WO2022058405A2 - Histone deacetylase inhibitors and uses thereof - Google Patents

Abstract

The present invention relates to histone deacetylase inhibitors and to pharmaceutical compositions comprising the same for use for the prevention and/or treatment of autism spectrum disorder, in particular 7Dup and other forms of intellectual disability and autism.

Description

Histone deacetylase inhibitors and uses thereof

TECHNICAL FIELD

The present invention relates to histone deacetylase inhibitors and their pharmaceutical compositions for use for the prevention and/or treatment of autism spectrum disorder, in particular 7Dup and other forms of intellectual disability and autism.

BACKGROUND ART

Autism spectrum disorder (ASD) comprises a highly prevalent group of neurodevelopmental disorders (NDD) affecting almost 1% of children. Children diagnosed with ASD exhibit impairments in language and social interaction coupled to stereotyped behaviors and, in many cases, the co-occurrence of varying degrees of intellectual disability [1],

Due to its extremely high prevalence and the lack of effective therapies, ASD represents a major unmet medical need.

Despite the phenotypic convergence of its core symptoms (modulated over an ample range of expressivity, whence the term spectrum), ASD is phenotypically and genetically highly heterogeneous with over 400 identified causal genetic alterations reinforcing the view of ASD as a collection of rare genetic conditions [2], The presence of similar core symptoms across the genetic spectrum of ASD suggests that few paradigmatic syndromes might make the understanding of ASD causes and therapeutic interventions feasible. As a matter of fact, duplication of a segment of chromosome 7 at 7ql 1.23 comprising 26-28 genes, one of the best characterized copy number variations (CNVs) underlying ASD (7Dup) [3, 4], might yield invaluable insights into ASD pathophysiology, also because it is symmetrically opposite to the hemideletion of the same interval that causes Williams-Beuren Syndrome (WBS), a multisystemic disease including hypersociability and selectively spared verbal abilities despite their mild to moderate intellectual disability (ID) and a severely compromised visual-spatial processing and planning [5], Almost all WBS patients have mild to moderate ID, while only a minority of 7Dup patients show ID. Moreover, both syndromes are characterized by anxiety and attention deficit hyperactivity disorder (ADHD). 7Dup patients show a range of ASD traits, especially in terms of varying degrees of language impairments_and social restriction. Even though 7Dup shares intellectual disability and developmental delay with the WBS, patients show the typical autistic traits, such as deficits in speech and social withdrawal (Fig. 1A). The combination of symmetrically opposite CNVs resulting into symmetrically opposite behavioral phenotypes offers unique opportunities to dissect the dosage-vulnerable circuits that affect language competence and sociability. Consequently, compounds that modulate gene dosage alterations may provide therapeutic options into ASD pathophysiology that so far has been notoriously difficult.

To date, several disease modeling studies have demonstrated that the use of different induced pluripotent stem cells (iPSCs)-derived cell types in different disease-relevant conditions is suitable for high-throughput screening (HTS), confirming the high potential of iPSCs and their differentiated derivatives in pharmacological research [6, 7, 8, 9], The process from basic research to bedside is very long, expensive, and poses a number of risks and difficulties along the way, with the result that the number of new potential therapeutic compounds that actually become drugs is very low.

The process of drug repositioning makes the drug discovery process much shorter because the initial phases of drug discovery have already been performed. Therefore, this represents a unique alternative tool for the unmet medical need related to many genetic diseases, including neurodevelopmental disorders [10, 11],

Among the different classes of existing drugs, histone deacetylase inhibitors (HDACi) are an interesting category of therapeutics with potential as anticancer drugs [12], There is a vast literature demonstrating the involvement of HDACs in suppressing critical genes in different types of cancer, including brain tumors [13, 14, 15], Interestingly, HDACi are now being considered as potential therapeutics also for neuropsychiatric disorders [16, 17], Among the genes of the 7ql 1.23 region, general transcription factor II-I (GTF2I) has key relevance: it mediates signal -dependent transcription and plays a prominent role in various signaling pathways [18], Most importantly, convergent evidence has implicated GTF2I as a major mediator of the cognitive-behavioral alterations in 7Dup [19, 20, 21], Using iPSCs from 7Dup patients, the inventors discovered that GTF2I is responsible for a large part of transcriptional dysregulation, evident at the pluripotent state, which is amplified upon differentiation into neural progenitors. Specifically, GTF2I recruits lysine demethylase 1 (LSD1) to repress transcription of critical neuronal genes, an effect that is rescued by inhibition of LSD1 [22], a potential target for therapeutic intervention. On the basis of these convergent lines of evidence, the identification, in patient-derived neurons, of compounds that restore normal expression of genes from CNV causative of ASD represents a promising upstream strategy to develop novel therapies for ASD. The inventors set out to identify compounds that can restore the expression levels of key genes from the WBSCR (WBS Critical Region), i.e. increasing or decreasing their expression in, respectively, WBS or 7Dup neurons. For the above reasons, the inventors selected as targets GTF2I along with three additional genes within the 7ql 1.23 region, namely BAZ1B, CLIP 2 and EIF4H, that emerged as critical for their role in the pathogenesis of WBS and 7Dup [23], In particular, BAZ1B is a chromatin remodeler that is involved in maintenance and migration of neural crest cells playing an important role in the evolution of modem human faces and thus being a prime candidate to study disease-associated craniofacial alterations [24, 25]; CLIP2 is a microtubule-binding protein abundantly expressed in neurons whose haploinsufficiency might contribute to the cerebellar and hippocampal dysfunctions observed in the WBS [26]; EIF4H is a translation initiation factor mediating protein synthesis that might be involved in growth retardation in EIF4H knockout mice [27, 28],

SUMMARY OF THE INVENTION

In the present invention a high-throughput screening (HTS) of 1478 compounds, including central nervous system (CNS) agents, epigenetic modulators and experimental substances was performed on patient-derived cortical glutamatergic neurons differentiated from inventors’ cohort of induced pluripotent stem cell lines (iPSCs), monitoring the transcriptional modulation of WBS interval genes, with a special focus on GTF2 in light of its overriding pathogenic role. Identified hits were validated by measuring gene expression by qRT-PCR and the results were confirmed by Western Blotting.

In particular, three histone deacetylase inhibitors (HDACi) that decreased the abnormal expression level of GTF2I in 7Dup cortical glutamatergic neurons differentiated from four genetically different iPSC lines were identified and selected. This effect was confirmed at the protein level.

The present data represent a unique opportunity for the development of a specific class of compounds for treating 7Dup and other forms of intellectual disability and autism.

Therefore, the present invention provides a histone deacetylase inhibitor for use in the treatment of autism spectrum disorder and/or intellectual disability, especially forms of the autism spectrum disorder and/or intellectual disability featuring an increase in GTF2I levels.

For “increase in GTF2I levels” it is intended an increase with respect to the level of GTF2I in a healthy subject or in a subject not affected by an autism spectrum disorder and/or intellectual disability.

GTF2I has emerged as critical gene within 7ql l.23 region CNV for its role in cognitive- behavioral defects observed in mouse model and human studies (Malenfant et al. 2011, Crespi et al. 2014). The levels of mRNA and protein of this transcription factor result altered compared with levels in healthy control -derived cells, mirroring the genetic dosage imbalance. GTF2I level is measured in in vitro patient-derived neuronal culture both at mRNA and at protein levels by RT-qPCR and Western Blot techniques, respectively. Then GTF2I levels may be measured at protein level and/or at mRNA level.

Preferably the autism spectrum disorder is 7Dup.

Preferably the inhibitor is a pan HD AC or HDAC1 and/or HDAC2 and/ or HDAC3 and/or HDAC6 inhibitor.

More preferably said inhibitor decreases GTF2I levels. GTF2I levels may be measured at protein level and/or at mRNA level in any biological sample obtained from the subject.

For “decrease GTF2I levels” it is intended that after administration of said HD AC inihibitor the levels of GTF2I in a sample obtained from the subject are decreased with respect to levels of GTF2I in a sample obtained from the same subject before HD AC inihibitor administration. Preferably said inhibitor is a hydroxamic acid, a benzamide or an aminobenzamide.

Still preferably said inhibitor is selected from the group consisting of: Vorinostat, Romidepsin , Panobinostat, Belinostat, Entinostat, Domatinostat, Resminostat, Rocilinostat, Trichostatin A, Valproic acid, Dacinostat, Bisthianostat, Quisinostat hydrochloride, CUDC-101 , Scriptaid, Tefinostat, Givinostat, Mocetinostat, Chidamide, Abexinostat, Pracinostat, Butyric acid, Pivanex, 4-phenylbutyric acid (or sodium phenylbutyrate), Tucidinostat (or chidamide), Nanatinostat, Fimepinostat, Remetinostat, Ricolinostat, Tinostamustine, JNJ-26481585, RG2833, M344, APH-0812, CG-745, CKD-506, CKD-581, CXD-101, FX-322, YPL-001, CFH- 367C, Tacedinaline, Citarinostat, CKD-504, CUDC-907, CUDC-908, HG-146, KA-2507, Lipocurc, MPT-0E028, NBM-BMX, OBP-801, OKI-179, RDN-929, VTR-297, REC-2282, CS- 3003, ACY-1035, ACY-1071, ACY-1083, ACY-738, ACY-775, ACY-957, ADV-300, AN-446, AP-001 (or Metavert), Arginine Butyrate, BMN-290, C-1A, CG-1521, CKD-509, CKD-L, CM- 414, Crocetin, CS-3158, CT-101, CX-1026, RCY-1410, SE-7552, SKLB-23bb, CY-190602, JBI-097, JBL 128, JMF-3086, KAN-0440262, KDAC-0001, Largazole, MPT-0B291, MPT- 0G211, MRx-0029, MRX-0573, MRX-1299, NHC-51, Nexturastat A, OKI-422, QTX-125, RCY-1305, SP-1161, SP-259, SRX-3636, ST-7612AA1, TJC-0545, Trichosic, YH-508, HSB- 501, NBM-1001, QTX-153, RDN-1201, ROD-119, ROD-1246, ROD-1275, ROD-1702, ROD- 2003, ROD-2089, ROD-702 and RTSV-5.

Preferably said inhibitor is Vorinostat, Mocetinostat or RG2833.

Preferably the inhibitor is combined with a further therapeutic agent. Preferably the further therapeutic agent is selected from the group consisting of: atypical antipsychotic drugs, psychostimulants, antidepressant agent, anti-epileptic agent, clonidine, rivastigmine, memantine, guanfacine, buspirone, atomoxetine, an epigenetic compound, a LSD1 inhibitor, a DNMT inhibitor, a histone methyltransferase (HMT) inhibitor, a EZH1/2 inhibitor, a PRMT inhibitor, a BET inhibitor, a DOT1L inhibitor, APTA-16, a Histone Lysine N Methyltransferase (EHMT2 or G9a) inhibitor, a dual inhibitor against G9a and DNMTs, a menin-MLLl (or KMT2A) interaction inhibitor, a SETD2 inhibitor.

