Fragile X syndrome (FXS) is a neurodevelopmental disorder associated with auditory hypersensitivity, circuit hyperexcitability, and seizures. Whether re-expression of the FMR1 gene and encoded Fragile X Messenger Ribonucleoprotein (FMRP) can restore sensory circuit dysfunction remains unclear. Here, we show that a viral AAV-FMR1 vector rescues audiogenic seizures in the Fmr1 -/y mouse model after both neonatal and adult delivery, indicating that auditory circuit dysfunction remains reversible. Local re-expression in the inferior colliculus (IC) is sufficient to reduce seizure susceptibility, identifying this region as a key site of FMRP-dependent circuit regulation. In the IC, Translating Ribosome Affinity Purification and RNA-seq (TRAP-seq) profiling reveals impaired induction of sound-evoked translation programs in Fmr1 -/y neurons, including those regulated by transcription factor Npas4. AAV-FMR1 restores a WT-like molecular response and normalizes unbalanced sound-evoked activation of VGLUT2+ excitatory neurons over VGAT+ inhibitory neurons in Fmr1 -/y IC. Together, these findings indicate altered translation of Npas4 in response to sound impairs recruitment of inhibition in the Fmr1 -/y IC, and this can be reversed with AAV-FMR1 administration. Moreover, the rescue of seizures after adult administration of AAV-FMR1 supports a gene therapy approach for FXS.

Background/Objectives: PTEN Hamartoma Tumour Syndrome (PHTS) is a rare inherited disorder caused by germline PTEN mutations, presenting with cancer predisposition and neurodevelopmental abnormalities, including macrocephaly. PTEN functions as a tumour suppressor by regulating the PI3K/AKT/mTOR pathway and contributes to cellular adhesion, migration, and genomic stability. As no curative therapy exists, gene therapy represents a promising avenue to correct the underlying genetic defect. Methods: In this study we evaluated the therapeutic potential of AAV9-mediated PTEN gene delivery in a PtenΔ5/+ mouse model, which is predominantly characterized by progressive lymphoid hyperplasia leading to lymph node tumour development, as well as hepatic focal lesions and macrocephaly. Results: Systemic vector delivery did not improve lymph node enlargement which represents the predominant disease phenotype in the PtenΔ5/+ mice at the doses tested, due to restricted vector biodistribution, but it did effectively rescue the hepatic lesions, including steatosis and steatohepatitis-like changes. Mechanistically, AAV-mediated PTEN expression significantly reduced elevated p-AKT levels, indicating attenuated hyperactivation of the PI3K/AKT/mTOR pathway. Additionally, intracranial delivery of the same vector in PtenΔ5/+ mouse neonates ameliorated macrocephaly without apparent adverse effects. Conclusions: These findings highlight the therapeutic potential of AAV9-mediated PTEN gene therapy in PHTS, particularly for hepatic and neuroanatomical manifestations. Optimization of vector biodistribution and transduction efficiency will be critical for clinical translation.

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by CGG trinucleotide repeat expansion in the fragile X messenger ribonucleoprotein 1 (FMR1) gene and the resulting loss of fragile X messenger ribonucleoprotein (FMRP). Gene therapy using recombinant adeno-associated virus (AAV) to restore FMRP expression, particularly in the brain, is a promising therapeutic strategy targeting the underlying cause of FXS. We examined the impact of AAV serotype 9 (AAV9)-mediated expression of a brain-abundant human FMRP isoform (isoform 7) driven by a fragment of the human FMR1 promoter on circuit and behavioral dysfunctions in the male Fmr1 knockout (KO) mouse, FXS model. Following intracerebroventricular (i.c.v.) injection of AAV9-NG276 into neonatal KO mice at a low (1e11 vg/animal) or high (3e11 vg/animal) dose, we assessed cortical phenotypes using electroencephalography (EEG) recordings and behavioral testing. High-dose AAV9-NG276 normalized baseline gamma power, improved sound-evoked responses, and reduced background neural activity. Analysis of behavioral deficits in adult KO mice showed that high-dose neonatal AAV9-NG276 delivery normalized exploratory behaviors, social preference, and probabilistic reversal learning. Thus, early AAV-mediated delivery of human FMR1 isoform 7 ameliorates cortical dysfunction and behavioral deficits in a murine FXS model and suggests that widespread cortical biodistribution is required for therapeutic benefit.

