to increased OPC differentiation into mature oligodendrocytes due to the non-cellular autonomy of WWOX in neurons. As expected, expression of WWOX via AAV9 in neurons increased the differentiation of OPCs into mature oligodendrocytes, as assessed by immunostaining with CC1 (a marker for mature oligodendrocytes) and anti-PDGFRα (a marker for OPCs) (Figure 9B). Quantification of CC1 and OPCs in the corpus callosum showed a significant increase in the number of mature oligodendrocytes in rescued mice compared to KO mice injected with a control virus in P17 (Figure 9C).

[0115] To further investigate the improvement in myelination observed after WWOX repair in neurons, electron microscopy (EM) analysis of the corpus callosum was performed in P17 and adult mice. Surprisingly, WWOX repair in neurons using AAV9-hSynI-mWwox resulted in an increase in the number of myelinated axons in the corpus callosum in P17 mice compared to KO mice. Furthermore, calculated g ratios indicated increased myelin thickness after WWOX repair in neurons compared to control KO mice. In addition, EM images of the corpus callosum and optic nerve in rescued adult (6-month-old) mice showed improved myelination. Notably, several differences in g ratios were observed when comparing the myelin thickness of the corpus callosum in KO+AAV-Wwox and WT mice in P17 and 6-month-old mice.

[0116] Repairing WWOX in neurons reduces anxiety and improves motor function. Next, we investigated behavioral changes in Wwox null mice after WWOX repair in their neurons. Unfortunately, the Wwox null mice were in poor condition and died prematurely, making it impossible to evaluate their behavior. To examine anxiety and motor coordination in the rescued mice, we performed an open-field elevated cruciform maze (EPM) test and a rotarod test (Figures 10A-E). Surprisingly, at weeks 8-10, the rescued mice (male and female) exhibited tracking patterns in the open field similar to those observed in wild-type mice, indicating that WWOX repair reduced anxiety in Wwox null mice. Furthermore, the speed and total distance traveled on the open-field track were very similar to those of wild-type mice (Figures 10B, C). In addition, the rescued female and male mice exhibited nearly normal behavior in the EPM (Figure 10D). The rotarod test was performed to check the motor coordination of the rescued mice. The results revealed that the mice rescued in Experiment 3 possessed motor coordination similar to that of WT mice, demonstrating the mice's learning ability. Overall, these results suggest that re-expressing WWOX in neurons of WWOX null mice restores activity and normal behavior.

[0117] Consideration This study aimed to repair WWOX in neurons and evaluate the therapeutic potential of this repair. In this study, we used an AAV9 vector to deliver WWOX target genes to mature neurons to treat complex neuronal disorders in a Wwox null mouse model. mWwox or hWWOX cDNA under the neuronal promoter synapsin-I was injected into the brains of neonatal Wwox null mice, demonstrating that this treatment could reverse the WWOX deficiency phenotype.

[0118] The role of WWOX in the regulation of CNS homeostasis has attracted attention as an important function of the WWOX gene. Deficiency of WWOX has been associated with many neurological diseases. 9,10 Particularly interesting is the WOREE syndrome, a severe and complex neurological disease that results in premature death with a median survival of 1 - 4 years. 9,10 Children with WOREE are refractory to current antiepileptic drugs (AEDs), challenging the medical and scientific communities to develop new treatment strategies. Delivery of AAV9 - WWOX to the brains of WOREE syndrome patients could be a novel gene - therapy approach to help these patients.

[0119] The effect of delivering AAV9 - SynI - WWOX to the brains of Wwox null mice was remarkable. First, delivery of WWOX neurons restored normal growth and survival rates of the mice without the occurrence of spontaneous seizures or ataxia. Furthermore, repair of WWOX in neurons was shown to reduce hyperexcitability in cell - adhesion recordings. Second, by repairing WWOX in neurons, myelination in all regions of the brain was improved, further confirming previous observations of the non - cell - autonomous function of WWOX in neurons for OPC maturation (Example 1). It should be noted that there are still some differences between the rescued mice and WT mice that may be due to oligodendrocyte - specific WWOX functions in the regulation of the myelination process. Third, WWOX repair improved the overall behavior of the rescued mice. These findings 9-11,33,34 suggest that the proposed role of WWOX in the regulation of autism

[0120] Another interesting result of WWOX delivery in neurons is the reversibility of hypoglycemia associated with WWOX deficiency in Wwox null mice. 35,36 These results are consistent with a central role of WWOX in glucose metabolism in the CNS and possibly other metabolic functions. 37-40Interestingly, targeted deletion of Wwox in skeletal muscle results in impaired glucose homeostasis. 41 This effect was associated with the cell-autonomous function of WWOX.

[0121] Another curious observation is that the rescued mice were also fertile and able to reproduce. Given that Wwox null mice have been shown to exhibit steroid production defects 16,29,42 current findings suggest that the function of WWOX in the CNS is superimposed on its tissue-level function. Overall, these findings suggest that WWOX may have multifaceted functions at both the organ and organism levels.

[0122] WWOX is ubiquitously expressed in all brain regions. 10,43,44 Current observations do not mean that WWOX expression in other brain cell types such as astrocytes and oligodendrocytes is unnecessary. Evidence associating WWOX function with oligodendrocyte pathology is beginning to emerge 45-49 but little is known about the cell-autonomous function of WWOX in oligodendrocytes. The fact that WWOX expression in neurons regulates oligodendrocyte maturation and antagonizes astrogliosis 50 suggests a complex function of WWOX in CNS physiology and pathophysiology and further detailed analysis is needed.

[0123] The WWOX gene was originally cloned as a putative tumor suppressor. 51,52 Indeed, many research activities in various animal models ( 15 summarized therein) and observations in human cancer patients 1,27,39,53-57Therefore, WWOX was proposed as a tumor suppressor. Given that WWOX repair is limited to the brain, it was hypothesized that other tissues lacking WWOX expression would be more susceptible to tumor development. Notably, no macroscopic tumorigenesis was detected in the limited number of adult Wwox null mice (6-8 months old, n=6) tested and treated with AAV9-hSynI-mWwox. This is not surprising, given that Wwox deletion in somatic cells in some tissues required other hits to promote tumorigenesis in animal models. 28,39,58,59

[0124] Wwox null mice had a limited lifespan and poor condition, forcing treatment at a very early stage of their life cycle (P0). However, attempts to treat postnatal Wwox null mice should be investigated in the future. Our current findings indicate that WWOX repair in neonatal mice using an AAV vector can reverse the phenotype associated with WWOX deficiency. This proof of concept is expected to lay the foundation for the potential of clinical trials of gene therapy for children suffering from WOREE syndrome, which is devastating and often refractory.

[0125] Materials and methods Plasmid vectors Mouse Wwox or human WWOX cDNA was cloned under the human synapsin I promoter of pAAV, and this vector was packaged with an AAV9 serotype (Vector Biolabs, Philadelphia, USA). Custom-made AAV9-hSynI-mWwox-IRES-EGFP, AAV9-hSynI-hWWOX-2A-EGFP, and AAV9-hSynI-EGFP viral particles were obtained from Vector Biolabs or the Vector Core Facility at Hebrew University of Jerusalem.

[0126] mouse Wwox null (- / -)The generation of mice (KO) has been reported previously. 16 These mice were maintained in an FVB background. Heterozygotes (+ / -) Mice were used for breeding to obtain Wwox null mice. The animals were kept in SPF units with free access to food and water in a 12-hour light / dark cycle. All animal-related experiments were conducted in accordance with the prior approval of the Institutional Animal Care Use Committee (HU-IACUC) of Hebrew University.

[0127] Intraventricular (ICV) injection of AAV particles into P0 Wwox null mice Freehand intracranial injection of AAV9-hSynI-mWwox-IRES-EGFP (AAV9-WWOX) or AAV9-hSynI-EGFP (AAV9-GFP) into Wwox null mice was performed according to a published protocol. 25 In short, Wwox null mice were identified by PCR genotyping at birth. Wwox null neonates were anesthetized by placing them on a dry, flat, cool surface. The heads of the anesthetized pup mice were gently wiped with a cotton swab soaked in 70% ethanol. 0.1% trypan blue was added to the virus to allow visualization of the dispensed solution. The injection site was located 2 / 5 of the distance from the lambda suture to each eye. The syringe (preloaded with virus) was held perpendicular to the surface of the skull, and the needle was inserted to a depth of approximately 3 mm. Approximately 1 μl (2 × 10⁻⁶) was dispensed. 10 The GC / hemisphere virus was dispensed using a NanoFil syringe equipped with a 33G bevel needle (World Precision Instruments). The other hemisphere was injected in the same manner. The injected pups were placed on a warming pad until they woke up, and then transferred to their mother's cage. Each injected mouse was carefully monitored for growth, mobility, seizures, ataxia, and general condition, and their phenotype was evaluated.

[0128] Weight and blood sugar levels As shown in the figure, the body weight of the mice was measured regularly. To monitor blood glucose levels, a small amount of blood was collected (mg / dL) by tearing the tip of the mouse's tail with scissors and measuring it using an Accu-Check blood glucose meter (Roche Diagnostics, Mannheim, Germany).

[0129] Immunofluorescence Mice of different genotypes and treatment groups (P17–P18) were euthanized with CO2 and transcardiacally perfused with 2% PFA / PBS. Dissected brains were fixed on ice for 30 minutes and then incubated overnight in 30% sucrose at 4°C. They were then embedded in OCT and sectioned using a cryostat (12–14 μm). Sagittal sections were washed with PBS, blocked with 5% goat serum containing 0.5% TritonX-100, incubated at room temperature for 1 hour, and then incubated overnight at 4°C with the primary antibody. Sections were then washed with PBS, incubated at room temperature for 1 hour with the secondary antibody tagged with the corresponding Alexa fluorophore, washed with PBS, and mounted with mounting medium.

[0130] Surgical procedures for electrophysiology Mice were anesthetized with ketamine / medetomidine (intraperitoneal; 100 and 83 mg / kg, respectively). The effectiveness of the anesthetic was confirmed by the absence of the toe-pinch reflex. Anesthesia was maintained during electrophysiological procedures by administering additional doses approximately every hour, using one-quarter of the initial dose. Body temperature was maintained using a heating pad (37°C) throughout all surgeries and experiments. The skin was removed to expose the skull. Custom-made metal pins were fixed to the skull using dental cement and connected to a custom stage. A small hole (craniotomy with a diameter of 3 mm) was made in the skull using a biopsy punch (Miltex, PA).