The further therapeutic agent may be any pharmacological intervention to treat ASD, in particular aspecific clinical issues associated with ASD (i.e. irritability, agitation, aggression, sleep disorders, seizures, hyperactivity, anxiety) and comprise different classes of drugs including: Atypical antipsychotic drugs: risperidone, aripiprazole, quetiapine, ziprasidone, olanzapine, Psychostimulants: methylphenidate, amphetamines, Adderall, dexmethylphenidate, Antidepressants: selective serotonine reputate SSRIs (Fluoxetine, sertraline, citalopram, escitalopram, and fluvoxamine, paroxetine), tryciclics (Mirtazapine), Anti-epileptic agent: lamotrigine, oxcarbazepine, diazepam, levetiracetam, divalproex Sodium, clonazepam, carbamazepine, gabapentin, topiramate, Other agents such as: clonidine, rivastigmine, memantine, guanfacine, buspirone, atomoxetine (LeClerc S et al Pharmacological Therapies for Autism Spectrum Disorder: A Review, P T. 2015; Sharma SR et al Autism Spectrum Disorder: Classification, diagnosis and therapy, Pharmacology & Therapeutics. 2018; Coleman DM et al Rating of the Effectiveness of 26 Psychiatric and Seizure Medications for Autism Spectrum Disorder: Results of a National Survey, J Child Adolesc Psychopharmacol. 2019). The further therapeutic agent may also be an epigenetic compound: a molecule that targets an epigenetic regulator or may itself be an epigenetic regulator. When said molecule targets an epigenetic regulator, said epigenetic regulator is preferably defined as any protein able to directly regulate genic transcription through interaction with DNA, RNA or chromatin. A molecule that targets an epigenetic regulator may be selected from the group consisting of: LSD1 inhibitors, DNMT inhibitors, histone methyltransferase (HMT) inhibitors such as EZH1/2 inhibitors or PRMT inhibitors, BET inhibitors.

The LSD1 inhibitor may be selected from the group consisting of: N-[4-[(lS,2R)-2- aminocyclopropyl]phenyl]-4-(4-methylpiperazin-l-yl)benzamide (DDP38003) and stereoisomers thereof such as N-[4-[(trans)-2-aminocyclopropyl]phenyl]-4-(4 methylpiperazin- l-yl)benzamide (DDP37368), ORY-1001 (or iadademstat), CC-90011, ORY- 2001 (or vafidemstat), any one or more of the compounds disclosed in WO2011131576, W02014086790, WO2015181380, WO2016034946, WO2017198780 or WO2019034774, all of which are herein enclosed by reference, GSK-2879552, IMG-7289 (or bomedemstat), INCB059872, 4SC-202 (or domatinostat), Seclidemstat, TAK-418, SYHA-1807, BEA-17, HM- 97211, HM-97346, JBI- 097, JBI-128, ORY-3001, RN-1, SP-2509, T-3775440, T-448, EPL110 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof. The DNMT inhibitor may be selected from the group consisting of: 5-azacytidine, 5-aza-2’- eoxycytidine, CC-486, 4'-thio-2'-deoxycytidine, 5aza-4'-thio-2'-deoxycytidine, Guadecitabine sodium (SGI-110), Zebularine, CP -4200, Flucytosine, Roducitabine, NSC-764276, EF-009, KM- 101, NTX-301, Sinefungin, an antisense oligonucleotide such as MG-98 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof. The HMT inhibitor may be: an EZH1/2 inhibitor selected from the group consisting of: Tazemetostat hydrobromide, Valemetostat, ZLD1039, GSK926, GSK126, PF-06821497, UNC1999, CPI-1205, MC-3629, CPI-0209, SHR-2554, CPI-169, EBI-2554, GSK-343, HM- 97594, IONISEZH-22.5Rx, JQEZ- 5, MS-1943, ORS-1, TBL-0404, KM-301, GSK2816126 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof, and/or a PRMT inhibitor, such as a PRMT5 inhibitor or a PRMT1 inhibitor, preferably said PRMT5 inhibitor is selected from the group consisting of: GSK3326595, JNJ- 64619178, PF-06939999, PRT-543, PRT-811, JBI-778, GSK-3235025 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof, preferably said PRMT1 inhibitor is GSK3368715 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof, and/or a DOT IL inhibitor, preferably Pinometostat (or EPZ-5676)or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof, and/or APTA-16 or a pharmaceutically acceptable salt, hydrate or solvate thereof, and/or a Histone Lysine N Methyltransferase (EHMT2 or G9a) inhibitor, preferably selected from the group consisting of: BIX-01294, a BIX-01294 analogue (or TM-2115) or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof, and/or a dual inhibitor against G9a and DNMTs, preferably CM-272 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof, and/or a menin-MLLl (or KMT2A) interaction inhibitor, preferably MI- 3454 and/or KO-539, or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof, a SETD2 inhibitor, preferably EPZ-040414 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof.

The BET inhibitor may be selected from the group consisting of: LBET762 (or molibresib), CPI- 0610, OTX015, RVX-280 (or apabetalone), ODM-207, PLX- 2853, ZEN-3694, ABBV-744, AZD-5153, BI-894999, JQ-1 BOS-475, CC-90010, CC-95775, Mivebresib, BPL23314, SYHA- 1801, ARV-771, CK-103, dBET-1, GSK-3358699, MA-2014, MS-417, NEO-2734, NHWD- 870, NUE-7770, OHM-581, PLX-51107, QCA-570, RVX-297, SF-2523, SF-2535, SRX-3177, SRX-3262, ZBC-260, DCBD-005, KM-601, MZ-1, SBX-1301, SRX-3225, ZL-0580, NUE- 19796, NUE-20798 or a pharmaceutically acceptable salt, hydrate or solvate thereof and/or combinations thereof. The invention also provides a pharmaceutical composition comprising a histone deacetylase inhibitor as defined above and at least one pharmaceutically acceptable carrier for use in the treatment of autism spectrum disorder and/or intellectual disability. Preferably the pharmaceutical composition comprises a further therapeutic agent as defined above.

The invention also provides a method to identify a subject to be treated with a histone deacetylase inhibitor for the treatment of autism spectrum disorder and/or intellectual disability comprising measuring the level of GTF2I in a biological sample of said subject and comparing said measured level to a control level. Control level may be the level of GTF2I in a healthy subject or a subject not affected by autism spectrum disorder and/or intellectual disability.

The invention also provides a method to monitor the efficacy of a histone deacetylase inhibitor for the treatment of autism spectrum disorder and/or intellectual disability in a subject comprising measuring the level of GTF2I in a biological sample of said subject and comparing said measured level to a control level. Control level may be the level of GTF2I in said subject before the start of the treatment with the inhibitor or the level of GTF2I in said subject at a different time point in respect to the measurement.

The definition of ASD may be taken from the DSM5 (Diagnostic and Statistical Manual of Mental Disorders) available for example at the following link: https://www.autismspeaks.org/autism-diagnosis-criteria-dsm-5. Autism spectrum disorder (ASD) refers to a highly prevalent range of neurodevelopmental disorders characterized by deficits in social communication and interaction, and restricted, repetitive patterns of behavior, interests, or activities. The OMIM entry for 7dup is: # 609757 at the following link: https://www.omim. org/entry/609757?search=7ql 1 ,23%20microduplication&highlight=7ql 123 %20microduplication

HDAC inhibition may be measured by any conventional methods known in the art, as well as described herein. HDAC inhibitors block the action of histone deacetylases, wich remove the acetyl groups from the lysine residues in core histones leading to a condensed and transcriptionally silenced chromatin (Dokmanovic M et al. 2007). The action of HDACi can result in either the up-regulation or the repression of gene expression. HDACi activity is analyzed at different HDAC inhibitor concentrations by measuring HDAC substrate fluorescence in in vitro assay. Test inhibitors are diluted to different concentrations and a cell nuclear extract, with fluorescent HDAC substrate are added. The reaction is allowed to proceed for 10 min at 25°C and the mixture is assayed for HDAC activity.

In the present invention a HDAC inhibitor is any known HDAC inhibitor, for instance as indicated in Table 1 below, or in Ho et al. J. Med. Chem, https://dx.doi.org/10.1021/acs.jmedchem.0c00830, such as Vorinostat (trade name; ZOLINZA®), Romidepsin (trade name; Istodax®), Panobinostat (trade name; FARYDAK®), Belinostat, Entinostat, Domatinostat, Resminostat, Rocilinostat, Trichostatin A, Valproic acid, Dacinostat, Bisthianostat, Quisinostat hydrochloride, CUDC-101 , Scriptaid, Tefinostat, Givinostat, Mocetinostat, Chidamide, Abexinostat, Pracinostat, Butyric acid, Pivanex, 4- phenylbutyric acid (or sodium phenylbutyrate), Tucidinostat (or chidamide, trade name; Epidaza®), Nanatinostat, Fimepinostat, Remetinostat, Ricolinostat, Tinostamustine, JNJ- 26481585, RG2833, M344, APH-0812, CG-745, CKD-506, CKD-581, CXD-101, FX-322, YPL- 001, CFH-367C, Tacedinaline, Citarinostat, CKD-504, CUDC-907, CUDC-908, HG-146, KA- 2507, Lipocurc, MPT-0E028, NBM-BMX, OBP-801, OKI-179, RDN-929, VTR-297, REC- 2282, CS-3003, ACY-1035, ACY-1071, ACY-1083, ACY-738, ACY-775, ACY-957, ADV-300, AN-446, AP-001 (or Metavert), Arginine Butyrate, BMN-290, C-1A, CG-1521, CKD-509, CKD-L, CM-414, Crocetin, CS-3158, CT-101, CX-1026, RCY-1410, SE-7552, SKLB-23bb, CY- 190602, JBI-097, JBI-128, JMF-3086, KAN-0440262, KDAC-0001, Largazole, MPT- 0B291, MPT-0G211, MRx-0029, MRX-0573, MRX-1299, NHC-51, Nexturastat A, OKI-422, QTX-125, RCY-1305, SP-1161, SP-259, SRX-3636, ST-7612AA1, TJC-0545, Trichosic, YH- 508, HSB-501, NBM-1001, QTX-153, RDN-1201, ROD-119, ROD-1246, ROD-1275, ROD- 1702, ROD-2003, ROD-2089, ROD-702 and RTSV-5 and their analogues or derivatives.

HD AC inhibitors, in particular those listed above, and pharmaceutical compositions comprising the same can be administered for the uses of the present invention by conventional methods and formulations well known in the art.

In particular, the skilled person knows how to choose the suitable administration mode according to the general knowledge in the field. Reference can be made for example to Remington’s Pharmaceutical Sciences, last edition.

The administration regime, dosage and posology will be determined by the physician according to his experience, the disease to be treated and the patient’s conditions.

According to the administration route chosen, the compositions will be in solid or liquid form, suitable for oral, parenteral, intravenous, intra-arterial or other suitable routes of administration. The pharmaceutical compositions according to the present invention contain, along with the active ingredient or ingredients, at least one pharmaceutically acceptable carrier and/or excipient. These may be particularly useful formulation coadjuvants, e.g. solubilising agents, dispersing agents, suspension agents, and emulsifying agents.

Average quantities of the active agent may vary and in particular should be based upon the recommendations and prescription of a qualified physician. Generally, the HDAC inhibitor is administered in a “pharmaceutically effective amount”. The amount of the compound actually administered will typically be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, drug combination, the age, body weight, and response of the individual patient, the severity of the patient's symptoms, and the like. Generally, an effective dose will be from 0.01 mg/kg to 100 mg/kg, preferably 0.05 mg/kg to 50 mg/kg. Compositions may be administered individually to a patient or may be administered in combination with other agents, drugs, hormones, irradiation or surgery. For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually mice, rats, guinea pigs, rabbits, dogs, monkeys or pigs.

The present invention will be described by means of non limiting examples in reference to the following figures.

Figure 1. Symmetric copy number variations at 7qll.23. A Schematic representation of genotype-phenotype correlation in WBS and 7Dup patients, compared to healthy control (CTL), emphasizing opposite and shared phenotypes. Genomic organization of WBS region with the 17 genes that are significantly expressed in neurons, in bold the four genes selected for their critical role in the pathogenesis of both WBS and 7Dup. B Lentiviral vector (top) and PiggyBac- based construct (bottom) used to induce iPSC differentiation into cortical neurons. Ubc: human Ubiquitin constitutive promoter, rtTA: TET transactivator promoter gene, NGN2: Neurogenin2 gene, Puro^: Puromicin resistance gene, Bsd^: Blasticidin resistance gene, white triangles represent terminal repeats of the transposon.

Figure 2. NGN2-mediated conversion of iPSCs to iNs. A Timeline of differentiation protocol for iPSC-derived cortical neurons. ROCKi: ROCK inhibitor; Doxy: doxycycline; Puro: puromycin. B RT-qPCR analysis of Nestin and Synaptophysin mRNA expression in NGN2 neurons at two, three and four weeks of differentiation. The expression level is normalized against GAPDH, and further standardized to iPSCs levels. C Day 28 WBS01CN3 neurons express mature excitatory cortical neuron markers: NeuN, TUBB3, Synapsin 1/2, MAP2, VGLUT1 and SATB2. D mRNA levels of genes in the WBS region, BAZ1B, CLIP2, EIF4H and GTF2I (mean ± S.D.), in iPSC lines (left) and in NGN2-induced neurons (right) in the three genotypes (WBS, CTL, 7Dup) (n=2). CTL: CtlOlC, Ctl08A; WBS: WBS01CN3, WBS02C; 7Dup: DUP01GN4, DupO2K. Relative expression was measured by RT-qPCR, to GAPDH and results were arbitrarily normalized to mRNA levels of CTL (asterisks indicate statistical significance according to a one way ANOVA test: *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001). Figure 3. HTS workflow outline. A Compounds were tested at 10 pM for 48 h on NGN2 neurons seeded in 96-well plates. After RNA extraction and cDNA preparation, custom TaqMan Array 384-well plates were assembled through an automated TEC AN Freedom EVO workstation. RT-qPCR were performed in QuantStudio™ 7 Flex Real-Time PCR System. B D API-stained (left) and GFP-positive (right) WBS01CN3 NGN2 neurons counted with Cellomics during differentiation. C Normalization panel for quantification of cell number (left) and GFP positive cells (right) in three different 96-well plates at DIV 28 (WBS01CN3 line). D Relative expression of BAZ1B, CLIP2, EIF4H and GTF2I mRNA (mean ± S.E.) in day 28 WBS01CN3 neurons was measured by RT-qPCR, upon treatment with different DMSO concentrations. Highlighted in bold the DMSO concentration chosen for the screening.

Figure 4. Primary screening of a pharmaceutical compound library. A Composition of the compound library (1478 compounds). B Robustness of primary HTS setup. For each batch of plates, control run statistics with average Ct values (Avg.) of GAPDH and SRSF9 housekeeping genes, their S.D. and CV are summarized. C Exclusion and inclusion criteria of the primary screening. D Scatter plot of the primary screening. Fold changes compared with DMSO control were plotted for each gene (BAZ1B, CLIP2, EIF4H, GTF2I) in WBS01CN3 NGN2 neurons. Selected hits are shown for GTF2I.

Figure 5. HDAC inhibitors lower the mRNA and the protein levels of GTF2I in 7Dup iNs. A Relative expression of BAZ1B, CLIP2, EIF4H and GTF2I mRNA (mean ± S.D.) in WBS01CN3 and DupO2K iNs (n=2) treated with Domatinostat compared to control (DMSO). Error bars represent variation between lines of the two genotypes (Holm-Sidak-corrected t test ***p < 0.001). B Relative expression of GTF2I mRNA (mean ± S.D.) in two 7Dup-derived iNs, DupO2K and DupOlG, treated with different classes of epigenetic compounds compared to control. Error bars represent variation between the two above-mentioned iN lines (one way ANOVA test: *P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001). Relative expression of GTF2I mRNA (mean ± S.D.) in Dup03B (C), DupO4A (E), DupOlG (G) and DupO2K (I) iNs treated with Vorinostat, Mocetinostat and RG2833 compared to control. Relative expression was measured by RT-qPCR, normalized against GAPDH-SRSF9-RPS18 geometric mean. In C, E, G and I, error bars represent variation between three technical replicates. Protein levels of GTF2I in Dup03B (D), DupO4A (F) and DupOlG (H) iNs treated with Vorinostat, Mocetinostat and RG2833 compared to control. Immunoblot (left) and densitometric analysis (right). L Protein levels of GTF2I in DupO4A iNs treated with different concentrations of Vorinostat compared to control. Immunoblot (left) and densitometric analysis (right). Figure 6. Neuronal marker expression and Sholl analysis in NGN2 neurons. A RT-qPCR analysis of neural markers by differentiated neurons according to the genotype. Relative expression of SATB2, Syntaxin 1A, MAP2, and Synapsin 1 to TBP levels. Results were arbitrarily normalized to mRNA levels of CTL neurons, (mean ± S.E., n=3, asterisks indicate statistical significance according to a one way ANOVA test: *P < 0.05, **P < 0.005, ***p < 0.0005, ****P < 0.0001). CTL: CtlOlC, Ctl08A; WBS: WBS01CN3, WBS02C; 7Dup: DUP01GN4, DupO2K. B Representative images of tracings from 7Dup, healthy CTL and WBS iPSC-derived neurons. C Sholl analysis of dendrites from WBS, 7Dup and CTL neurons revealed no significant alterations in dendritic morphology (mean ± S.D., Two-way ANOVA test shows no statistical significant differences). Lines used CTL: CtlOlC; WBS: WBS01C, WBS02C; 7Dup: DupOlG, Dup03B.

Figure 7. Effect of HD AC inhibitors on the expression levels of WBSCR genes. A Schematic representation of WBSCR genes analyzed after treatment of 7Dup iNs with the three HDACi indicated below. B Relative expression levels of 17 genes in WBSCR in Dup03B and DupO4A iNs treated with Vorinostat, Mocetinostat and RG2833 compared to DMSO controls (mean ± S.D. asterisks indicate statistical significance according to a one way ANOVA test: *P < 0.05, **P < 0.005).

Figure 8. HDAC inhibitors slightly impact on cell viability and lower GTF2I protein levels. Cell viability, Histone H3 acetylation and GTF2I protein levels were measured in NGN2-induced neurons DIV28 exposed to the indicated doses of the selected HDAC inhibitors. A. Cell viability of four iN DUP lines after being treated with Vorinostat, Mocetinostat, or RG2833 for 48 h. B. Representative immunoblot showing levels of GTF2I, PARP1 and ac-H3 in DUP4A line; GAPDH, loading control; cf., cleaved form. C. Representative immunoblot showing levels of GTF2I and ac-H3 in four different DUP lines treated with Vorinostat lOuM for 48h. D. Quantification of GTF2I level in four different DUP lines treated with HDACi for 48h (mean ± SE).

Figure 9. HDAC inhibitors lower GTF2I protein levels in 7Dup cortical organoids. GTF2I protein levels were measured in cortical organoids DIV100 exposed to the indicated doses of the selected HDAC inhibitors for 15 days. A. Representative immunoblot showing levels of GTF2I, and ac-H3 in DUP04A cortical organoids; GAPDH, loading control. B. Quantification from two 7Dup lines in two different rounds of differentiation treated with HDACi for 15 days relative to mock control (mean ± SE).

DETAILED DESCRIPTION OF THE INVENTION Methods iPSCs lentiviral infection and PiggyBac system

Patient-derived iPSC lines were infected with an activator lentivirus, containing the reverse tetracycline transactivator (rtTA) constitutively expressed under the control of the UbC promoter, and an effector lentivirus, containing an NGN2-P2A-EGFP-T2A-Puro cDNA under the control of the tetracycline responsive element [29] (Fig. IB, top). Infected iPSCs were sorted as single cells in 96-well plates, selected based on the round morphology of colonies and gradually expanded. Selected lines were then induced for one day adding doxycycline to the medium. GFP- positive lines were then selected and expanded, further being stabilized and characterized. Through this system the inventors generated the iPSC monoclonal line WBS01CN3 (WBS) and DUP01GN4 (7Dup).

To establish a robust and rapid neuronal differentiation method, the inventors utilized a direct conversion technology. Mouse Ngn2 cDNA, under tetracycline-inducible promoter (tetO), was transfected into iPSCs by a newly developed enhanced PiggyBac (ePB) transposon system [9, 30, 31] (Fig. IB, bottom). 4xl0⁵ iPSCs, for each line, were electroporated with 2,25 pg of the ePB construct carrying the inducible Neurogenin-2 (NGN2) overexpression cassette and 250 ng of the plasmid encoding transposase for the genomic integration of the inducible cassette. Electroporations were performed using the Neon Transfection System (MPK 10096, Thermo Fisher Scientific). iPSCs were selected using blasticidin 5 pg/ml (R21001, Gibco) for five days and stable iPSC lines were stocked. Through the ePB system the inventors generated the following polyclonal lines: CtlOlC, Ctl08A (CTL): WBS01C , WBS02C (WBS); Dup03B, DupO4A, DupOlG, DupO2K (7Dup).

NGN2 differentiation into cortical glutamatergic neurons

In order to obtain cortical glutamatergic neurons (iNs), on day -1 iPSCs were dissociated with Accutase (GIBCO, Thermo Fisher Scientific) and seeded in plates coated with 2,5 % (v/v) Matrigel (Coming) in mTeSR™ supplemented with ROCK inhibitor (STEMCELL Technologies). iPSCs were then cultured in MEM1, composed by DMEM/F12 1 :1 (Euroclone/Gibco) supplemented with NEAA 1%, N2 1%, BDNF 10 ng/ml, NT-3 10 ng/ml, Laminin 0,2 pg/ml and 2 pg/ml doxycycline hydrochloride, from day 0 to day 1. On day 1, puromycin 1 pg/ml was added to MEM1 and, on day 2, the medium was changed with NBM Plus, composed by Neurobasal Plus (Thermo Fisher Scientific) supplemented with 50x B27 Plus supplement (GIBCO, Thermo Fisher Scientific), Glutamax 0.25% (Thermo Fisher Scientific) and 2 pg/ml doxycycline hydrochloride. On day 7, differentiated neuronal cells were dissociated with Accutase and seeded into poly-D-lysine coated 96-well plates (3842, Corning) at a density of 20.000 cells/well in NBM Plus; culture medium was then changed 50% once a week until day

28.

Immunocytochemistry

Neurons were fixed in 4% paraformaldehyde in PBS for 15 min. at room temperature immediately after removal of culture medium, and pipetting was done slowly to prevent dislodging cells from coverslips. The cells were then washed 3 times for 5 min. with PBS, permeabilized with 0.1% Triton X-100 in PBS for 15 min., and blocked in 5 % donkey serum in PBS for 30 min. After blocking, the cells were incubated with primary antibodies diluted in blocking solution overnight at +4°C. The cells were washed 3 times with PBS for 5 min. and incubated with secondary antibodies at room temperature for 1 h. Nuclei were then stained with DAPI solution 1 :5000 at room temperature for 10 min. Coverslips were rinsed in sterile water and mounted on a glass slide with 7-8 pl of Mowiol mounting medium.