[0131] Cell adhesion records Cell adhesion records were acquired using the blind patch-clamp recording method. Electrodes (approximately 7 MOhm) were extracted from filamentous, thin-walled borosilicate glass (outer diameter 1.5 mm, inner diameter 0.86 mm, Hilgenberg GmbH, Malsfeld, Germany) using a vertical two-stage puller (PC-12, Narishige, EastMeadow, NY). The electrodes were filled with an internal solution containing: 140 mM K-gluconate, 10 mM KCl, 10 mM HEPES, 10 mM Na2-phosphocreatine, and 0.5 mM EGTA, adjusted to pH 7.25 with KOH. The electrodes were inserted at a 45° angle to a depth of 300 μm. The electrode placement targeted the brain surface located 1.6–2 mm posterior to Bregma and 4 mm lateral to the midline. During electrode placement, action potentials (spikes) were generated in most cases when the pipette resistance increased from 10 to 200 MOhm. Detection of a single spike was the criterion for initiating recording. All recordings were acquired with an intracellular amplifier in current-clamp mode (MultiClamp 700B, Molecular Devices) at a sampling rate of 10 kHz (CED Micro1401-3, Cambridge Electronic Design Limited) and filtered with a high-pass filter. For the calculation of mean firing rate, the firing rate over a 4-minute recording time was calculated for each recorded cell. A two-sample t-test was used to assess statistical significance between recorded groups.

[0132] Electron microscopy Mice were anesthetized and perfused with a fixative containing 2% paraformaldehyde and 2.5% glutaraldehyde (EM grade) in 0.1 M sodium cacodylate buffer, pH 7.3. Brains were isolated and incubated in the same fixative at room temperature for 2 hours, then stored at 4°C until processing. The recovered tissues (corpus callosum, optic nerve) were washed four times with sodium cacodylate, post-fixed for 1 hour with 1% osmium tetroxide and 1.5% potassium ferricyanide in sodium cacodylate, and washed four times with the same buffer. The tissue samples were then dehydrated for 10 minutes each with a series of stepwise ethanol solutions (30%, 50%, 70%, 80%, 90%, 95%), followed by three 20-minute rinses each with 100% ethanol, and then two rinses with propylene oxide. Next, tissue samples were impregnated with a series of epoxy resins (25%, 50%, 75%, and 100%) for 24 hours each, and polymerized in an oven at 60°C for 48 hours. The blocks were sectioned using an ultramicrotome (Ultracut E, Riechert-Jung), and 80 nm sections were obtained and stained with uranyl acetate and lead citrate. The sections were observed using a Jeol JEM 1400 Plus transmission electron microscope, and photographs were taken using a Gatan Orius CCD camera. The EM micrographs were analyzed using computer-aided ImageJ analysis software. To calculate the g ratio, myelinated axons (approximately 300, 100 axons per mouse, n=3 per genotype) from EM images derived from the corpus callosum were analyzed by dividing the inner diameter of the axon by the total diameter of the axons.

[0133] Open field testing The open field trial was conducted according to a previously published protocol. 60 In short, mice were placed in a corner of a 50 x 50 x 33 cm arena and allowed to explore freely for 6 minutes. The center of the arena was defined as a 25 x 25 cm square in the middle of the arena. Speed ​​and time spent in the center and around the circumference of the arena were measured. Mice tested in the open field were recorded using a video camera connected to a computer equipped with tracking software (Ethovision 12).

[0134] Elevated Cross Maze Test The test apparatus consisted of two closed arms (30 x 5 cm) facing each other, bordered by a 1 cm high edge and bordered by a 16 cm high edge, and two open arms perpendicular to them, all at a height of 75 cm from the floor. Mice were placed in the maze and allowed to explore for 5 minutes. The time spent in both the open and closed arms was recorded. 60

[0135] Rotor rod test Each animal was placed on a rotating rod with a rotation speed increased from 5 revolutions per minute (rpm) to 40 rpm for 99 seconds. Each animal's test consisted of three trials at 20-minute intervals. The time it took for the animal to fall from the device (latency) was recorded for each animal's trial. If an animal did not fall from the device within 240 seconds of the start of the test, the test was terminated. 61

[0136] Image acquisition and analysis Immunostained sections were imaged using a panoramic digital slide scanner or an Olympus FV1000 confocal laser scanning microscope or a Nikon A1R+ confocal microscope. Acquired images were processed using the relevant microscopy software programs, namely CaseViewer, F-10-ASW Viewer, and NIS elements, respectively. Images were analyzed using ImageJ software. Images were analyzed with genotype blinding, and processing included overall changes in brightness and contrast.

[0137] statistical analysis All graphs and statistical analyses were performed using either Excel or GraphPad Prism5. Experimental results are shown as mean ± SEM. Statistical significance was tested using a two-tailed independent Student t-test. Results were considered significant if P < 0.05, otherwise expressed as ns (not significant). Data analysis was performed openly against genotypes. Sample size and p-values ​​are shown in the legend of the figures.

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Abdeen, S.K., Salah, Z., Khawaled, S., and Aqeilan, R.I. (2013). Characterization of WWOX inactivation in murine mammary gland development. Journal of cellular physiology 228, 1391-1396. 10.1002 / jcp.24310. 59. Ferguson, BW, Gao, 60. Wolf, G., Lifschytz, T., Ben-Ari, H., Tatarskyy, P., Merzel, TK, Lotan, A., and Lerer, B. (2018). Effect of chronic unpredictable stress on mice with developmental under-expression of the Ahi1 gene: behavioral manifestations and neurobiological correlates. Transl Psychiatry 8, 124. 10.1038 / s41398-018-0171-1. 61. Greenbaum, L., Lifschytz, T., Zozulinsky, P., Broner, EC, Slonimsky, A., Kohn, Y., and Lerer, B. (2012). Alteration in RGS2 expression level is associated with changes in haloperidol induced extrapyramidal features in a mutant mouse model. Eur Neuropsychopharmacol 22, 379-386. 10.1016 / j.euroneuro.2011.09.006.

[0139] Example 3: Modeling of hereditary epileptic encephalopathy using brain organoids preface Epilepsy is a neurological disorder characterized by a chronic predisposition to recurrent seizures (Fisher et al, 2014; Aaberg et al, 2017). Approximately 50 million people worldwide are affected by epilepsy, making it the most common chronic neurological disorder in children (Aaberg et al, 2017; Blumcke et al, 2017). About 40% of early infancy seizures are caused by developmental epileptic encephalopathy (DEE), formerly known as early infantile epileptic encephalopathy (EIEE) (Howell et al, 2021). These are pathologies of the developing brain, characterized by intractable epilepsy-like activity and impairments in brain and cognitive function (Lado et al, 2013; Shao & Stafstrom, 2016; Nashabat et al, 2019; Howell et al, 2021). Several genes are thought to be involved in the cause of DEE (McTague et al, 2016). In recent years, the role of autosomal recessive mutations in the WWOX gene in the pathogenesis of DEE has been increasingly recognized (Piard et al, 2018; Nashabat et al, 2019). WWOX, a tumor suppressor spanning the chromosomal fragile region FRA16D, is highly expressed in the brain and is suggested to play an important role in central nervous system (CNS) homeostasis (Abu-Remaileh et al, 2015). In 2014, WWOX was identified as being associated with autosomal recessive spinocerebellar ataxia-12 (SCAR12) (Gribaa et al, 2007; Mallaret et al, 2014) and WWOX-associated epileptic encephalopathy (also known as WOREE syndrome or DEE28) (Abdel-Salam et al, 2014; Ben-Salem et al, 2015; Mignot et al, 2015). Both disorders are associated with a variety of neurological symptoms, including seizures, intellectual disability, developmental delay, and spasticity, but differ in severity, onset, and the type of underlying mutation. WOREE syndrome is considered more aggressive, appears as early as 1.5 months of age, and is associated with more extreme genetic changes (Banne et al, 2021).These observations may suggest that both syndromes can be viewed as a continuum. In addition to seizures, patients with WOREE syndrome may present with overall developmental delay, progressive microcephaly, atrophy of specific CNS components, and premature death. However, it is important to note that the phenotype of WOREE syndrome is broad, and symptoms vary from patient to patient. For example, microcephaly is observed in some patients, but not in many others (Piard et al, 2018).

[0140] While modeling WWOX loss in rodents has revealed the role of WWOX in the mammalian brain (Aqeilan et al, 2007, 2008; Suzuki et al, 2009; Mallaret et al, 2014; Tanna & Aqeilan, 2018; Tochigi et al, 2019), the genetic background and brain development of specific patients cannot be modeled in mice, although they are inherent and preserved in patient-derived induced pluripotent stem cells (iPSCs). To circumvent the comprehensive lack of availability of DEE brain samples, including those from patients with WWOX mutations, genome editing and reprogramming techniques were used to reproduce the genetic changes observed in patients with WOREE and SCAR12 syndrome in human PSCs. Subsequently, many aspects of brain spatial organization and cell type formation were reproduced, and brain organoids and 3D neuron cultures with neuronal function were generated in vitro (Amin & Pasca, 2018; Sidhaye & Knoblich, 2020). This allowed us to model the development and maturation characteristics of the CNS and its complex circuits in a system that is more representative of human physiology in vivo than 2D cell cultures. Using this platform, we identified critical deficits in neuronal populations, cortical formation, and electrical activity, and tested possible rescue strategies. This approach leads to a deeper understanding of the physiology and pathophysiology of WWOX in the CNS, laying the foundation for developing more appropriate therapies and supporting the concept of using human brain organoids to model other human epileptic disorders.

[0141] result Generation and characterization of WWOX knockout brain organoids To shed light on the pathogenesis of DEE, WOREE syndrome was studied using a prototype model with brain organoids. The role of WWOX in human brain development in a controlled genetic background was investigated by generating WWOX knockout (KO) clones of the WiBR3 hESC line using a CRISPR / Cas9 system (Abdeen et al, 2018). WWOX expression in these lines was evaluated using immunoblotting. Two clones, WWOX-KO line 1B (WKO-1B, hereafter KO1) and WKO-A2 (hereafter KO2), which consistently showed undetectable protein levels of WWOX throughout validation, were selected for further study. These clones were evaluated for genetic stability and pluripotency using karyotype analysis and teratoma assays, respectively. Sanger sequencing confirmed WWOX editing in exon 1. Furthermore, to confirm the cell-autonomous function of WWOX, WWOX cDNA was restored to the endogenous AAVS locus of the WWOX-KO1 hESC line, and phenotypic reversibility was examined. The KO1-AAV4 strain was selected to produce CO with robust and stable WWOX expression throughout the validation process and will be referred to as W-AAV hereafter. These strains were virtually indistinguishable from the parent cell strain (WiBR3 WT) in terms of morphology and growth throughout the culture period.