Cellomics

Neurons in 96-well plates were fixed in 4% paraformaldehyde in PBS, permeabilized with 0.1 % Triton X-100 and then counterstained with DAPI (1 :5000) to enable autofocusing of the automated Thermo Scientific ArrayScan VTI High-content screening microscope (Cellomics). Cell counting of validated objects was done in the DAPI channel and in the GFP channel.

Automation protocol

All liquid handling was done in an automated manner by a TECAN Freedom EVO automated platform under control of EVOware® software. The TECAN has been programmed to prepare up to 20 96-well plates in a single run, which would produce 1080 individual datapoints. The modular robotic scripts were designed as building blocks for users with minimal automation programming experience to assemble an automated process from cells preparation to sample analysis. The inventors prepared scripts for compound treatment, RNA and cDNA dilution (and predilution if necessary), reagent addition (Cells-to-CT, RT), and sample re-positioning in prespotted 384-well plates. Each module contained user-friendly interfaces for inputs of assay variables, such as volumes, dilution factors and plate maps. The liquid-handling robot used in this work is a Tecan Freedom EVO-2 150 liquid handling unit equipped with a 96-well headadapter with filter tips; the pipetting volume range was from 10 to 1000 pl. The Freedom EVO worktable was loaded with three solution reservoir carriers (1 x Trough 100 ml, 3 Pos. and 2 x Trough 25 ml, 3 Pos.), two 96-well plate carriers (96-well, 6 Pos.), and one 384-well plate carrier (96-well, 3 Pos.).

Quantitative RT-PCR A custom TaqMan Cells-to-CT™ kit (Invitrogen AMI 729) was used to extract the RNA and perform reverse transcription to obtain cDNA, according to the manufacturer’s instructions. After media aspiration, 30 pl of 2x lysis solution, with diluted DNasel, were added to 30 pl of the remaining buffer in each well; then the plate was incubated for 5 min. at room temperature. Subsequently Stop solution (3 pl) was added and the solution was incubated at room temperature for 2 min. Then, 30 pl of lysates were transferred to a new PCR plate with 40 pL of reverse transcription enzyme mix previously added to each well. The thermal cycling conditions were: 60 min. at 42°C, and 5 min. at 85 °C. cDNA was diluted with 50 pl of water and then a 5 -pl aliquot of each cDNA reaction was added to 5 pl of each TaqMan master mix reaction into pre-spotted custom 384-well plates. A QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) was utilized to determine the Ct values. Relative mRNA expression levels were normalized and analyzed through the comparative delta-delta Ct method using the QBase Biogazelle software.

Hit selection

The inventors used a strategy based on fold-difference analysis of target genes to housekeeping genes, comparing compound- to DMSO control-treated wells. Hits were defined as more than 2- fold increase or less than 0,5-fold decrease in at least three out of four genes, or in at least GTF2I. Thirty -five compounds fulfilled the first criteria and 36 compounds the second one in the primary screening.

Antibodies

The following antibodies were used for Western Blot analysis: pAb anti-GTF2I 1 : 1000 (A301- 330A, Bethyl Laboratories), pAb anti-GAPDH 1 :5000 (ABS16, Merck Millipore), Histone H3K9ac 1 : 1000 (Abeam ab4441), Cleaved PARP (Asp214) 1 : 1000 (CellSignal. 5625). and secondary antibody horseradish peroxidase-conjugated donkey anti-rabbit (Pierce). The following antibodies were used for immunocytochemistry analyses: NeuN 1 :500 (MABN140, Sigma-Aldrich), TUBB3 1 : 1000 (PRB-435P, BioLegend), MAP2 1 :500 (M9942, Sigma- Aldrich), vGlutl 1 : 1000 (135303, Synaptic Systems), SATB2 1 :200 (ab51502, Abeam), Synapsin 1/2 1 : 1000 (106004, Synaptic Systems).

Chemicals

Epigenetic compound library: Selleckchem Cat. N° LI 900; Bioactive compound library: Food and Drug Administration (FDA) approved and clinical compounds selected from the Library of Pharmacologically Active Compounds (LOPAC, Sigma) and the Spectrum Collection (MicroSource Inc).

Protein extraction and immunoblotting Proteins were extracted from iNs grown in 10 cm or 6-well plates by washing the cells with ice- cold PBS, followed by immersion in lysis buffer (25 mM Hepes pH 7.5, 300 mM NaCl, 10% glycerol, 1% NP-40) supplemented with cOmplete™ protease inhibitor cocktail (Sigma). Lysates were sonicated using the Bioruptor Sonication System (UCD200) for three cycles of 30 s with 60-s breaks at high power and then centrifuged at 13.000g for 15 min. Protein quantification was performed using the Bradford protein assay (Bio-Rad) following the manufacturer's instructions. Protein extracts (10-20 pg per sample) were run on a precast NuPAGE 4-12% Bis-Tris Gel (NP0335BOX, Life Technologies), transferred to a nitrocellulose membrane and blocked in TBST (50 mM Tris, pH 7.5, 150 mM NaCl and 0,1% Tween-20) and 5% milk at room temperature for 1 h. Primary and secondary antibodies were diluted in TBST and 5% milk. The immunoreactive bands were detected by ECL (GE Healthcare) and imaged with a ChemiDoc XRS system (Bio-Rad Laboratories). Densitometric analysis was performed using the ImageLab 4.1 Software (Bio-Rad Laboratories).

Proliferation Assay

Number of viable cells was estimated by automatic counting with Fiji. In brief, 3xl0⁵ cells/well were plated in poly-D-lysine coated 6-well flat bottom plates (Coming) with 4 replicates per condition and induced to differentiate into neurons for 28 days. Cells were treated with HDACi for 48h and neurons images were acquired at 20x magnification using the ScanR Microscope. Image acquisition was done in automated manner, recording 9 field each well; two channels were acquired per batch for GFP and visible light. Measurements were performed every 24 h. Each data point was normalized to a DMSO treated well.

Cortical organoid protocol

Cortical organoids were differentiated as previously described (Lopez-Tobon 2019). Briefly 2x10^A5 iPSCs cells per well were plated into ultra-low-attachment 96well plastic plates (Corning) in FGF2-free knockout serum medium. For the first 24 h (day 0), the medium was supplemented with the ROCK inhibitor Y-27632 (EMD Chemicals). For neural induction, dorsomorphin (Merck, 5 pM) and SB-431542 (Tocris, 10 pM) were added to the medium until day 5. From day 6 onward, organoids were moved to neural medium (NM) containing Neurobasal (Invitrogen 10888), B-27 serum substitute without vitamin A (Invitrogen 12587), GlutaMax 1 : 100 (Fisher 35050071), 100 U/ml penicillin and streptomycin (Invitrogen) and 50 mM b -Mercaptoethanol (Gibco 31350010). The NM was supplemented with 20 ng/ml FGF2 (Thermo) and 20 ng/ml EGF (Tocris) for 19 days with daily medium change in the first 10 days, and every other day for the subsequent 9 days. On day 12, floating organoids were moved to orbital shaker (VWR Standard Orbital Shaker, Model 1000) and kept on constant shaking at 50 rpm to promote nutrient and oxygen exchange. To induce neurogenesis, FGF2 and EGF were replaced with 20 ng/ml BDNF (Peprotech) and 20 ng/ml NT3 (Peprotech) starting at day 25, while from day 43 onwards only NM without growth factors was used for medium changes every other day. Organoids were treated at day 100 for 15 days, drugs were refreshed every 3 days.

Virus preparation for Sholl analysis

Five million HEK 293T cells were plated in 10 cm plates and grown in 10% fetal bovine serum in DMEM. On the next day, cells were transfected with plasmids for gag-pol (10 pg), rev (10 pg), VSV-G (5 pg) and the target construct (15 pg) CaMKIIa-mK02, using the calcium phosphate method [79], On the next day, the medium was changed. On the day after, the medium was spun down in a high-speed centrifuge at 30,000g, at 4 °C for 2 h. The supernatant was discarded and 100 pl of PBS were added to the pellet and left overnight at 4 °C. On the next day, the solution was triturated, distributed into 10-pl aliquots and frozen at -80 °C.

Neuronal infection

At day 4 of the differentiation protocol, neurons were infected with virus bearing CaMKIIa- mK02; specifically, 10 pl virus per 6cm plate from a standard preparation (see Virus preparation). At this time, an appropriate number of vials of mouse astrocytes were thawed into a 10 cm plate, in order to obtain at least 1.25 million of astrocytes at day 8. At day 8, infected neurons were digested with accutase for 5 min., washed with PBS, counted, and seeded at a total density of at least 30.000 cells/cm² (300.000 cells/well in a 6-well plate) in a 1 :50 ratio with not infected neurons and in a 1 :1 ratio with mouse astrocytes, in poly-D-lysine-coated coverslips. Over the following weeks, the coverslips were monitored and those with at least 10 visible individual neurons were kept for image acquisition.

Image acquisition

For morphometric analysis, images of neurons were acquired at lOx magnification using the Leica DM6 Multifluo Fluorescence Microscope. Image acquisition was done in a semi-automated manner, with manual picking of individual neurons and batch acquisition. Two channels were acquired per batch for GFP and mK02.

Morphological analysis

All analyses were completed in Fiji (ImageJ). Dendrite length was characterized using the Simple Neurite Tracer ImageJ plugin. For Sholl analysis, the center of concentric spheres was defined as the center of the soma, and a 10 pm radius interval was used. In order to compare the Sholl analysis curves between genotypes, a two-way ANOVA test was performed. P-values < 0.05 were considered statistically significant.

Data analysis Qbase+ software version 3.0 (Biogazelle, Zwijnaarde, Belgium) was used to analyze the variability of the genes tested and to determine the hit compounds. The geometric mean of the cycle threshold value of the endogenous control genes GAPDH, SRSF9 and RPS18 was used to normalize the data, and the DMSO-treated samples were used as calibrator.

Statistical analysis

Statistical analyses were performed using PRISM (GraphPad, version 6.0). Results are expressed as means ± S.D or means ± S.E._Statistical significance was determined according Holm-Sidak- corrected t test, considering each iPSC line as biological replicates (n) or according to a one way ANOVA test as indicated in figure legends. Asterisks indicate statistical significance (*P < 0.05, **P < 0.005, ***P < 0.0005, ****P < 0.0001).

EXAMPLES

EXAMPLE 1

NGN2-driven neurogenesis retains the defining transcriptional imbalances of 7qll.23 CNV With the goal of identifying compounds capable of restoring the physiological expression levels of the four aforementioned genes from the WBSCR, the inventors set out to establish HTS-proof conditions for the differentiation and maintenance of patient-derived cortical neurons, starting off with the NGN2-driven system of iPSC differentiation [29] and adapting it to HTS as follows. First, the inventors reasoned that, in a HTS setting inherently prone to fluctuations in numerous technical variables, the use of lines with a fixed number of integrations would help reduce the confounding variables inherent to the differentiation of polyclonal batches with an unchecked diversity of copy number integrations of the NGN2 transgene. Thus, the inventors used an NGN2 expressing lentivirus to generate a stable monoclonal iPSC line originally reprogrammed from a patient harboring the WBS deletion (hereafter WBS01CN3 line). Second, since the original NGN2-driven protocol [29] relied on astrocytes to support neuronal growth but their presence would have interfered with gene expression analysis in neurons, the inventors used a new formulation medium (NBM Plus) that allowed to replace astrocytes and minimize media changes, thus also reducing the automation complexity of the HTS. Third, the inventors adapted the differentiation protocol to a HTS platform by first seeding the iPSCs and culturing them in large batches on Matrigel-coated 15 cm dishes and then detaching them for seeding on poly-D-lysine coated 96-well plates (Figure 2A). The inventors validated the robustness of this protocol by both immunocytochemistry and qRT-PCR. Forced NGN2 expression converted iPSCs into mature neuronal morphology in 28 days with a rapid decline of the neural progenitor marker Nestin and an increase in the expression of the synaptic marker Synaptophysin (Figure 2B). iPSC-derived NGN2-induced neurons (iNs) expressed glutamatergic markers like vGLUTl, cortical markers such as SATB2 and the expected combination of both early neuron markers like TUBB3 and mature neuron markers as MAP2, NeuN and the synaptic marker Synapsin 1/2 (Figure 2C). The inventors also analyzed the expression levels of SATB2,MAP2 and SYN1 in both WBS and 7Dup iNs compared to healthy control (CTL) iNs, using SyntaxinlA (STX1A), a WBSCR gene, as internal control of symmetrical dosage imbalance. While SATB2 and SYN1 show an increased expression in both 7Dup and WBS iNs compared to CTL, MAP2 does not show 7ql 1.23 dosagedependent alterations in expression levels (Figure 6 A). Moreover, as a baseline evaluation of HTS-relevant neuronal morphology, the inventors performed a morphometric Sholl analysis of dendrites in WBS, 7Dup and CTL iNs, plotting the number of intersections with circles centered on the soma against the distance from the cell body. Detailed analysis of neurons revealed unaltered complexity for both basal and apical dendrites across the three genotypes (Figure 6 B, C).

Importantly, the inventors confirmed that the transcriptional levels of BAZ1B, CLIP2, EIF4H and GTF2I mirrored the symmetrical gene dosage in both WBS and 7Dup iNs compared to healthy control (CTL) iNs, confirming that the gene dosage imbalance is maintained upon cortical neuronal differentiation (Figure 2D) and hence that it represents a rational target for a mechanistically-based therapeutic intervention.

EXAMPLE 2

Establishment of an in vitro platform for drug screening

In order to screen large chemical libraries, the inventors defined disease-relevant models for WBS and 7Dup suitable for HTS. The inventors tested the differentiation protocol with a WBS line (WBS01CN3) to establish the proper conditions for adaptation to a miniaturized HTS format (Figure 3A). The inventors obtained optimal cell-plating settings for 96-well plates using poly- D-lysine coated plates by seeding 20.000 cells/well. Cellular growth and percentage of GFP positive cells were monitored by automated cell counting after Dapi-nuclear staining through five weeks of differentiation. An expected slight decrease of total cell number was observed over the course of differentiation, while GFP positive cells percentage remained stable around a 70%. On these bases, 28 DIV (days in vitro) has been chosen as time point to perform the in vitro assay (Figure 3B). Good consistency and reproducibility have been found once assessed cell plating consistency for three plates from a single round of differentiation considering both cell number (Figure 3C, left panel) and GFP positivity (Figure 3C, right panel). Optimal morphology and proliferation characteristics of iPSC lines were checked periodically, and the lines were kept in culture for no more than two months, the duration of the screening. To develop an iN-based HTS assay, considering the relevance that could have for both WBS and 7Dup in correcting the genetic imbalance, the inventors envisaged that promising compounds could restore mRNA levels of four genes in the WBS region, namely GTF2I, BAZ1B, CLIP 2 and EIF4H. Therefore, the inventors selected TaqMan qRT-PCR assays for measuring the expression levels of these genes against their internal controls GAPDH, SRSF9 and RPS18. Transcript levels were measured after cell lysis, RNA extraction, qRT-PCR and data quantification. DMSO had no major impact on growth of iNs and on mRNA levels up to 0,5 % DMSO (v/v) (Figure 3D). Finally, having checked all the parameters, the inventors proceeded with a moderate-sized screening of around 100 96-well plates of iNs. The inventors screened a library of 1478 small molecules in biological triplicate (4434 treatment conditions in total). The present screening library comprises an extensive variety of compounds, including e.g. central nervous system (CNS) agents, natural compounds, hormonal agents, epigenetic and immune system modulators, antioxidants (Figure 4A). The compounds were selected analysing an internally available Chemical Collection of more than 200.000 compounds composed by FDA approved drugs, bioactive compounds (including preclinical and clinical compounds), a kinase target library, a fragment library and a commercially available screening library. During the process of selection, pain assay interference compounds and molecules presenting known reactive and/or toxic moieties have been filtered and removed. Among the remaining compounds, approved drugs, preclinical and clinical molecules have been preferred with the aim of accelerating the path from discovery to patients. Epigenetic compounds have been largely represented inside the preclinical compounds envisaging a relevant role of chromatin perturbation and epigenetic modifications for the considered pathology. Compounds were screened at 10 pM in 0,1% DMSO, with each plate containing DMSO control wells. The inventors used the WBS patient-derived monoclonal line WBS01CN3 for the first HTS, looking for molecules that restore the gene dosage of BAZ1B, CLIP2, EIF4H and GTF2I n patient-specific iNs. To validate their qRT-PCR assay, the inventors measured parameters of the screening workflow, as coefficient of variation (CV), to demonstrate consistency in Ct values in the four batches of cell plates. Differences in Ct values were minimal among replicate wells of the same batch giving a CV < 20% (Figure 4B). The strategy the inventors used to nominate candidate hits out of the 1478 compounds tested in triplicate was based on fold-difference analysis of WBS genes to housekeeping genes, comparing compound wells to DMSO control -treated wells. Compounds that gave rise to increased GAPDH Ct values > 3 S.D., compared to DMSO values, or to GAPDH Ct values > 30 were excluded from the analysis, as revealing high cellular toxicity (Figure 4C). Hits were defined as more than 2-fold increase or less than 0,5-fold decrease in at least one out of the four genes (Figure 4C, D). Thirty- five compounds fulfilled the first criteria and were further analyzed under standard 6-well plate culture conditions, but the inventors did not confirm any compound able to increase more than 2-fold the expression levels of the target genes in WBS neurons.

EXAMPLE 3

HDAC inhibitors specifically lower the mRNA and the protein levels of GTF2I in 7Dup induced neurons.

While the above results uncover the WBS gene dosage as particularly resilient to any attempt at positive transcriptional modulation, at least within the chemical universe the inventors explored in this screening, the inventors noticed negative transcriptional modulation by specific compounds such as domatinostat, which decreased the expression levels of GTF2I, in the WBS genetic background, without any significant transcriptional modulation of the other three genes BAZ1B, CLIP2 and EIF4H compared to the vehicle control (DMSO) (Figure 5 A). The inventors thus reasoned that such compounds that are able to further lower, even in a haploinsufficient context, critical WBSCR genes such as GTF2I could prove particularly useful to rescue the transcriptional imbalance of the symmetrical 7ql 1.23 syndrome. For this purpose, the inventors generated several polyclonal lines, i.e. Dup03B, DupO4A, DupOlG, DupO2K, using the ePB based system containing the same NGN2 cassette that the inventors used for the monoclonal lines. The inventors thus tested domatinostat at the same concentration (10 pM for 48 h) in 28 day-old 7Dup iNs (i.e. harboring the symmetrically opposite genetic lesion) and confirmed the specific effect of lowering GTF2I levels (Figure 5A). The inventors thus set out to expand this observation to other compounds within the epigenetic subset of the present HTS library. The inventors observed that 20, out of 22 epigenetic compounds tested in the validation process, lowered GTF2I levels in 7Dup iNs, and, interestingly, they all belong to the class of HDAC inhibitors (Figure 5B). Indeed, both JNJ-7706621 and UNC0379, which are, respectively, a CDK inhibitor and a histone methyltransferase inhibitor, have no effect on GTF2I mRNA (Figure 5B). In order to characterize these hits in greater detail and prioritize them, the inventors carried out an analysis of their selectivity profile as well as of the chemical diversity and of their pharmacokinetic properties. This led us to select the following three compounds, vorinostat, mocetinostat and RG2833, according to the parameters of (i) Blood-Brain Barrier (BBB) penetration ability, (ii) FDA approval, and (iii) HDAC class/type selectivity (Table 1).

Table 1. Selection criteria for HDACi. Among the HDACi tested, reported in table, three HDACi chosen for further studies, according to HD AC class/type selectivity, FDA approval and BBB penetration ability, are marked in bold.

The inventors thus went on to validate the G7F2/-lowering effect of the three selected compounds on multiple 7Dup patient-derived lines, so as to secure the generalizability of their findings across a heterogeneity of human backgrounds harboring the 7ql l.23 duplication. The inventors confirmed that the three selected HDACi lower the expression levels of GTF2I in 7Dup 28 day- old neurons derived from four genetically different iPSC lines, i.e. Dup03B (Figure 5C), DupO4A (Figure 5E), DupOlG (Figure 5G) and DupO2K (Figure 51). The effect of HDACi on GTF2I was confirmed also at a protein level in Dup03B (Figure 5D), DupO4A (Figure 5F) and DupOlG (Figure 5H) iNs. Specifically, vorinostat reduced consistently also the protein levels of GTF2I, to a degree comparable to the transcriptional read-out and in a reproducible manner across different patient-derived iNs; mocetinostat and RG2833 showed more variable correspondence between transcript and protein level assays across patient-derived iNs. In order to define the compounds’ effect across the 7ql l.23 interval, the inventors expanded our gene expression analysis testing the effect of Vorinostat, Mocetinostat and RG2833 on 13 additional genes of the WBS region, prioritizing those most relevant to the neuronal pathophysiology of the 7Dup (Figure 7A) [23], Interestingly, the inventors observed that the three compounds decrease the expression levels of GTF2IRD1 and VPS371). along with GTF2I (Figure 7B, right panel), while Vorinostat and Mocetinostat slightly decrease the expression level also of CLIP2. Four genes show a trend of increase upon treatment while most of the remaining genes in the region show no major changes in expression levels (Figure 7B, left and central panel).

Given the key role played by GTF2I in the pathophysiology of both syndromes [19, 20, 21, 32, 33, 34], alongside initial evidence linking its polymorphisms to sociability metrics in the wider population [35], the largely selective effect of the three compounds provides thus a promising basis for the translation of these findings, especially in the context of a chronic treatment meant to provide cognitive/behavioral amelioration (GTF2I dosage-dependent) while leaving largely unaffected the regulation of other 7ql 1.23 genes with pleiotropic functions.

Combining these observations, the inventors treated DupO4A neurons with different concentrations of vorinostat to define a dose-response range, finding that the reduction of GTF2I protein levels was maintained down to 1 pM (Figure 5L).

EXAMPLE 4

HDAC inhibitors have minor effect on cell viability and lower protein levels of GTF2I in 7Dup cortical organoids.