[0142] To investigate how WWOX depletion affects brain development in a 3D context, hESCs were differentiated into brain organoids (COs) using an established protocol (Lancaster et al, 2013; Lancaster & Knoblich, 2014). COs from all genotypes showed comparable macroscopic morphology and development at all stages. Next, the expression patterns of WWOX in the developing brain at various time points were investigated by co-staining with neuronal progenitor cells and neuronal markers, two major populations found in organoids. At 10 weeks, WWOX expression was found to be related to SOX2, which corresponds to radial glial cells (RGs), brain progenitor cells.+ WWOX expression was specifically localized in the ventricular zone (VZ) composed of cells, but not in surrounding cells. This finding is consistent with previous studies showing limited WWOX expression in the early stages of mouse cortical development (Chen et al, 2004). To confirm the identity of WWOX-expressing cells within the VZ, we co-stained for crystallin αB (CRYAB), which is specifically expressed in radial glial cells (vRGs) of the ventricles (Pollen et al, 2015), and confirmed WWOX expression in these cells (Figure 11B). Furthermore, even at later stages, such as 24 weeks when the VZ structure is lost, WWOX expression was primarily SOX2-mediated. + This was observed in cells. Importantly, WWOX expression was not detected in CO cells generated from the WWOX-KO line, but similar levels of expression were observed for other markers, such as SOX2 and neuron-specific class III β-tubulin (TUBB3 or TUJ1) (Figure 11B). Interestingly, W-AAV CO cells, in which WWOX expression is driven by the human ubiquitin promoter (UBP), showed high WWOX levels in the VZ, as expected, but other cell populations also showed significant WWOX expression.

[0143] Next, to address the microcephaly phenotype observed in some patients, organoid diameters were measured throughout the culture period, but no significant differences were found. Therefore, the development of histological brain structure was investigated. In WT organoids, SOX2 + VZ, which consists of cells, is composed of intermediate progenitor cells (IP;TBR2 + It is surrounded by cells (also called EOEMS), which indicates the presence of the subventricular zone (SVZ). Outside this layer is the cortical plate (CP), which is mainly composed of neurons (NeuN). +The cells consisted of (1) cells. At 10 weeks, no visible differences were observed in the composition or formation of the vesicle zone (VZ) and surrounding structures in CO cells (Figure 11C), suggesting that these populations were similar in proportion. This was further supported by measuring the RNA expression levels of progenitor cell markers (SOX2 and PAX6) and the neuronal marker TUBB3 (Figure 11C). This surprising observation led to further investigation of the two major neuronal subpopulations found in CO cells: glutamatergic neurons (indicated by vesicle glutamate transporter 1, VGLUT1) and GABAergic neurons (indicated by glutamate decarboxylase 67, GAD67). Immunostaining revealed that VGLUT1 expression remained similar in KO CO cells compared to WT cells, but GAD67 expression showed a significant increase. In contrast, WWOX repair (W-AAV) significantly reversed this imbalance (Figure 11D). The RNA levels of SLC17A6 (VGLUT2), SLC17A7 (VGLUT1), GAD1 (GAD67), and GAD2 (GAD65) showed the same trend.

[0144] These findings suggest that during human embryonic development, WWOX expression was limited to cells in the apical layer of the ventricle zonater (VZ), and WWOX depletion did not affect the VZ-SVZ-CP structure, but disrupted the balance between glutamatergic and GABAergic neurons.

[0145] Brain organoids depleted of WWOX showed hyperexcitability and epileptic-like activity. Brain organoids generate neurons whose electrophysiological function has been previously demonstrated (Trujillo et al, 2019). To characterize the functional properties of WWOX-KO CO, local aggregate potential (LFP) recordings were performed on CO sections at week 7. Electrodes were placed 150 μm away from the edges of the section (data not shown) to avoid areas that could be damaged by section preparation. Sample traces of WT and KO CO revealed visible differences between the two lines under baseline conditions (Figure 12A, left). The mean spectral power of the electric field recordings showed an overall increase in power for KO CO at 0.25–1 Hz, typically labeled as slow-wave oscillations (SWO, <1 Hz) (Figure 12B), and a decrease in the high-frequency range of 30–79.9 Hz. Oscillatory power (OP) was quantified by the area under the curve, which was significantly higher than that of the WT line under baseline conditions (Figure 12C). Over time, the OP (operational dysplasia) rate in the KO (knockout) lineage decreased significantly, while the OP rate in the WT (wild-type) lineage remained the same, suggesting a developmental delay in the KO lineage.

[0146] To further measure the hyperexcitability of the KO strain, 100 μM 4-AP, a convulsive drug commonly used to induce seizures, was inoculated onto sections during recording. 4-AP showed alterations in LFP recordings of both the WT and KO strains (Figure 12A, right), while the KO strain showed significantly increased activity, which was not observed in the WT trace. The effect of 4-AP on spectral power became apparent 5 minutes after its addition. Cross-frequency coupling of sample traces for both the WT and KO strains in the presence of 4-AP revealed an increase in the δ:HFO frequency pair (an attribute previously used to characterize and classify the metastable state of seizures (Guirgis et al, 2013)). Importantly, transduction of lentiviral DNA containing WWOX cDNA resulted in a recovery of the KO strain in terms of mean power spectral density (Figures 12D and E).

[0147] Brain organoids depleted of WWOX showed impaired astrogenesis and DNA damage response. While it is widely accepted that an imbalance between excitatory and inhibitory activity in the brain is a major mechanism of seizures, this does not necessarily mean that neurons are the only population involved. Brain samples from epilepsy patients are well known to show signs of inflammation, astrocyte activation, and gliosis (Cohen-Gadol et al, 2004; Thom, 2009), and in some cases this may be the only histopathological finding (Blumcke et al, 2017). Whether this phenomenon is a consequence of or a cause of acute seizures remains debated (Vezzani et al, 2011; Robel et al, 2015; Rossini et al, 2017; Patel et al, 2019). Furthermore, recent studies have shown the presence of astrogliosis in the brains of Wwox null mice (Hussain et al, 2019).

[0148] To address this, immunofluorescence staining was used to visualize the astrocyte markers glial fibrillary acidic protein (GFAP) and S100 calcium-binding protein B (S100β) in CO at weeks 15 and 24 (Figure 13A-C). This revealed that the significant increase in astrocytes in WWOX-KO CO progressed over time, which was partially reversed in W-AAV CO. This was further supported by immunoblotting analysis of CO at week 20. It is noteworthy that GFAP also exhibits RG (Middeldorp et al, 2010), which is abundant at week 15 but decreases in number at week 24, potentially causing noise in the early stages.

[0149] Astrocytes originate from two distinct cell populations in the brain: RG cells, which switch from neurogenesis to astrogenesis, or astrocyte progenitor cells (APCs) (Zhang et al, 2016; Blair et al, 2018). To track these differences in astrocyte markers, we compared COs at 6 weeks and 10 weeks of age. Significant expression of S100β was observed in the VZ of COs at 6 weeks, where no astrocyte markers were detected in WT organoids (Figure 13D). At 10 weeks, S100β expression was observed in both WT and KO organoids, but no significant difference was detected in double-positive cells suggesting similar glial proliferation when co-stained with the cell proliferation marker Ki67. + Nuclear SOX2 + When quantified along with the nucleus, the proportion of SOX2 remained intact in WT compared to WWOX-KO (18.5% in WT, 95% CI = 14.2-22.81; 19.5% in KO, 95% CI = 15.29-22.81), but the proportion of Ki67 in proliferating cells (9.5% in WT, 95% CI = 6.63-12.35; 4.9% in KO, 95% CI = 2.76-7.13) and Ki67 showed differences. + / SOX2 + The proportion of double-positive cells (51.83% in WT, 95% CI = 41.18–62.48; 27.09% in KO, 95% CI = 14.1–40.1) was reduced. These findings indicate that SOX2 is affected by WWOX loss. + This means that while overall cell proliferation decreases, the total amount of RG outside the VZ remains unaffected, raising questions about the source of astrocytes.

[0150] This unique behavior of vRG in KO CO allowed us to investigate its function in detail by examining the physiological DNA damage response (DDR), a signaling pathway in which WWOX is known to be directly involved (Abu-Odeh et al, 2014b; Abu-Odeh et al, 2016). For this purpose, we stained for γH2AX and 53BP1, which are surrogate markers for DNA double-strand breaks. SOX2 in the innermost layer of VZ +Significant accumulation of γH2AX and 53BP1 lesions was observed in the cell nuclei, with mean 1.5 lesions / nucleus [95% CI = 1.33~1.74] and 1.2 lesions / nucleus [95% CI = 1~1.38] in WWOX-KO, respectively. This is comparable to 0.78 γH2AX lesions / nucleus [95% CI = 0.55~1.02] and 0.62 53BP1 lesions / nucleus in WT CO of the same age, and 0.58 lesions / nucleus [95% CI = 0.38~0.77] and 0.56 lesions / nucleus [95% CI = 0.37~0.76] in W-AAV CO of the same age (Figures 13E and F). These findings are consistent with the direct role of WWOX in DDR signaling (Aqeilan et al, 2014; Hazan et al, 2016). Importantly, W-AAV CO showed improved DDR. Interestingly, in highly proliferating cells in the VZ, a greater number of γH2AX lesions were observed, which were seen by co-staining with Ki67 and SOX2 + 18.6% of cells were double-positive in KO CO (compared to 11.9% [95% CI = 8-15%] [95% CI = 15-22%]). This suggested that continued proliferation of damaged cells might be accompanied by reduced apoptosis, which could indicate a loss of checkpoint inhibition. This hypothesis was resolved by staining caspase-3 cleaved in VZ, which revealed that apoptosis in these cells was reduced during WWOX-KO, but rescued in W-AAV CO.

[0151] In conclusion, the number of astrocytes gradually increases in WWOX-KO CO, likely due to enhanced RG differentiation and increased DNA damage in neural progenitor cells.