The inventors relied on the effect obtained at the protein level and on cell viability, to evaluate the compounds capable of fine-tuning the level of GTF2I, avoiding excessive decreases that might spill into the WBS dosage range. The inventors examined the effects of HDAC inhibitors on cell viability in iPSC-derived NGN2-induced neurons. To do this, iN at day 28 were treated once with increasing concentrations (0.1-10 pM) of Vorinostat, Mocetinostat and RG2388 for 48 h (Figure 8 A) and living cells were counted 48 hours post-treatment in four different lines. Treatment with Vorinostat, Mocetinostat and RG2388 at 0.1 and 1 pM had no significant effect on cell viability (Figure 8 A). Vorinostat and Mocetinostat at highest concentration induced a 20- 30% reduction of cell viability (Figure 8 A) and have raised apoptosis as confirmed by Cleaved- PARP accumulation (Figure 8 B). The effect on GTF2I level was confirmed as dose dependent in four different patient-derived iN lines (Figure 8 B, D).

Then the potential of each molecule to increase the levels of histone acetylation was evaluated, using western blot analysis for histone acetylation (acH3-K9), as a readout of HDAC inhibition. All tested molecules induce a dose-dependent H3 acetylation accumulation (Figure 8 B, C).

To confirm the ability to rescue the transcriptional imbalance of GTF2I protein in a model of cortical development the inventors treated cerebral organoids at day 100 with increasing concentration of above mentioned HDAC inhibitors. Upon a 15 days treatment, the results confirmed a concentration-dependent reduction of GTF2I protein in cortical organoids (Figure 9 A, B) induced by the three tested inhibitors.

Discussion

WBS and 7Dup are two paradigmatic neurodevelopmental disorders whose unique alignment of symmetrically opposite CNV and symmetrically opposite phenotypes in sociality and language provides unique glimpses into the molecular architecture of ASD. This first exploration, viaHTS, of a large chemical space in search of clinically relevant compounds to restore the transcriptional dosage of key WBSCR genes led us to the following results.

First, the inventors introduced an adaptation of the NGN2-driven conversion of iPSCs into functional iNs [29, 36] to an automation-intensive HTS format, which can serve as template to streamline further drug screening and/or repurposing campaigns targeting cortical glutamatergic neurons. Specifically, this entailed benchmarking of HTS-proof conditions attuned to the specific challenges of patient-derived iPSCs and iNs, including comparison of culture conditions or modes of NGN2 transgene insertion (exposing the value of the monoclonal line used in the primary HTS campaign to minimize confounding variables, followed by validation in polyclonal lines derived from multiple patients through the easily scalable polyclonal format).

Second, the inventors identified HDAC inhibition as a powerful and surprisingly specific chromatin intervention for rescuing the aberrant transcriptional levels of GTF2I, the cardinal gene involved in by 7ql 1.23 CNV. HDACi prevent the deacetylation of histones thereby facilitating gene expression. Intensively studied for treatments of different malignancies, from hematological entities to solid tumors [37, 38], HDACi have also been probed in models of neurodegenerative disorders, such as Alzheimer's [39, 40], Parkinson’s [41] and Huntington's diseases [42], and diabetic neuropathic pain [43], but their application to neurodevelopmental disorders, and ASD in particular, remains to be fully explored. Indeed, although previous studies highlighted a link between HDAC inhibition and improvement of social cognition in different mouse models of ASD [44, 45, 17], and functional recovery in cortical neurons in MECP2 duplication syndrome [46], the use of HDACi in 7Dup patients has never been anticipated.

Here the inventors identified and confirmed three HDACi (vorinostat, mocetinostat and RG2833) that are able to reduce GTF2I expression both at a transcription and at the protein level in 7Dup iNs.

In particular, vorinostat is an FDA-approved Pan HDAC -inhibitor that crosses the BBB [48]; mocetinostat is a class I selective HDACi that passes the BBB in mice [48], and RG2833 is a brain-penetrant HDACi with a specificity for HDACI and HDAC3 [49] (Table 1). The common characteristic of these compounds, which grounded their rational for selecting them for validation amongst the other HDACs leads emerged from the HTS, is the ability to pass the BBB, an obviously crucial aspect for neurodevelopmental disorders. Importantly, at present vorinostat is among four HDACi, along with panobinostat, belinostat and depsipeptide (romidepsin), that have already received FDA approval for the treatment of a number of conditions, including refractory cutaneous T-cell lymphoma, refractory multiple myeloma and peripheral T-cell lymphoma, respectively [50, 51, 52, 53], Besides existing approval, the present results provide additional support for vorinostat as the most promising HDACi amongst the ones the inventors identified. Specifically, the inventors probed the effect of the three compounds also at the protein level, aiming at scoring the best performance on two criteria: i) the narrow range of the effect, i.e. privileging the compound best capable of fine-tuning the level of GTF2I, thus avoiding an excessive decrease that might spill into the WBS dosage range; and ii) the robusteness of this fine-tuned effect across different patients. On this basis, the inventors observed that inhibitors of the invention reduce the protein levels of GTF2I in iNs derived from different patients. Finally, its effect is maintained down to 1 pM, the dose corresponding to the clinically active tolerated relevant concentration approved for oncology indications [54],

Third, while the effect of HDACi on GTF2I is arguably indirect, it is very specific with respect to the other three genes the inventors had scored as targets in their screening. This is consistent with the observation, as summarized in Table 1, that the most represented HDACi specificities among the compounds the inventors identified are for different classes of HDAC: HDAC 1, 3 and 6. HDAC 1 and 3 are included in class I HDAC, while HDAC 6 belongs to another class (lib). Specifically, HDAC 1 is expressed primarily in neurons and it mainly functions in combination with HDAC2 in several repressor complexes; HDAC3 is the most highly expressed class I HDAC in the brain and it is also predominantly expressed in neurons, playing an essential role in brain development [55]; lastly, HDAC6 is involved in processes related to neurodegeneration, binding to ubiquitinated protein aggregates [56],

This diversity of pathways whose inhibition converges on GTF2I is not surprising given the observations from several studies demonstrating how HDAC inhibitors can cause both up- and down-regulation of gene expression patterns [57, 58, 59, 60], pointing to the fact that HDAC inhibitions also alter the expression of additional enzymes or co-factors which in turn will act as activators or repressors of other downstream genes.

Finally, the specificity of effect on GTF2I underscores the possibility that even in disorders caused by fairly large CNV encompassing multiple genes, it is possible to identify compounds that, albeit acting through major regulatory pathways such as histone deacetylation, end up exerting, in the context of patient-derived disease-relevant cell types, a exquisitely specific effect. For clinical translation this is potentially highly relevant, since in multi-gene CNV disorders for which one gene is particularly critical (as the case of GTF2I for 7ql 1.23 CNV), selective therapies may likely have fewer side effects than those modulating the expression of the entire CNV.

This underscores the possibility that even in disorders caused by fairly large CNV encompassing multiple genes, it is possible to identify compounds that, albeit acting through major regulatory pathways such as histone deacetylation, end up exerting, in the context of patient-derived diseaserelevant cell types, a exquisitely specific effect. For clinical translation this is potentially highly relevant, since in multi-gene CNV disorders for which one gene is particularly critical (as the case of GTF2I for 7ql 1.23 CNV), selective therapies may likely have fewer side effects than those modulating the expression of the entire CNV.

Together, the present results establish the power of ASD patient-specific neurons for drug discovery and/or repositioning through HTS and identify HDACi, and especially vorinostat, as particularly promising repurposed compounds for 7Dup.

Drug repositioning has the potential to provide new therapeutic alternatives for patients as well as “new” innovative use for “old” drugs thus delivering relevant clinical improvement while reducing their clinical development time compared to de novo development of new chemical entities.

Considered the unmet medical need in the ASD field, the present HTS-derived results represent a unique opportunity to develop first-in-class therapeutic agents for the 7Dup syndrome and possibly other neurodevelopmental conditions and an intriguing prospect to investigate the link between HDAC inhibition and GTF2I regulation. Finally, effective treatments of 7Dup core symptoms will also help to reduce the staggering physical and mental stress on patients’ caregivers, along with the financial burden involved in managing this disease, conferring a great benefit to the society.

BIBLIOGRAPHY

1. Lai M-C, Lombardo MV, Baron-Cohen S. Autism. Lancet. 2014 Mar 8;383(9920):896- 910.

2. Ronemus M, lossifov I, Levy D, Wigler M. The role of de novo mutations in the genetics of autism spectrum disorders. Nat Rev Genet. 2014 Feb;15(2): 133-41.

3. Van der Aa N, Rooms L, Vandeweyer G, van den Ende J, Reyniers E, Fichera M, et al. Fourteen new cases contribute to the characterization of the 7ql 1.23 microduplication syndrome. Eur J Med Genet. 2009 Jun;52(2-3):94-100.

4. Sanders SJ, Ercan-Sencicek AG, Hus V, Luo R, Murtha MT, Moreno-De-Luca D, et al. Multiple recurrent de novo CNVs, including duplications of the 7ql l.23 Williams syndrome region, are strongly associated with autism. Neuron. 2011 Jun 9;70(5):863-85.

5. Pober BR. Williams-Beuren syndrome. N Engl J Med. 2010 Jan 21;362(3):239-52. 6. Lee G, Ramirez CN, Kim H, Zeltner N, Liu B, Radu C, et al. Large-scale screening using familial dysautonomia induced pluripotent stem cells identifies compounds that rescue IKBKAP expression. Nat Biotechnol. 2012 Dec;30(12): 1244-8.

7. Li J, Ma J, Meng G, Lin H, Wu S, Wang J, et al. BET bromodomain inhibition promotes neurogenesis while inhibiting gliogenesis in neural progenitor cells. Stem Cell Res. 2016 Sep;17(2):212-21.

8. Cayo MA, Mallanna SK, Di Furio F, Jing R, Tolliver LB, Bures M, et al. A Drug Screen using Human iPSC-Derived Hepatocyte-like Cells Reveals Cardiac Glycosides as a Potential Treatment for Hypercholesterolemia. Cell Stem Cell. 2017 Apr 6;20(4):478- 489. e5.

9. Kondo T, Imamura K, Funayama M, Tsukita K, Miyake M, Ohta A, et al. iPSC-Based Compound Screening and In Vitro Trials Identify a Synergistic Anti-amyloid P Combination for Alzheimer’s Disease. Cell Rep. 2017 Nov 21;21(8):2304-12.

10. Tranfaglia MR, Thibodeaux C, Mason DJ, Brown D, Roberts I, Smith R, et al. Repurposing available drugs for neurodevelopmental disorders: The fragile X experience. Neuropharmacology. 2018 May 4;147:74-86.

11. Gogliotti RG, Niswender CM. A coordinated attack: rett syndrome therapeutic development. Trends Pharmacol Sci. 2019;40(4):233-6.

12. Ververis K, Hiong A, Karagiannis TC, Licciardi PV. Histone deacetylase inhibitors (HDACIs): multitargeted anticancer agents. Biologies. 2013 Feb 25;7:47-60.

13. Bolden JE, Peart MJ, Johnstone RW. Anticancer activities of histone deacetylase inhibitors. Nat Rev Drug Discov. 2006 Sep;5(9):769-84.

14. Rasheed WK, Johnstone RW, Prince HM. Histone deacetylase inhibitors in cancer therapy. Expert Opin Investig Drugs. 2007 May;16(5):659-78.

15. Ecker J, Witt O, Milde T. Targeting of histone deacetylases in brain tumors. CNS Oncol. 2013 Jul;2(4):359-76.

16. Simonini MV, Camargo LM, Dong E, Maloku E, Veldic M, Costa E, et al. The benzamide MS-275 is a potent, long-lasting brain region- selective inhibitor of histone deacetylases. Proc Natl Acad Sci USA. 2006 Jan 31 ; 103(5): 1587-92.