[0152] RNA sequencing of WWOX-depleted brain organoids revealed major differentiation defects. To investigate molecular characteristics, whole transcriptome RNA sequencing (RNA-seq) analysis was performed on WT and KO CO at week 15. Despite the known heterogeneity of brain organoids, principal component analysis (PCA) separated the samples into two distinct clusters. This analysis revealed 15,370 differentially expressed genes, of which 1,246 were upregulated in WWOX-KO CO, showing a fold change of more than 1.2 (FC>1.2) and a significant P-value (P<0.01), while 1,021 genes were downregulated (FC<1 / 1.2, P<0.01). Among the top 100 upregulated genes, we identified genes associated with neuronal populations such as GABAergic neurons (GAD1, GRM7, LHX5) and astrocytes (AGT, S100A1, GJA1, OTX2), as well as genes related to neuronal processes such as calcium signaling (HRC, GRIN2A, ERBB3, P2RX3, HTR2C, PDGFRA) and axonal guidance (GATA3, DRGX, ATOH1, NTN1, SHH, RELN, OTX2, SLIT3, GBX2, LHX5). Among the top 100 downregulated genes, we found genes associated with GABA receptors (GABRB3, GABRB2), autophagy (IFI16, MDM2, RB1, PLAT, RB1CC1), and the mTOR pathway (EIF4EBP1, PIK3CA, RB1CC1).

[0153] Gene set enrichment (GSEA) and gene ontology (GO) enrichment analyses of the top 3,000 differentially expressed genes revealed inhibition of processes related to ATP synthesis-coupled electron transport and oxidative phosphorylation, among other things, all consistent with the previously reported function of WWOX in mouse models (Abu-Remaileh & Aqeilan, 2014, 2015; Abu-Remaileh et al, 2018). Downregulation of genes related to negative regulation of the cell cycle was observed, consistent with previously reported reductions in checkpoint inhibition (Abu-Odeh et al, 2014b; Abu-Odeh et al, 2016). On the other hand, significant enrichment was observed in pathways related to region formation, neuronal fate determination and fate identification, axis identification (ventral-dorsal and anterior-posterior), and glycolysis and gluconeogenesis, some of which are supported by previous studies (Wang et al, 2012; Abu-Remaileh & Aqeilan, 2014). As expected, the upregulated genes were associated with developmental pathways such as the Wnt pathway (e.g., WNT1, WNT2B, WNT3, WNT3A, WNT5A, WNT8B, LEF1, AXIN2, GBX2, ROR2, LRP4, NKD1, IRX3, CDH1) and the Shh pathway (e.g., SHH, GLI1, LRP2, PTCH1, HHIP, PAX1, PAX2).

[0154] Since WWOX had previously been identified as being involved in the Wnt signaling pathway (Bouteille et al, 2009; Wang et al, 2012; Abu-Odeh et al, 2014a; Cheng et al, 2020; Khawaled et al, 2020), we decided to further investigate this using our CO model. First, we used RNA-seq data to examine the expression of various members of the WNT signaling pathway (WNT1, WNT3, WNT5A, WNT8B, etc.), standard targets (Axin2, TCF7L2, LEF1, TCF7L1, etc.), brain-specific targets (IRX3, ITGA9, GATA2, FRAS1, SP5), and receptors (ROR2, FZD2, FZD10, FZD1). Next, we validated some of these genes using qPCR (Figure 14A). Furthermore, downregulation of several Wnt-related genes in W-AAV CO, including WNT3, WNT3A, WNT1, and ROR2, was observed (Figure 14A). To further demonstrate Wnt activation, we showed the translocation of β-catenin to the nucleus, a characteristic of the standard Wnt pathway. For this purpose, CO at week 16 was subdivided into cytoplasmic and nuclear fractions and immunoblotted (Figure 14B). An approximately 1.7-fold increase in the normalized intensity of β-catenin in the nucleus of WWOX-KO CO was observed, supporting the concept of Wnt pathway activation after WWOX loss. This was further supported by examining the dynamic expression of several Wnt-related genes (WNT3, AXIN2, LEF1, and TCF7) from weeks 6 to 24, revealing chronic Wnt activation in KO CO compared to the gradual depletion of WT CO.

[0155] Recent evidence has shown that activation of Wnt in developing forebrain organoids leads to disruption of neuronal identification and cortical layer formation (Qian et al, 2020). To investigate whether this occurs in WWOX-KO CO, we examined the expression levels of cortical layer markers using RNA-seq data (Qian et al, 2016). Interestingly, changes were observed in all six layers, with decreased expression in layers I-IV (indicated by TBR1, BCL11B, SATB2, and POU3F2) and a significant increase in superficial layers V-VI (indicated by CUX1 and RELN). This pattern was also confirmed by qPCR. Interestingly, when protein levels were examined using immunofluorescence staining, a disabling expression pattern and stratification, as well as TBR1, were observed in WWOX-KO CO. + CTIP2 + (BCL11B), and SATB2 + Neuronal mixing was also observed (Figures 17D and E). This defect was progressive and worsened at week 24. Surprisingly, when the effects of ectopic WWOX expression were examined, a less distinct phenotype was observed. CTIP2 in W-AAV CO compared to WWOX-KO CO + Cells and SATB2 + Cell number recovered and stratification improved, but RNA levels only partially improved. In contrast, the expression of surface markers CUX1 and RELN, which were upregulated in WWOX-KO, was decreased in W-AAV along with the upper layer marker POU3F2(BRN2). Importantly, when examining the expression of dorsal and ventral genes in CO, organoids were observed to be dorsal identity, as assessed by high RNA readout counts. Notably, no statistically significant differences were observed between WT and KO in the expression of these markers.

[0156] Overall, RNA-seq revealed impaired spatial patterning, axis formation, and cortical layer formation in WWOX-KO CO cells, which correlate with disruption of cellular pathways and activation of Wnt signaling. Reintroduction of WWOX prevented these changes to some extent, further supporting its potential significance in gene therapy.

[0157] Brain organoids of patient-derived WWOX-related developmental and epileptic encephalopathy Disease modeling using CRISPR-edited cells is a widely used tool, but it has been criticized for not modeling the complete genetic background of human patients. Therefore, we reprogrammed peripheral blood mononuclear cells (PBMCs) from two families with WWOX-related diseases of different severity. The first family carries the c.517-2A>G splice site mutation (Weisz-Hubshman et al, 2019) which results in the WOREE syndrome (DEE28) phenotype in homozygous patients (referred to as the WSM family), and the second family carries the c.1114G>C(G372R) mutation (Mallaret et al, 2014) which results in the SCAR12 phenotype in homozygous patients (referred to as the WPM family). All iPSC lineages showed normal morphology of primed hPSCs and self-renewal capacity, and these were evaluated for the expression of pluripotency markers.

[0158] Next, CO was generated from iPSCs isolated from healthy heterozygous parents of the WSM family (the father is called WSM F1, the mother is called WSM M2, collectively called WSM P) and diseased homozygous sons (lines WSM S2 and S5, collectively called WSM S). Furthermore, the rescue approach described for W-AAV CO was employed to reintroduce WWOX into the WSM S5 line (called WSM S5 W-AAV3 and W-AAV6, collectively called WSM S W-AAV). These organoids were then examined for neuronal differentiation and VZ formation (Figure 15A). Similar to hESC-derived CO, WWOX was mainly expressed in the VZs of WSM F1 and WSM M2. β3 tubulin+ Positive cells and SOX2 + The number of positive cells was similar, WSM S CO did not show detectable levels of WWOX, while WSM S W-AAV CO expressed WWOX overall.

[0159] Next, to evaluate the neuronal hyperexcitability of WSM S CO, cell adhesion recordings were performed on 41 WSM S CO neurons, as well as 24 neurons derived from WSM P CO and 40 neurons derived from WSM S W-AAV organoids. Tracing the samples with spontaneous firing of action potentials from WSM S, WSM P, and WSM S W-AAV CO revealed visible differences among the three groups recorded under the same conditions. WSM S CO showed increased action potential bursts and overall neuronal activity compared to WSM P and WSM S W-AAV organoids (Figure 15B). Neurons from WSM S CO were compared to neurons from WSM P (P<0.0001) and WSM S W-AAV (P<0.0001) CO (Figure 15C). There was no significant difference in firing rates between WSM P and WSM S W-AAV CO neurons (P=0.7681). Importantly, no significant difference was observed when comparing CO of the WSM F1 lineage with that of the WSM M2 lineage (P=0.0952). Overall, these results indicate neuronal hyperexcitability in 7-week-old organoids derived from patients with WOREE syndrome compared to their parents.

[0160] Consistent with other findings, WSM S CO at week 10 showed increased GAD67 expression compared to WSM F1 and WSM M2, and this phenotype was reversed in WSM S W-AAV CO (Figures 15D and E). VGLUT1 expression did not change between different lineages. Next, the expression of astrocyte markers was evaluated, and increased expression of GFAP and S100β was found in WSM S compared to same-age controls (Figure 16A). DDR deficiency in the vRG of WSM S CO was also evident (Figures 16B and C). The status of the Wnt pathway was also evaluated using qPCR, and findings suggesting activation were obtained in WSM S CO at week 10 compared to same-age WSM P and WSM S W-AAV (Figure 16D). Finally, cortical laminarization was evaluated using immunofluorescence, and the cortical marker CTIP2 was found in WSM S CO. + and SATB2 + We found a decrease in the expression of [the substance] (Figures 16E and F).

[0161] Since the majority of the phenotype was observed in the cortical regions of CO, we decided to adopt a cortical-specific protocol to generate forebrain organoids (FOs), considering the demonstrated role of WWOX in the cortex (Qian et al, 2016, 2018). First, to verify reproducibility, we generated FOs from WSM F1 and WSM S5 and found equivalent phenotypes of WSM CO.

[0162] Next, we investigated whether the WPM SCAR12 family, which exhibits a milder patient phenotype, shows a similar phenotype to CO and FO in WOREE syndrome. FOs were generated from healthy heterozygous parents (WPM F2 and WPM M3) and their affected homozygous daughters and sons (WPM D1 and WPM S1). As expected, the FOs were indistinguishable in terms of morphology, growth, and β3-tubulin and SOX2 expression. However, WWOX was detected in the VZ of WPM F2 and WPM M3, while little signaling was observed in WPM D1 and S1, which was consistent with WWOX levels in iPSCs. Dorsal forebrain identity was verified by PAX6 staining. Surprisingly, there were no significant differences in the ratio of glutamatergic to GABAergic neurons in the transcript expression levels of neuronal markers. While some differences were observed between organoids (FOs) from similar genotyped strains, cortical marker expression levels were comparable between healthy iPSC strains (WPM F2 and WPM M3) and diseased strains (WPM D1 and WPM S1), supporting the concept of normal neuronal and cortical development. Interestingly, Wnt gene RNA levels showed a pattern suggestive of Wnt pathway activation, raising questions about its role in the pathogenesis of milder diseases. Furthermore, immunohistochemistry and qPCR analysis at the astrocyte level did not reveal significant differences. Finally, analysis of DDR signaling in the VZ of FOs showed no significant differences in the accumulation of DNA damage lesions between healthy and diseased SCAR12 individuals. Overall, these data suggest that developmental outcomes differ between SCAR12 and organoids derived from WOREE syndrome.