17. Basu T, O’Riordan KJ, Schoenike BA, Khan NN, Wallace EP, Rodriguez G, et al. Histone deacetylase inhibitors restore normal hippocampal synaptic plasticity and seizure threshold in a mouse model of Tuberous Sclerosis Complex. Sci Rep. 2019 Mar 27;9(1):5266.

18. Roy AL. Biochemistry and biology of the inducible multifunctional transcription factor TFII-I: 10 years later. Gene. 2012 Jan 15;492(1):32-41.

19. Malenfant P, Liu X, Hudson ML, Qiao Y, Hrynchak M, Riendeau N, et al. Association of GTF2i in the Williams-Beuren syndrome critical region with autism spectrum disorders. J Autism Dev Disord. 2012 Jul;42(7): 1459-69.

20. Mervis CB, Dida J, Lam E, Crawford-Zelli NA, Young EJ, Henderson DR, et al. Duplication of GTF2I results in separation anxiety in mice and humans. Am J Hum Genet. 2012 Jun 8;90(6): 1064-70.

21. Antonell A, Del Campo M, Magano LF, Kaufmann L, de la Iglesia JM, Gallastegui F, et al. Partial 7ql l.23 deletions further implicate GTF2I and GTF2IRD1 as the main genes responsible for the Williams-Beuren syndrome neurocognitive profile. J Med Genet. 2010 May;47(5):312-20.

22. Adamo A, Atashpaz S, Germain P-L, Zanella M, D’Agostino G, Albertin V, et al. 7ql 1.23 dosage-dependent dysregulation in human pluripotent stem cells affects transcriptional programs in disease-relevant lineages. Nat Genet. 2015 Feb;47(2): 132-41.

23. Meria G, Brunetti -Pierri N, Micale L, Fusco C. Copy number variants at Williams-Beuren syndrome 7ql l.23 region. Hum Genet. 2010 Jul;128(l):3-26.

24. Lalli MA, Jang J, Park J-HC, Wang Y, Guzman E, Zhou H, et al. Haploinsufficiency of BAZ1B contributes to Williams syndrome through transcriptional dysregulation of neurodevelopmental pathways. Hum Mol Genet. 2016 Apr 1;25(7): 1294-306.

25. Zanella M, Vitriolo A, Andirko A, Martins PT, Sturm S, O’Rourke T, et al. Dosage analysis of the 7ql 1.23 Williams region identifies BAZ1B as a major human gene patterning the modern human face and underlying self-domestication. Sci Adv. 2019 Dec 4;5(12):eaaw7908.

26. Schubert C. The genomic basis of the Williams-Beuren syndrome. Cell Mol Life Sci. 2009 Apr;66(7): 1178-97.

27. Sonenberg N, Hinnebusch AG. Regulation of translation initiation in eukaryotes: mechanisms and biological targets. Cell. 2009 Feb 20;136(4):731-45.

28. Capossela S, Muzio L, Bertolo A, Bianchi V, Dati G, Chaabane L, et al. Growth defects and impaired cognitive-behavioral abilities in mice with knockout for Eif4h, a gene located in the mouse homolog of the Williams-Beuren syndrome critical region. Am J Pathol. 2012 Mar;180(3):1121-35.

29. Zhang Y, Pak C, Han Y, Ahlenius H, Zhang Z, Chanda S, et al. Rapid single-step induction of functional neurons from human pluripotent stem cells. Neuron. 2013 Jun 5;78(5):785— 98.

30. Kim S-I, Oceguera- Yanez F, Sakurai C, Nakagawa M, Yamanaka S, Woltjen K. Inducible Transgene Expression in Human iPS Cells Using Versatile All-in-One piggyBac Transposons. Methods Mol Biol. 2015 May 30;

31. Lenzi J, Pagani F, De Santis R, Limatola C, Bozzoni I, Di Angelantonio S, et al. Differentiation of control and ALS mutant human iPSCs into functional skeletal muscle cells, a tool for the study of neuromuscolar diseases. Stem Cell Res. 2016 Jul; 17(1): 140-7.

32. Morris CA, Mervis CB, Hobart HH, Gregg RG, Bertrand J, Ensing GJ, et al. GTF2I hemizygosity implicated in mental retardation in Williams syndrome: genotype-phenotype analysis of five families with deletions in the Williams syndrome region. Am J Med Genet A. 2003 Nov 15;123A(l):45-59.

33. Borralleras C, Sahun I, Perez- Jurado LA, Campuzano V. Intraci sternal Gtf2i Gene Therapy Ameliorates Deficits in Cognition and Synaptic Plasticity of a Mouse Model of Williams- Beuren Syndrome. Mol Ther. 2015 Nov;23(l l): 1691-9.

34. Young EJ, Lipina T, Tam E, Mandel A, Clapcote SJ, Bechard AR, et al. Reduced fear and aggression and altered serotonin metabolism in Gtf2irdl -targeted mice. Genes Brain Behav. 2008 Mar; 7(2): 224-34.

35. Crespi BJ, Hurd PL. Cognitive-behavioral phenotypes of Williams syndrome are associated with genetic variation in the GTF2I gene, in a healthy population. BMC Neurosci. 2014 Nov 28; 15: 127.

36. Ho S-M, Hartley BJ, Tew J, Beaumont M, Stafford K, Slesinger PA, et al. Rapid Ngn2- induction of excitatory neurons from hiPSC-derived neural progenitor cells. Methods. 2016 May 15;101 : l 13-24.

37. Lane AA, Chabner BA. Histone deacetylase inhibitors in cancer therapy. J Clin Oncol. 2009 Nov 10;27(32):5459-68.

38. Slingerland M, Guchelaar H-J, Gelderblom H. Histone deacetylase inhibitors: an overview of the clinical studies in solid tumors. Anticancer Drugs. 2014 Feb;25(2): 140-9.

39. Cuadrado-Tejedor M, Perez-Gonzalez M, Garcia-Munoz C, Muruzabal D, Garcia-Barroso C, Rabal O, et al. Taking advantage of the selectivity of histone deacetylases and phosphodiesterase inhibitors to design better therapeutic strategies to treat alzheimer’s disease. Front Aging Neurosci. 2019 Jun 21;11 :149.

40. Guan J-S, Haggarty SJ, Giacometti E, Dannenberg J-H, Joseph N, Gao J, et al. HDAC2 negatively regulates memory formation and synaptic plasticity. Nature. 2009 May 7;459(7243):55-60.

41. Harrison IF, Smith AD, Dexter DT. Pathological histone acetylation in Parkinson’s disease: Neuroprotection and inhibition of microglial activation through SIRT 2 inhibition. Neurosci Lett. 2018 Feb 14;666:48-57.

42. Gundersen BB, Blendy JA. Effects of the histone deacetylase inhibitor sodium butyrate in models of depression and anxiety. Neuropharmacology. 2009 Jul;57(l):67-74.

43. Denk F, Huang W, Sidders B, Bithell A, Crow M, Grist J, et al. HD AC inhibitors attenuate the development of hypersensitivity in models of neuropathic pain. Pain. 2013 Sep;154(9): 1668-79.

44. Foley AG, Gannon S, Rombach-Mullan N, Prendergast A, Barry C, Cassidy AW, et al. Class I histone deacetylase inhibition ameliorates social cognition and cell adhesion molecule plasticity deficits in a rodent model of autism spectrum disorder. Neuropharmacology. 2012 Sep;63(4):750-60.

45. Ma K, Qin L, Matas E, Duffney LJ, Liu A, Yan Z. Histone deacetylase inhibitor MS-275 restores social and synaptic function in a Shank3 -deficient mouse model of autism. Neuropsychopharmacology . 2018;43(8): 1779-88.

46. Nageshappa S, Carromeu C, Trujillo CA, Mesci P, Espuny-Camacho I, Pasciuto E, et al. Altered neuronal network and rescue in a human MECP2 duplication model. Mol Psychiatry. 2016 Feb;21(2): 178-88.

47. Palmieri D, Lockman PR, Thomas FC, Hua E, Herring J, Hargrave E, et al. Vorinostat inhibits brain metastatic colonization in a model of triple-negative breast cancer and induces DNA double-strand breaks. Clin Cancer Res. 2009 Oct 1;15(19):6148— 57.

48. Coni S, Mancuso AB, Di Magno L, Sdruscia G, Manni S, Serrao SM, et al. Selective targeting of HD AC 1/2 elicits anticancer effects through Glil acetylation in preclinical models of SHH Medulloblastoma. Sci Rep. 2017 Mar 9;7:44079.

49. Rai M, Soragni E, Chou CJ, Barnes G, Jones S, Rusche JR, et al. Two new pimelic diphenylamide HDAC inhibitors induce sustained frataxin upregulation in cells from Friedreich’s ataxia patients and in a mouse model. PLoS One. 2010 Jan 21 ;5(l):e8825.

50. Marks PA, Breslow R. Dimethyl sulfoxide to vorinostat: development of this histone deacetylase inhibitor as an anticancer drug. Nat Biotechnol. 2007 Jan;25(l):84-90.

51. Wahaib K, Beggs AE, Campbell H, Kodali L, Ford PD. Panobinostat: A histone deacetylase inhibitor for the treatment of relapsed or refractory multiple myeloma. Am J Health Syst Pharm. 2016 Apr l;73(7):441-50.

52. Lee H-Z, Kwitkowski VE, Del Valle PL, Ricci MS, Saber H, Habtemariam BA, et al. FDA Approval: Belinostat for the Treatment of Patients with Relapsed or Refractory Peripheral T-cell Lymphoma. Clin Cancer Res. 2015 Mar 23;21(12):2666-70.

53. Jain S, Zain J. Romidepsin in the treatment of cutaneous T-cell lymphoma. J Blood Med. 2011 Apr 4;2:37-47.

54. Liston DR, Davis M. Clinically relevant concentrations of anticancer drugs: A guide for nonclinical studies. Clin Cancer Res. 2017 Jul 15;23(14):3489— 98.

55. Thomas EA. Involvement of HDAC1 and HDAC3 in the pathology of polyglutamine disorders: therapeutic implications for selective HDAC1/HDAC3 inhibitors. Pharmaceuticals (Basel). 2014 May 26;7(6):634-61.

56. Simoes-Pires C, Zwick V, Nurisso A, Schenker E, Carrupt P-A, Cuendet M. HDAC6 as a target for neurodegenerative diseases: what makes it different from the other HDACs? Mol Neurodegener. 2013 Jan 29;8:7.

57. Thomas EA, Coppola G, Desplats PA, Tang B, Soragni E, Burnett R, et al. The HDAC inhibitor 4b ameliorates the disease phenotype and transcriptional abnormalities in Huntington’s disease transgenic mice. Proc Natl Acad Sci USA. 2008 Oct 7;105(40): 15564-9.

58. Steffan JS, Bodai L, Pallos J, Poelman M, McCampbell A, Apostol BL, et al. Histone deacetylase inhibitors arrest polyglutamine-dependent neurodegeneration in Drosophila. Nature. 2001 Oct 18;413(6857):739-43.

59. Peart MJ, Smyth GK, van Laar RK, Bowtell DD, Richon VM, Marks PA, et al. Identification and functional significance of genes regulated by structurally different histone deacetylase inhibitors. Proc Natl Acad Sci USA. 2005 Mar 8;102(10):3697-702.