[0163] Consideration DEE is a group of severe neurological syndromes whose underlying molecular pathology is unknown (Howell et al, 2021). It is not surprising that current medical treatments are inadequate, coupled with a lack of access to human samples. Our research, utilizing major technological advancements in developmental biology, aimed to model refractory human DEE in a tissue-relevant context, along with the role of WWOX in severe WOREE syndrome. By utilizing genetic manipulation and reprogramming in conjunction with electrophysiology, hyperexcitability was observed and epilepsy-like activity was successfully demonstrated in both CRISPR-edited brain organoids with WWOX and patient-derived brain organoids. We then further investigated cellular and molecular changes that highlight possible mechanisms of the disease's pathophysiology. First, we noticed that while the neuronal population was quantitatively almost intact, GABAergic markers were significantly increased. This finding is even more surprising considering the decrease in GABA receptor components observed in RNA-seq. This may indicate a disruption in the development of a normal, balanced neuronal network, supporting the increased electrical activity observed in these organoids. It should be noted that several pieces of evidence suggest that GABAergic synapses have a depolarizing effect during development (Obata et al, 1978; Ben-Ari et al, 2007; Murata & Colonnese, 2020). The seizure dynamics in developmental epilepsy are related to GABA. AIt is known that the depolarization of the GABA response depends not on hyperpolarization due to receptor activation, but particularly on the accumulation of intracellular chlorides that leads to a depolarized chloride reversal potential (Khalilov et al. 2005; Ben-Ari et al. 2007). Evidence of increased mean spectral power in WWOX-depleted CO and WSM FO, and their recovery in the presence of lentiviruses including WWOX, further strengthens the idea that depolarized GABA plays a crucial role in seizure susceptibility. These findings shed new light on the lack of efficacy of conventional anticonvulsant therapies in immature neurons (Khalilov et al. 2005; Murata & Colonnese, 2020), and WWOX-depleted CO provides a useful model for testing and studying novel therapies targeting excitatory GABAergic responses. Increased SWO has long been associated with various stages of the seizure cycle (onset, during seizure, and termination) (Bragin & Engel, 2008). Previous studies have shown that SWO modulates cortical excitability (Vanhatalo et al, 2004) and can localize the seizure onset zone during the pre-seizure period (Miller et al, 2007). Furthermore, slow waves have been identified as a characteristic feature of seizure activity on electroencephalograms in full-term and premature infants (Patrizi et al, 2003). The mechanism of SWO during seizure onset is not well understood. However, several hypotheses suggest increased extracellular potassium, pH, glial cell dysfunction, and / or changes in blood-brain barrier function (Bragin & Engel, 2008). Investigating this mechanism is beyond the scope of this paper, but it is an interesting direction to explore in the future.

[0164] On the other hand, Figures 12A-C show a decrease in higher frequency activity, namely beta (12-30 Hz) and gamma (>30 Hz) oscillations. Higher frequency activity, particularly gamma oscillations, is known to increase before seizure onset in specific lesion areas and has been studied as a possible determinant of epilepsy development (Medvedev et al, 2011), but our data do not support this for WWOX-related seizures. One possible explanation for this is the maturity of the CO at 7 weeks of age. Gamma oscillations are related to functional connectivity, integrating neural networks within and across brain structures (Kheiri et al, 2013; Ahnaou et al, 2017). The low output in these high-frequency ranges may be due to delayed development and reduced functional connectivity of the WWOX-KO organoid. An interesting observation in the WSM FO at 12 weeks was the enhancement of activity in the WSM S5 FO in the high-frequency range. This contrast may be due to age, developmental protocol, or the underlying mechanisms of the detected epilepsy-like activity. These, in particular the underlying mechanisms, need to be further investigated using detailed single-cell analysis and targeted channel blockers and drugs.

[0165] Next, a detailed examination of other populations found in brain organoids revealed an increase in astrocyte markers, but the RG population expressing high levels of WWOX appeared to maintain a normal ratio. This pattern was detected early and appeared to originate from vRG rather than APC. A possible explanation is the disruptive DDR signaling observed in WWOX-depleted organoids. Previous studies in both ESC-derived and primary mouse neural stem cells (NSCs) have shown that the accumulation of DNA damage lesions in the nucleus or mitochondrial DNA leads to the differentiation of NSCs into astrocytes (Wang et al, 2011; Schneider et al, 2013). In the CNS, physiological DNA breaks can be formed by replication stress (primarily in progenitor cell division), oxidative and metabolic stress as a result of reactive oxygen species (ROS) accumulation, and even neuronal activity (as part of development and learning) (Suberbielle et al, 2013; Madabhushi et al, 2014; Madabhushi et al, 2015). Impaired repair of these breaks is associated with the development of CNS pathology and neurodegeneration (Suberbielle et al, 2013; Madabhushi et al, 2014; Shanbhag et al, 2019). These findings suggest a constitutive role of WWOX in vRGs, where WWOX maintains proper DDR signaling under physiological conditions and prevents the accumulation of DNA damage associated with differentiation disorders. While we chose to focus on vRGs in our DDR analysis, it is important to note that because they highly express WWOX, the data do not suggest that these breaks are particularly accumulated in vRGs and may persist in offspring.

[0166] The ability of brain organoids to develop functional synapses and the dynamics of complex neural networks has been rapidly established through intensive research (Trujillo et al, 2019; Sidhaye & Knoblich, 2020), but their ability to model epilepsy-like activity has only recently been studied (preprint: Samarasinghe et al, 2019; Sun et al, 2019). Sun et al (2019) used brain organoids to model Angelman syndrome using UBE3A-KO hESCs, reproducing hyperactive neuronal firing, abnormal network synchronization, and underlying channel dysfunction observed in 2D and mouse models (Sun et al, 2019). Samarasinghe et al (2019) used organoid fusion to generate inhibitory interneuron-rich organoids from iPSCs of Rett syndrome patients. In organoids with the disease, increased sensitivity to hyperexcitability, reduced microcircuit clusters, recurrent epileptic-like spikes, and altered frequency oscillations were observed, all of which can be traced back to dysfunctional inhibitory neurons (preprint: Samarasinghe et al, 2019). Furthermore, using this model, treatment options were tested by treating mutant organoids with valproic acid (VPA) or the TP53 inhibitor pifislin-α (PFT). Compared to vehicle-based treatment, improvements in neuronal activity were observed, with PFT yielding better results than VPA. While these studies were pioneering, they focused on the electrophysiological changes observed in disease-modeling organoids. Considering the lack of macroscopic neurohistochemical changes in epilepsy patients to guide mechanical studies (Blumcke et al, 2017), our research sought to enhance the use of brain organoids for molecular studies of epilepsy. This objective was highlighted by bulk RNA-seq analysis, which showed a loss of regional identity acquisition, cortical layer disruption, and activation of Wnt signaling.The latter is particularly interesting in light of the intended role of the Wnt signaling pathway as a regulator of seizure-inducing effects on the brain, and therefore could be a therapeutic target (Yang et al, 2016; Qu et al, 2017; Hodges & Lugo, 2018). The aforementioned cortical dysplasia is reminiscent of cortical dysplasia, which has a well-known role in the pathogenesis of drug-resistant epilepsy (Tassi et al, 2002; Fauser et al, 2006; Kobow et al, 2019).

[0167] Consistent with these findings, a recent study examining brain tissue from fetuses with WOREE syndrome reported abnormal migration of the outer granular layer within the molecular layer of the cortex, a phenotype also validated in a WWOX spontaneous mutant rat model (Iacomino et al, 2020). This observation was further supported by transcriptome analysis performed by Kosla et al (2019) on human neuronal progenitor cells (hNPCs) after silencing WWOX using shRNA. This study found that knockdown of WWOX resulted in the loss of enrichment of genes associated with neural crest differentiation and migration, as well as cell-cell adhesion, present in WT hNPCs. The authors also reported decreased mitochondrial redox potential, enhanced cell adhesion to the growth surface, and decreased expression of MMP2 and MMP9. Iacomino et al (2020) reanalyzed this transcriptome data, focusing on genes associated with neuronal migration and differentiation, and found decreased expression of several neuronal migration-related genes, including microtubule proteins and kinesin family proteins. In particular, cortical layer formation has been found to be influenced by the state of the Wnt pathway, a pathway in which WWOX is involved via its binding partners (Qian et al, 2020). For example, WWOX is known to bind to the Disheveled proteins Dvl1 and Dvl2, and the latter is inhibited by WWOX, thus attenuating the Wnt pathway (Bouteille et al, 2009; Abu-Odeh et al, 2014a). Our research further highlights the possibility of crosstalk between Wnt activation and DNA damage, a phenomenon previously described (Elyada et al, 2011). This is very consistent with the previously described multifaceted function of WWOX (Abu-Remaileh et al, 2015) and the reduction in negative regulation of the cell cycle and MDM2 levels observed in RNA-seq. Ki67 in VZ of KO CO + An accumulation of DNA breaks was observed in the cells, which may be explained by Wnt activation, which promotes proliferation and possibly replication stress.

[0168] In addition to disease modeling in brain organoids, we attempted to rescue the observed phenotype by reintroducing WWOX into the hESC genome. This resulted in hyperphysiological expression and partial rescue of WWOX in all cell populations observed in CO. These results provide proof of concept for successful WWOX reintroduction as a means of modifying the phenotype and possibly as a therapeutic intervention. Nevertheless, these findings suggest that optimizing population-targeted delivery and fine-tuning expression levels are crucial for successful gene therapy approaches to WOREE syndrome patients.

[0169] Finally, FOs were generated from patients with WOREE syndrome (WSM) and the relatively milder SCAR12 (WPM) phenotype. Our findings show that both brain organoid culture protocols (CO and FO) yielded similar results, verifying the WWOX deficiency phenotype and indicating its cortical origin. Interestingly, the same developmental abnormalities observed in WOREE organoids were not observed when modeling the SCAR12 family. SCAR12 FOs showed very slight, if any, differences in forebrain neuron development, astrocyte development, and DDR signaling. This strengthens the system's ability to model differences between syndromes and highlights the need to investigate the rare SCAR12 syndrome and the multifaceted functions of WWOX in more detail (Abu-Remaileh et al, 2015; Banne et al, 2021). It is noteworthy that while there are significant differences in WWOX expression among healthy heterozygous parents of different families, the differences observed in affected homozygous patients are very small. These results raise the question of whether disease severity correlates with WWOX function levels rather than total expression levels.

[0170] Overall, our data demonstrate the ability of brain organoids to model childhood epileptic encephalopathy, while also elucidating the pathological changes observed in patients with germline mutations in WWOX and potential approaches for therapeutic development.

[0171] Materials and methods Cell culture and plasmids Under FGF / KOSR conditions, WiBR3 hES cell lines and the resulting iPS cell lines were maintained on a irradiated DR4 mouse embryonic fibroblast (MEF) feeder layer under 5% CO2 conditions: DMEM / F12 (Gibco; 21331-020 or Biological Industries; 01-170-1A) supplemented with 15% knockout serum replacement (KOSR, Gibco; 10828-028), 1% GlutaMAX (Gibco; 35050-038), 1% MEM non-essential amino acids (NEAA, Biological Industries; 01-340-1B), 1% sodium pyruvate (Biological Industries; 03-042-1B), 1% penicillin-streptomycin (Biological Industries; 03-031-113), and 8 ng / ml bFGF (PeproTech; 100-18B). The culture medium was changed daily, and the cultures were subcultured every 5–7 days either manually or by trypsin treatment with type C trypsin (Biological Industries; 03-053-1B). For the first 24–48 hours after subculturing at a concentration of 10 μM, a Rho-related kinase inhibitor (ROCKi, also known as Y27632) (Cayman; 10005583) was added.

[0172] For hESC transfection, cells were cultured in 10 μM ROCKi 24 hours prior to electroporation. Cells were detached using trypsin C solution and mixed with a total of 100 μg of DNA constructs in PBS (Ca 2+ and Mg 2+The cells were resuspended in (containing) and electroporated using a Gene Pulser Xcell System (Bio-Rad; 250V, 500μF, 0.4cm cuvette). Subsequently, the cells were seeded onto an MEF feeder layer in FGF / KOSR medium supplemented with ROCKi. In the case of WWOX-KO, the px330 plasmid containing sgRNA targeting exon 1 was co-electroporated with pNTK-GFP in a 1:5 ratio. After 48 hours, GFP-positive cells were selected and then sparsely seeded onto MEF feeder plates for colony isolation approximately 10 days later (2,000 cells per 10cm plate). For WWOX reintroduction, the pAAVS-2aNeo-UBp-IRES-GFP plasmid, cloned to contain the WWOX coding sequence, was co-electroporated with px330 targeting the AAVS1 locus (Guernet et al, 2016). GFP was then selected, and colonies were isolated using 0.5 μg / ml puromycin. Gene editing was validated by Western blotting. The sgRNA sequences are listed in Table EV3.

[0173] To isolate RNA or protein, hPSCs were subcultured on Matrigel-coated plates (Corning; 356231) as described above, and cultured in NutriStem hPSC XF medium (Biological Industries; 05-100-1A).

[0174] Brain organoid generation, culture, and lentiviral infection Brain organoids were generated from hESCs as described above, with the following modifications (Lancaster et al, 2013; Lancaster & Knoblich, 2014; Bagley et al, 2017; Lancaster et al, 2018): Human WiBR3 cells and WSM iPSCs were maintained on mitotic-inactivated MEFs. Four to seven days before the start of the protocol, cells were passaged onto 60 mm plates coated with MEF or Matrigel (Corning; FAL356231) and grown until a concentration of 70-80% was reached. On day 0, hESC colonies were detached from the MEF using 0.7 mg / ml collagenase D solution (Sigma; 11088858001) and dissociated into single-cell suspensions using rapid treatment with type C trypsin for 2 minutes. For cells cultured on Matrigel, collagenase D treatment was skipped, and cells were immediately dissociated with type C trypsin; there were no further variations in the protocol from this point onward. Although only empirically observed, no significant differences were found in the final results. However, hPSCs cultured on MEFs appeared to have a higher success rate in neural induction and were therefore preferred.

[0175] After dissociation, cells were counted and suspended in hESC medium consisting of DMEM / F12 supplemented with 20% KOSR, 3% USDA-certified hESC-quality FBS (Biological Industries), 1% GlutaMAX, 1% NEAA, 100 μM 2-mercaptoethanol (Sigma; M3148), 4 ng / ml bFGF, and 10 μM Rocki. For embryoid body (EB) formation, 9,000 cells were seeded into each well of an ultra-low adhesion V-bottom 96-well plate (S-Bio Prime; MS-9096VZ). The EBs were further nourished every 5 days, with fresh bFGF and ROCKi added during the first exchange. On day 6, the culture medium was replaced with Neural Induction (NI) medium (Bagley et al, 2017) consisting of DMEM / F12, 1% N2 supplement (Gibco; 17502048), 1% GlutaMAX, 1% MEM-NEAA, and 1 μg / ml heparin solution (Sigma; H3149). The NI medium was replaced every other day until the neuroepithelium was established (usually on days 11-12), with quality control performed as shown (Lancaster & Knoblich, 2014; Bagley et al, 2017), and the well-developed EBs were embedded in Matrigel droplets (Lancaster & Knoblich, 2014; Bagley et al, 2017). The droplets were transferred to a 90 mm sterile, untreated culture dish (Miniplast; 825-090-15-017) containing Cerebral Differentiation Medium (CDM) consisting of a 1:1 mixture of DMEM / F12 and Neuro-basal Medium (Gibco; 21103049 or Biological Industries; 06-1055110-1A), a 0.5% N2 supplement, a 1% B27 supplement without vitamin A (Gibco; 12587010), 1% GlutaMAX, 1% penicillin / streptomycin, 0.5% NEAA, 50 μM 2-mercaptoethanol, 2.5 μg / ml human recombinant insulin (Biological Industries; 41-975-100), and 3 μM CHIR-99021 (Axon Medchem; 1386). The medium was changed every other day.From day 16 onward, organoids were cultured on an orbital shaker in Cerebral Maturation Medium (CMM) (Lancaster et al, 2018) at 37°C and 5% CO2. The medium was configured similarly to CDM, but the B27 supplement was changed to a B27 supplement containing vitamin A (Gibco; 17504044), without CHIR-99021, and containing 400 μM vitamin C (Sigma; A4403) and 12.5 mM HEPES buffer (Biological Industries; 03-025-1B). The medium was changed every 2–4 days. From week 6, 1% Matrigel was added to the medium. To reduce the possibility of contamination, organoids were transferred to fresh sterile plates every 30 days. All media described were filtered through a 0.22 μm filter and stored at 4°C until use. Unless otherwise specified, the same batch of organoids was used for all analyses.

[0176] Lentiviral transduction of WWOX was performed as previously published (Deverman et al, 2016; Khawaled et al, 2019). Briefly, viruses containing WWOX were generated from the pDEST12.2™ destination vector (Gateway Cloning Technology). After ultracentrifugation, titers were empirically determined by infecting 293T cells. On day 35 of culture, individual CO cells were transferred to Eppendorf tubes containing CMM, 1:100 virus-containing medium, and 5 μg / ml polyblen (Merck; TR-1003-6), and incubated overnight. The following day, the organoids were returned to shaken culture in fresh medium.

[0177] Reprogramming of somatic cells Blood samples from family members with WOREE and SCAR12 syndrome were provided solely for research purposes with informed consent from all human subjects, under the approval of the Kaplan Medical Center Helsinki Committee, and all experiments adhered to the principles set forth in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.

[0178] iPSCs were directly induced from PBMCs by infection with Yamanaka factor and Sendai virus CytoTune-iPS 2.0 Kit, following the manufacturer's instructions. Briefly, blood samples from PBMCs were isolated by Ficoll gradient and cultured in StemPro-34® medium (Gibco; 10639-011) supplemented with StemPro-34 Nutrient Supplement (Gibco; 10639-011), 100 ng / ml human SCF (PeproTech; 300-07), 100 ng / ml human FLT-3 ligand (R&D Systems; 308-FKE), 20 ng / ml human IL-3 (PeproTech; 200-03), and 10 ng / ml human IL-6 (PeproTech; 200-06). After 24 hours, half of the medium was replaced. 24 hours later, on day 0 of the protocol, the cells were transferred to a 6-well plate, the reprogramming virus mixture was added, and the plate was centrifuged at 1,000 × g for 30 minutes at room temperature. The cells were resuspended and returned to the incubator overnight. The following day, the cells were centrifuged and washed to remove any remaining virus, and resuspended in fully supplemented StemPro-34 medium, with additional medium added on day 2. On day 3, the cells were transferred to a 10 cm MEF-coated plate, half of the medium was replaced with cytokine-free complete StemPro-34, and the other half of the medium was replaced every other day. By day 7, cells at various stages of reprogramming were observed, and to prevent reprogramming-related apoptosis, the medium was gradually changed to mTeSR supplemented with 10 μM ROCKi. On day 16, colonies with normal morphology and growth rate were harvested, grown, the expression of pluripotency markers was verified, and the cells were sequenced for WWOX mutations.