60. Chueh AC, Tse JWT, Tbgel L, Mariadason JM. Mechanisms of Histone Deacetylase Inhibitor-Regulated Gene Expression in Cancer Cells. Antioxid Redox Signal. 2015 Jul l;23(l):66-84.

61. Lopez-Tobon A, Villa CE, Cheroni C, Trattaro S, Caporale N, Conforti P, et al. Human cortical organoids expose a differential function of GSK3 on cortical neurogenesis. Stem Cell Rep. 2019 Oct 1;

62. Connolly RM, Rudek MA, Piekarz R. Entinostat: a promising treatment option for patients with advanced breast cancer. Future Oncol. 2017 Jun; 13(13): 1137-48.

63. Venugopal B, Baird R, Kristeleit RS, Plummer R, Cowan R, Stewart A, et al. A phase I study of quisinostat (JNJ-26481585), an oral hydroxamate histone deacetylase inhibitor with evidence of target modulation and antitumor activity, in patients with advanced solid tumors. Clin Cancer Res. 2013 Aug l;19(15):4262-72.

64. Razak ARA, Hotte SJ, Siu LL, Chen EX, Hirte HW, Powers J, et al. Phase I clinical, pharmacokinetic and pharmacodynamic study of SB939, an oral histone deacetylase (HD AC) inhibitor, in patients with advanced solid tumours. Br J Cancer. 2011 Mar l;104(5):756-62.

65. Mu S, Kuroda Y, Shibayama H, Hino M, Tajima T, Corrado C, et al. Panobinostat PK/PD profile in combination with bortezomib and dexamethasone in patients with relapsed and relapsed/refractory multiple myeloma. Eur J Clin Pharmacol. 2016 Feb;72(2): 153-61.

66. Steele NL, Plumb JA, Vidal L, Tjornelund J, Knoblauch P, Buhl-Jensen P, et al. Pharmacokinetic and pharmacodynamic properties of an oral formulation of the histone deacetylase inhibitor Belinostat (PXD101). Cancer Chemother Pharmacol. 2011 Jun;67(6): 1273-9.

67. Furlan A, Monzani V, Reznikov LL, Leoni F, Fossati G, Modena D, et al. Pharmacokinetics, safety and inducible cytokine responses during a phase 1 trial of the oral histone deacetylase inhibitor ITF2357 (givinostat). Mol Med. 2011 Jun;17(5-6):353-62.

68. Moj D, Britz H, Burhenne J, Stewart CF, Egerer G, Haefeli WE, et al. A physiologically based pharmacokinetic and pharmacodynamic (PBPK/PD) model of the histone deacetylase (HD AC) inhibitor vorinostat for pediatric and adult patients and its application for dose specification. Cancer Chemother Pharmacol. 2017 Oct 7;

69. Ribrag V, Kim WS, Bouabdallah R, Lim ST, Coiffier B, Illes A, et al. Safety and efficacy of abexinostat, a pan-histone deacetylase inhibitor, in non-Hodgkin lymphoma and chronic lymphocytic leukemia: Results of a phase 2 study. Haematologica. 2017 Jan 25;

70. Shimizu T, LoRusso PM, Papadopoulos K, Patnaik A, Beeram M, Smith LS, et al. Phase I first-in-human study of CUDC-101, a multi -targeted inhibitor of HDACs, EGFR and HER2 in patients with advanced solid tumors. Clin Cancer Res. 2014 Aug 8;20(19):5032-40.

71. de Bono JS, Kristeleit R, Tolcher A, Fong P, Pacey S, Karavasilis V, et al. Phase I pharmacokinetic and pharmacodynamic study of LAQ824, a hydroxamate histone deacetylase inhibitor with a heat shock protein-90 inhibitory profile, in patients with advanced solid tumors. Clin Cancer Res. 2008 Oct 15;14(20):6663-73.

72. Boumber Y, Younes A, Garcia-Manero G. Mocetinostat (MGCD0103): a review of an isotype-specific histone deacetylase inhibitor. Expert Opin Investig Drugs. 2011 Jun;20(6):823-9.

73. Oki Y, Kelly KR, Flinn I, Patel MR, Gharavi R, Ma A, et al. CUDC-907 in relapsed/refractory diffuse large B-cell lymphoma, including patients with MYC- alterations: results from an expanded phase I trial. Haematologica. 2017 Aug 31;102(l l): 1923-30.

74. Ikeda M, Ohno I, Ueno H, Mitsunaga S, Hashimoto Y, Okusaka T, et al. Phase I study of resminostat, an HD AC inhibitor, combined with S-l in patients with pre-treated biliary tract or pancreatic cancer. Invest New Drugs. 2018 Jul 11 ;37(1): 1- 9.

75. Undevia SD, Kindler HL, Janisch L, Olson SC, Schilsky RL, Vogelzang NJ, et al. A phase I study of the oral combination of CI-994, a putative histone deacetylase inhibitor, and capecitabine. Ann Oncol. 2004 Nov 1;15(11): 1705— 11. 76. Soragni E, Miao W, ludicello M, Jacoby D, De Mercanti S, Clerico M, et al. Epigenetic therapy for Friedreich ataxia. Ann Neurol. 2014 Oct;76(4):489-508.

77. von Tresckow B, Sayehli C, Aulitzky WE, Goebeler M-E, Schwab M, Braz E, et al. Phase I Study of Domatinostat (4SC-202), a class I Histone Deacetylase Inhibitor in Patients with Advanced Hematological Malignancies. Eur J Haematol. 2018 Oct 22; 102(2): 163-73. 78. Cosenza M, Pozzi S. The therapeutic strategy of HDAC6 inhibitors in lymphoproliferative disease. Int J Mol Sci. 2018 Aug 9;19(8).

79. Zaslavsky K, Zhang W-B, McCready FP, Rodrigues DC, Deneault E, Loo C, et al. SHANK2 mutations associated with autism spectrum disorder cause hyperconnectivity of human neurons. Nat Neurosci. 2019 Mar 25;22(4):556-64.

Claims

1. A histone deacetylase inhibitor for use in the treatment of autism spectrum disorder and/or intellectual disability.

2. The inhibitor for use according to claim 1 wherein the autism spectrum disorder and/or intellectual disability is characterized by an increase in GTF2I levels.

3. The inhibitor for use according to any one of claim 1 or 2 wherein the autism spectrum disorder and/or intellectual disability is 7Dup.

4. The inhibitor for use according to any one of previous claims wherein said inhibitor is a pan HD AC or HDAC1 and/or HDAC2 and/ or HDAC3 and/or HDAC6 inhibitor.

5. The inhibitor for use according to any one of previous claims wherein said inhibitor decreases GTF2I levels.

6. The inhibitor for use according to any one of previous claims wherein said inhibitor is a hydroxamic acid, a benzamide or an aminobenzamide.

7. The inhibitor for use according to any one of previous claims wherein said inhibitor is selected from the group consisting of: Vorinostat, Romidepsin , Panobinostat, Belinostat, Entinostat, Domatinostat, Resminostat, Rocilinostat, Trichostatin A, Valproic acid, Dacinostat, Bisthianostat, Quisinostat hydrochloride, CUDC-101 , Scriptaid, Tefinostat, Givinostat, Mocetinostat, Chidamide, Abexinostat, Pracinostat, Butyric acid, Pivanex, 4- phenylbutyric acid (or sodium phenylbutyrate), Tucidinostat (or chidamide), Nanatinostat, Fimepinostat, Remetinostat, Ricolinostat, Tinostamustine, JNJ-26481585, RG2833, M344, APH-0812, CG-745, CKD-506, CKD-581, CXD-101, FX-322, YPL- 001, CFH-367C, Tacedinaline, Citarinostat, CKD-504, CUDC-907, CUDC-908, HG- 146, KA-2507, Lipocurc, MPT-0E028, NBM-BMX, OBP-801, OKI-179, RDN-929, VTR-297, REC-2282, CS-3003, ACY-1035, ACY-1071, ACY-1083, ACY-738, ACY- 775, ACY-957, ADV-300, AN-446, AP-001 (or Metavert), Arginine Butyrate, BMN- 290, C-1A, CG-1521, CKD-509, CKD-L, CM-414, Crocetin, CS-3158, CT-101, CX- 1026, RCY-1410, SE-7552, SKLB-23bb, CY-190602, JBI-097, JBI-128, JMF-3086, KAN-0440262, KDAC-0001, Largazole, MPT-0B291, MPT-0G211, MRx-0029, MRX- 0573, MRX-1299, NHC-51, Nexturastat A, OKI-422, QTX-125, RCY-1305, SP-1161, SP-259, SRX-3636, ST-7612AA1, TJC-0545, Trichosic, YH-508, HSB-501, NBM-1001, QTX-153, RDN-1201, ROD-119, ROD-1246, ROD-1275, ROD-1702, ROD-2003, ROD-2089, ROD-702 and RTSV-5.

32

8. The inhibitor for use according to any one of previous claims wherein said inhibitor is Vorinostat, Mocetinostat or RG2833.

9. The inhibitor for use according to any one of previous claims wherein said inhibitor is combined with a further therapeutic agent.

10. The inhibitor for use according to claim 9 wherein said further therapeutic agent is selected from the group consisting of: atypical antipsychotic drugs, psychostimulants, antidepressant agent, anti-epileptic agent, clonidine, rivastigmine, memantine, guanfacine, buspirone, atomoxetine, an epigenetic compound, a LSDl inhibitor, a DNMT inhibitor, a histone methyltransferase (HMT) inhibitor, a EZH1/2 inhibitor, a PRMT inhibitor, a BET inhibitor, a DOT1L inhibitor, APTA-16, a Histone Lysine N Methyltransferase (EHMT2 or G9a) inhibitor, a dual inhibitor against G9a and DNMTs, a menin-MLLl (or KMT2A) interaction inhibitor and a SETD2 inhibitor.

11. A pharmaceutical composition comprising a histone deacetylase inhibitor as defined in any one of claims 4 to 8 and at least one pharmaceutically acceptable carrier for use in the treatment of autism spectrum disorder and/or intellectual disability.

12. The pharmaceutical composition according to claim 11 comprising a further therapeutic agent selected from the group consisting of: atypical antipsychotic drugs, psychostimulants, antidepressant agent, anti-epileptic agent, clonidine, rivastigmine, memantine, guanfacine, buspirone, atomoxetine, an epigenetic compound, a LSD1 inhibitor, a DNMT inhibitor, a histone methyltransferase (HMT) inhibitor, a EZH1/2 inhibitor, a PRMT inhibitor, a BET inhibitor, a DOT IL inhibitor, APTA-16, a Histone Lysine N Methyltransferase (EHMT2 or G9a) inhibitor, a dual inhibitor against G9a and DNMTs, a menin-MLLl (or KMT2A) interaction inhibitor and a SETD2 inhibitor.

13. A method to identify a subject to be treated with a histone deacetylase inhibitor for the treatment of autism spectrum disorder and/or intellectual disability comprising measuring the level of GTF2I in a biological sample of said subject and comparing said measured level to a control level.

14. A method to monitor the efficacy of a histone deacetylase inhibitor for the treatment of autism spectrum disorder and/or intellectual disability in a subject comprising measuring the level of GTF2I in a biological sample of said subject and comparing said measured level to a control level.

33