[0179] Forebral organoid generation and culture Forebral organoids were generated from iPSCs as previously described, with the following modifications (Qian et al, 2016, 2018): iPSC cells were maintained on mitotically inactivated MEFs. Four to seven days before the start of the protocol, cells were subculturified on MEF-coated 60 mm plates and cultured to a maximum concentration of 70–80%. On day 0, iPSC colonies were detached, dissociated, and counted, as with CO, and resuspended in hPSC medium containing DMEM / F12, 20% KOSR, 1% GlutaMax, 1% MEM-NEAA, 1% penicillin / streptomycin, and 100 μM 2-mercaptoethanol. 9,000 cells per well were seeded into V-bottom 96-well plates. On day 1, the culture medium was changed to Neuroectoderm Medium (NEM), an hPSC medium supplemented with 2 μM A83 (Axon Medchem; 1421) and 100 nM LDN-193189 (Axon Medchem; 1527), and this was changed every other day. On days 5 and 6, half of the medium was aspirated and replaced with Neural Induction Medium (NIM) consisting of DMEM / F12, 1% N2 supplement, 1% GlutaMax, 1% penicillin / streptomycin, 1% NEAA, 10 μg / ml heparin, 1 μM CHIR-99021 (Axon Medchem; 1386), and 1 μM SB-431542 (Sigma; S4317). On day 7, quality control and Matrigel embedding were performed as described (Qian et al, 2018), and EB cells were continued to be cultured in NIM with the medium changed every other day. On day 14, Matrigel removal was performed (Qian et al, 2018), and the medium was changed to Forebrain Differentiation Medium (FDM) consisting of DMEM / F12, 1% N2 supplement, 1% B27 containing vitamin A, 1% NEAA, 1% GlutaMax, 1% penicillin / streptomycin, 50 μM 2-mercaptoethanol, and 2.5 μg / ml insulin, and transferred to an orbital shaker at 37°C and 5% CO2. The medium was changed every 2-3 days.On day 71, the culture medium was changed to Forebrain Maturation Medium (FMM) containing Neurobasal medium, 1% B27 supplement with vitamin A, 1% GlutaMax, 1% penicillin / streptomycin, 50 μM 2-mercaptoethanol, 200 μM vitamin C, 20 ng / ml human recombinant BDNF (Pepro-Tech; 450-02), 20 ng / ml human recombinant GDNF (Pepro-Tech; 450-10), 1 μM dibutyryl-cAMP (Sigma; D0627), and 1 ng / mL TGF-β1 (Pepro-Tech; 100-21C). The medium was changed every 2-3 days.

[0180] Immunofluorescence Organoid fixation and immunostaining were performed as previously described (Mansour et al, 2018). Briefly, the organoids were washed three times with PBS, then transferred to 4% ice-cold paraformaldehyde for 45 minutes for fixation, washed three times with cold PBS, and equilibrated overnight in 30% sucrose solution for cryoprotection. The following day, the organoids were embedded in OCT, snap-frozen with dry ice, and sectioned to 10 μm using a Leica CM1950 cryostat.

[0181] For immunofluorescence staining, sections were warmed to room temperature, washed with PBS for rehydration, permeabilized with 0.1% TritonX-100 (PBT) in PBS, and then blocked for 1 hour in blocking buffer containing 5% normal goat serum (NGS) and 0.5% BSA in PBT. The sections were then incubated overnight at 4°C with primary antibody diluted in blocking solution. The following day, the sections were washed three times with shaking in PBS (PBST) containing 0.05% Tween-20, and incubated for 1.5 hours at room temperature with secondary antibody diluted in blocking buffer and Hoechst 33258 solution. The slides were washed four times with shaking in PBST, and the coverslips were mounted using Immunofluorescence Mounting Medium (Dako; s3023). The sections were imaged with an Olympus FLUOVIEW FV1000 confocal laser scanning microscope and processed using the accompanying Olympus FLUOVIEW software. γH2AX-positive nuclei were manually counted using NIH ImageJ and statistically analyzed as described below.

[0182] Electrophysiological recording The organoids were embedded in 3% cold-gelled agarose (at approximately 36°C) and incubated on ice for 5 minutes. Then, using a Leica 1200S Vibratome, they were sectioned to 400 μm in sucrose solution (mM: 87 NaCl, 25 NaHCO3, 2.5 KCl, 25 glucose, 0.5 CaCl2, 7 MgCl2, 1.25 NaHPO4, and 75 sucrose) at 4°C. The sections were incubated in artificial cerebrospinal fluid (ACSF, mM: 125 NaCl, 25 NaHCO3, 2.5 KCl, 10 glucose, 2.5 CaCl2, 1.5 MgCl2, pH 7.38, and 300 mOsm) at 37°C for 30 minutes, followed by 1 hour at room temperature. During recording, sections were incubated in ACSF at 37°C with perfusion carbogen (95% O2, 5% CO2) under baseline conditions. Local collective potential (LFP) and whole-cell patch-clamp recordings were performed using electrodes withdrawn from borosilicate capillary glass and placed 150 μm from the outer edge of each section. The LFP electrodes were filled with ACSF, while the patch electrodes were filled with internal solution. Data were recorded using MultiClamp software at a sampling rate of 25,000 Hz. Data were analyzed using MATLAB software. Traces were filtered by (i) removing noise using a 60-notch filter (5th harmonic) and (ii) removing variations from the recording setup using a 0.1 Hz high-pass IIR filter. Large fluctuations in the signal were then removed using the trend removal function (using a Hamming window), and normalized spectral power was calculated using the Fast Fourier Transform. The area under the curve of the power spectral density plot was calculated by obtaining the sum of frequencies binned over a specific frequency range.

[0183] Cell adhesion records Cell adhesion records were acquired using the blind patch-clamp recording method. Spontaneous neuronal activity derived from organoid neuronal populations was recorded. Electrodes (approximately 7 MOhm) were extracted from filamentous, thin-walled borosilicate glass (outer diameter 1.5 mm, inner diameter 0.86 mm, Hilgenberg GmbH) using a vertical two-stage puller (PC-12, Narishige). The electrodes were filled with an internal solution containing the following (in mM): 140 K-gluconate, 10 KCl, 10 HEPES, 10 Na2-phosphocreatine, and 0.5 EGTA, and the pH was adjusted to 7.25 with KOH.

[0184] Electrodes were inserted at a 45° angle to the organoid surface. During recording, the organoid was held in a Matrigel-free CMM at 35°C. Spikes occurred in most cases as the pipette resistance increased from 10 to 200 MOhm. Detection of a single spike was the criterion for initiating recording. All recordings were acquired with an intracellular amplifier in current-clamp mode (MultiClamp 700B, Molecular Devices) at a sampling rate of 10 kHz (CED Micro1401-3, Cambridge Electronic Design Limited), and filtered with a high-pass filter to eliminate aggregate potentials and retain neuronal spikes.

[0185] Data analysis of cell adhesion recordings was performed using custom-written code in MATLAB (The MathWorks). Spikes recorded in cell adhesion mode were extracted from the raw voltage traces by applying a threshold (the spike threshold was placed well above the peak of the background noise level). For the calculation of the average firing rate, the firing rate over a 4-minute recording time was calculated for each recorded cell.

[0186] Immunoblot analysis and intracellular fractionation For all proteins, organoids were homogenized with a lysis buffer containing 50 mM Tris (pH 7.5), 150 mM NaCl, 10% glycerol, and 0.5% Nonidet P-40 (NP-40) supplemented with protease and phosphatase inhibitors. To separate the cytoplasmic fraction, organoids were pulverized with hypotonic lysis buffer [10 mmol / l HEPES (pH 7.9), 10 mmol / l KCl, 0.1 mmol / l EDTA] supplemented with 1 mmol / l DTT, protease and phosphatase inhibitors. After swelling the cells on ice for 15 minutes, 0.5% NP-40 was added, and the cells were lysed by vortexing. After centrifugation, the cytoplasmic fraction was collected. Subsequently, the remaining pellet was incubated with 1 mmol / l DTT in a hypertonic nucleus extraction buffer [20 mmol / l HEPES (pH 7.9), 0.42 mol / l KCl, 1 mmol / l EDTA] at 4°C for 15 minutes with shaking to obtain the nuclear fraction. The sample was centrifuged and the liquid phase was collected.

[0187] Western blotting was performed under standard conditions using 40–50 μg of protein in each sample. The blotting was repeated 2–3 times per experiment, and the results were quantified using Bio-Rad's Image Lab software.

[0188] RNA extraction, reverse transcription PCR, and qPCR Total RNA was isolated using Bio-Tri reagent (Biolab; 9010233100) as described by the manufacturer for the phenol / chloroform-based method. cDNA was synthesized using 0.5–1 μg of RNA with the qScript cDNA Synthesis Kit (QuantaBio; 95047). qRT-PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems; AB4367659). All measurements were performed three times and standardized to either HPRT or UBC levels.

[0189] Library preparation and RNA sequencing Library preparation and RNA sequencing were performed by the Genomic Applications Laboratory at the Hebrew University Core Research Facility following standard procedures. Briefly, RNA quality was evaluated using the RNA ScreenTape Kit (Agilent Technologies; 5067-5576), D1000 ScreenTape Kit (Agilent Technologies; 5067-5582), Qubit® RNA HS Assay Kit (Invitrogen; Q32852), and Qubit® DNA HS Assay Kit (Invitrogen; 32854).

[0190] For mRNA library preparation, 1 μg of RNA per sample was processed using the KAPA Stranded mRNA-Seq Kit with mRNA Capture Beads (Kapa Biosystems; KK8421). The library was eluted with 20 μl of elution buffer, adjusted to 10 mM, and 10 μl (50%) was collected from each sample and pooled in a single tube. The multiplex sample pool (1.5 pM, containing 1.5% PhiX) was loaded into a NextSeq 500 / 550 High Output v2 Kit (75 cycles) cartridge (Illumina; FC-404-1005) and loaded into a NextSeq 500 System Machine (Illumina) under 75 cycles and single-read sequencing conditions.

[0191] For library quality control, Fastq files were tested with FastQC (ver. 0.11.8), and trimming was performed for remaining adapters, low-quality bases (Q=20), and read lengths (20 bases). Trimming was performed with trim galore (ver. 0.6.1). Read counts were high, approximately 30-50M per sample, and hardly decreased after filtering. Transcriptome mapping was performed using Salmon (ver. 1.2.1) in mapping-based mode with both validation mapping mode and gc bias correction enabled. Before alignment, a Salmon index was created based on HS GRCh38 CDNA release 99 (November 2019) using a KMER size of 25. Salmon mapping reports both raw transcript counts and TPM counts. The resulting mapping rate was high, at 80-90%. A total of eight CO samples (four WT COs and four KO COs) were sequenced. One WT sample failed preliminary quality control (low read count and low transcriptome mapping rate). Another WT sample (neither WWOX-KO nor WT) that was not clustered with any of the other samples was evident in both PCA and dendrogram analysis. These two samples were extracted for further analysis, and a total of six samples were used for further analysis. For differential gene expression determination (KO vs WT), raw transcription counts were filtered by a minimum total count of 10 for all six samples, imported using the R package tximport (ver. 1.16.1), and analyzed with DEeq2 (ver. 1.28.1). Counts were normalized by DEeq2, differential gene expression was filtered, and α was set to 0.01. Mean-based and contraction-based magnification changes were calculated based on apeglm (ver. 1.10.0).

[0192] To create the heatmaps shown in Figures 16A and C, the list of differentially expressed genes was divided into sublists of upregulated (WWOX-KO expression was higher than WT expression) and downregulated genes. Each sublist was sorted by magnification change, and the top 100 genes were selected from each sublist. For each selected gene, the log2 normalized count was scaled, and the results were plotted as a heatmap using the heatmap.2 function of the R package gplots (ver. 3.0.3).

[0193] The heatmap shown in Figure 14F displays the scaled log2-normalized counts of each of the six cortical gene markers in heatmap format using the R package gplots' heatmap.2. Gene set enrichment analysis was performed using Broad Institute GSEA software (ver. 4.0.3). The input included 15,348 genes ranked by log2 of the magnification change. The GO set is Broad Institute set c5.all.v7.0. Acceptable sets are those with between 15 and 500 genes. The gene set is GO Biological Process. Acceptable sets for this analysis are those with between 10 and 500 genes. PCA plots of the first two components were calculated and plotted using the base R function. The calculations are based on log2-transformed and log2-normalized counts with a pseudo-count of 1 added.

[0194] statistics Experimental results were presented as mean ± SEM, or as box plots showing the first and third quartiles, minimum and maximum values, and median. First, normality was determined using the Wilk-Shapiro test. For normally distributed samples, a two-tailed independent Student t-test with Welch's correction was used to compare values ​​between the test and control samples. For non-normally distributed samples, the non-parametric Mann-Whitney test was used. For comparisons between two or more samples, one-way ANOVA was used, and multiple comparisons were corrected using Tukey's multiple comparison test. For non-normally distributed samples, the Kruskal-Wallis test was used in conjunction with Dunn's multiple comparison test. In kinetic experiments, the analysis for multiple t-tests was corrected using the Holm-Sidak method without assuming equal standard deviations (SDs). The p-value cutoff for statistically significant results is as follows: ns (not significant). * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Statistical analysis and visual data display were performed using GraphPad Prism8. Randomization and blinding were not applied in this study. Experiments were conducted with several biological replicas, using at least two hPSC lines for each genotype (except for the WiBR3 WT line). Unless otherwise specified, experiments were performed with multiple batches of organoids.

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[0196] Sequence SEQ ID NO:1: Human WWOX cDNA (coding) sequence (NCBI reference sequence NM_016373.4) atggc agcgctgcgc tacgcggggc tggacgacac ggacagtgag gacgagctgc ctccgggctg ggaggagaga accaccaagg acggctgggt ttactacgcc aatcacaccg aggagaagac tcagtgggaa catccaaaaa ctggaaaaag aaaacgagtg gcaggagatt tgccatacgg atgggaacaa gaaactgatg agaacggaca agtgtttttt gttgaccata taaataaaag aaccacctac ttggacccaa gactggcgtt tactgtggat gataatccga ccaagccaac cacccggcaa agatacgacg gcagcaccac tgccatggaa attctccagg gccgggattt cactggcaaa gtggttgtgg tcactggagc taattcagga atagggttcg aaaccgccaa gtcttttgcc ctccatggtg cacatgtgat cttggcctgc aggaacatgg caagggcgag tgaagcagtg tcacgcattt tagaagaatg gcataaagcc aaggtagaag caatgaccct ggacctcgct ctgctccgta gcgtgcagca ttttgctgaa gcattcaagg ccaagaatgt gcctcttcat gtgcttgtgt gcaacgcagc aacttttgct ctaccctgga gtctcaccaa agatggcctg gagaccacct ttcaagtgaa tcatctgggg cacttctacc ttgtccagct cctccaggat gttttgtgcc gctcagctcc tgcccgtgtc attgtggtct cctcagagtc ccatcgattt acagatatta acgactcctt gggaaaactg gacttcagtc gcctctctcc aacaaaaaac gactattggg cgatgctggc ttataacagg tccaagctct gcaacatcct cttctccaac gagctgcacc gtcgcctctc cccacgcggg gtcacgtcga acgcagtgca tcctggaaat atgatgtact ccaacattca tcgcagctgg tgggtgtaca cactgctgtt taccttggcg aggcctttca ccaagtccat gcaacaggga gctgccacca ccgtgtactg tgctgctgtc ccagaactgg agggtctggg agggatgtac ttcaacaact gctgccgctg catgccctca ccagaagctc agagcgaaga gacggcccgg accctgtggg cgctcagcga gaggctgatc caagaacggc ttggcagcca gtccggctaa Sequence number 2: Human WWOX amino acid sequence MAALRYAGLDDTDSEDELPPGWEERTKDGWVYYANHTEEKTQWEHPKTGKRKRVAGDLPYGWEQETDENGQVFFVDHINKRTTYLDPRLAFTVDDNPTKPTT RQRYDGSTTAMEILQGRDFTGKVVVVTGANSGIGFETAKSFALHGAHVILACRNMARASEAVSRILEEWHKAKVEAMTLDLALLRSVQHFAEAFKAKNVPLHVL VCNAATFALPWSLTKDGLETTFQVNHLGHFYLVQLLQDVLCRSAPARVIVVSSESHRFTDINDSLGKLDFSRLSPTKNDYWAMLAYNRSKLCNILFSNELHRR LSPRGVTSNAVHPGNMMYSNIHRSWWVYTLLFTLARPFTKSMQQGAATTVYCAAVPELEGLGGMYFNNCCRCMPSPEAQSEETARTLWALSERLIQERLGSQSG Sequence ID 3: Human WWOX cDNA (whole) Sequence ID 4: Sequence of the human synapsin I promoter ac tacaaaccga gtatctgcag agggccctgc gtatgagtgc aagtgggttt taggaccagg atgaggcggg gtgggggtgc ctacctgacg accgaccccg acccactgga caagcaccca acccccattc cccaaattgc gcatccccta tcagagaggg ggaggggaaa caggatgcgg cgaggcgcgt gcgcactgcc agcttcagca ccgcggacag tgccttcgcc cccgcctggc ggcgcgcgcc accgccgcct cagcactgaa ggcgcgctga cgtcactcgc cggtcccccg caaactcccc ttcccggcca ccttggtcgc gtccgcgccg ccgccggccc agccggaccg caccacgcga ggcgcgagat aggggggcac gggcgcgacc atctgcgctg cggcgccggc gactcagcgc tgcctcagtc tgcggtgggc agcggaggag tcgtgtcgtg cctgagagcg cagctgtgct cctgggcacc gcgcagtccg cccccgcggc tcctggccag accacccta ggaccccctg ccccaagtcg cagccttcga Sequence ID 5: Synapsin-1 / WWOX cDNA construct (bold font - minimal SynI promoter, underlined - human WWOX cDNA, normal font - AAV9 vector) [ka] [ka] [ka]

Claims

1. A composition for administering to a patient's brain to treat WW domain-containing oxidodriductase (WWOX)-related CNS disease, the composition comprising a WWOX wild-type gene under the control of a regulatory element that expresses WWOX in the brain.

2. The composition according to claim 1, wherein the WWOX-related CNS disease is selected from WWOX-related epileptic encephalopathy (WOREE) syndrome, spinocerebellar ataxia, autosomal recessive syndrome 12 (SCAR12), Alzheimer's disease, West syndrome, autism, multiple sclerosis, and disorders of sex development (DSD).

3. The composition according to claim 2, wherein the WWOX-related CNS disease is WOREE syndrome or SCAR12.

4. The composition according to claim 1, wherein the regulatory element is a promoter that directs the expression of the WWOX gene in a neuron.

5. The composition according to claim 4, wherein the promoter is a universal promoter.

6. The composition according to claim 5, wherein the promoter is a CMV promoter, an E2F1 promoter, a U1snRNA promoter, or a derivative thereof.

7. The composition according to claim 4, wherein the regulatory element is a promoter that is specifically expressed in neurons.

8. The composition according to claim 7, wherein the promoter is selected from the synapsin I promoter, the CamKII promoter, the MeCP2 promoter, the NSE promoter, and the Hb9 promoter, or derivatives thereof.

9. The composition according to claim 7, wherein the promoter is not expressed in glial cells or is expressed at a lower level.

10. The composition according to claim 9, wherein the promoter is not expressed or is expressed at a lower level in oligodendrocytes and / or astrocytes.

11. The composition according to claim 7, wherein the regulatory element is a synapsin I promoter or a derivative thereof.

12. The composition according to claim 1, wherein the regulatory element is a promoter that directs the expression of the WWOX gene in an oligodendrocyte.

13. The composition according to claim 12, wherein the promoter is selected from the MBP promoter, the PLP1 promoter, and the CNP promoter, or derivatives thereof.

14. The composition according to claim 1, wherein the regulatory element is a promoter that directs the expression of the WWOX gene in astrocytes.

15. The composition according to any one of claims 1 to 14, wherein the WWOX wild-type gene is delivered by a viral vector.

16. The composition according to claim 15, wherein the viral vector is an adeno-associated virus (AAV) delivery system.

17. The composition according to claim 16, wherein the AAV delivery system is AAV9.

18. The composition according to any one of claims 1 to 14, wherein the WWOX wild-type gene encodes the amino acid sequence of Sequence ID No.

2.

19. The composition according to claim 18, wherein the WWOX wild-type gene under the control of a regulatory element comprises a nucleotide sequence substantially described in SEQ ID NO: 1 or 3.

20. The composition according to claim 1, wherein the administration is by a route selected from direct injection into the parenchyma, injection into the cerebrospinal fluid via the ventricles, and an intrathecal (cisternus or lumbar) route.

21. A composition for administering to a patient's brain to treat WOREE syndrome or SCAR12, the composition comprising an AAV9 gene delivery system containing the WWOX wild-type gene under the control of the synapsin-1 promoter.

22. The composition according to claim 21, wherein the AAV9 delivery system comprises a nucleotide sequence substantially described in SEQ ID NO: 1 or SEQ ID NO:

3.

23. An expression construct containing the WWOX wild-type gene, which is under the expression control of a neuron-specific promoter.

24. The expression construct according to claim 23, wherein the promoter is synapsin 1 or a derivative thereof.

25. An expression construct according to claim 24, comprising a nucleotide sequence substantially described in SEQ ID NO: 1, 3, 4, or 5.

26. The expression construct according to any one of claims 23 to 25, wherein the expression construct is a viral vector.

27. The expression construct according to claim 26, wherein the viral vector is adeno-associated virus (AAV).

28. The expression construct according to claim 27, wherein the AAV is AAV9.

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