Mechanistic understanding of how gene activity is regulated has focussed on the roles of transcription factors at promoters and enhancers, whereas mechanisms capable of globally fine-tuning gene expression through dispersed binding across large genomic regions have received less attention. Here we provide evidence that the essential stem cell transcription factor SALL4 modulates gene expression according to DNA base composition by reading the frequency of its AT-rich target motifs. Using an acute depletion strategy, we establish that SALL4-repressed genes localise to AT-rich genomic domains with high levels of dispersed SALL4 occupancy. While SALL4 is localised within peaks and distributed broadly across the genome, explainable machine learning revealed that its occupancy across the gene body is a strong predictor of transcriptional output. We observed rapid increases in chromatin accessibility and histone acetylation independent of transcriptional activity, suggesting that SALL4 primarily acts upon chromatin, while transcriptional changes are secondary. Accordingly, preventing SALL4 from recruiting the histone deacetylase and nucleosome remodelling corepressor NuRD mimicked a Sall4 -null phenotype in stem cells and animal models. Our findings reveal that SALL4’s interpretation of DNA sequence optimises the global epigenome and transcriptome, a process integral to maintaining the stem cell gene expression programme. ### Competing Interest Statement The authors have declared no competing interest. European Research Council, https://ror.org/0472cxd90, 694295 Wellcome Trust, https://ror.org/029chgv08, 107930, 203149, 226791

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Like other genetic neurodevelopmental disorders, it lacks molecular biomarkers to evaluate disease and therapeutic outcomes. We present a strategy to define biomarkers of MeCP2 dysfunction in brain with potential to delineate mechanisms and monitor therapeutic interventions. This strategy relies on a library of proteins responsive to Mecp2 gene dosage and correlated with molecular and clinical outcomes after AAV9-mediated MECP2 gene therapy in Mecp2 -KO mice. Gene rescue restored MeCP2 in brain, improved clinical phenotypes, and reverted transcriptome and proteome abnormalities. We identified 327 shared proteins among 1852 cortical and hippocampal proteins responsive to Mecp2 / MECP2 . Of these, 119 also displayed Mecp2/MECP2-dependent transcript changes. Both the Mecp2-responsive proteome and transcript–protein pairs were enriched in synaptic and metabolic pathways, including central carbon and NAD+ metabolism. We used this therapy-responsive protein library to guide selection of candidate cerebrospinal fluid (CSF) biomarkers in RTT. CSF composition from neurotypical and RTT groups was analyzed using ultrasensitive nucleic acid-based multiplexed ELISA. Twenty-eight proteins were altered in RTT, nine overlapping with Mecp2 dosage- and therapy-sensitive proteins. Multivariate regression linked several candidates to Mecp2 / MeCP2 abundance and phenotypic improvement in mice. This paradigm provides a rigorous molecular systems-level framework integrating genetics, preclinical gene therapy, and clinical metrics to define robust cross-species biomarkers and mechanisms in RTT, with potential applicability to other neurodevelopmental disorders. One Sentence Summary Genetic Identification of cross-species biomarkers and mechanisms in Rett Syndrome ### Competing Interest Statement W.K. was the Chief Scientific Officer of Anavex Life Sciences Corp. He received funding from the International Rett Syndrome Foundation, the National Institutes of Health and the Centers for Disease Control and Prevention, and he has been a consultant for Anavex, AveXis, Acadia, Compass, EryDel/Quince, Neuren Pharmaceuticals, Newron, GW Pharmaceuticals, Marinus, Biohaven, Zynerba, Ovid Therapeutics, Stalicla and Tetra. He has conducted clinical trials with Neuren and Ipsen. S.C. is currently the Chief Scientific Officer at Neurogene Inc. He received research funding from the Rett Syndrome Research Trust, Simons Initiative for the Developing Brain, Neurogene and Rettco Inc. He has received patent royalties relating to gene therapy products being developed for Rett syndrome. Rett Syndrome Research Trust, https://ror.org/03s455144

Conventional methods of gene transfer lead to inconsistent transgene expression within cells. This variability can be problematic, particularly in conditions like Rett syndrome (RTT), a neurological disorder caused by mutations in the MECP2 (methyl-CpG binding protein 2) gene, because overexpression of MECP2 can also cause adverse effects. To address these challenges, we devised a gene regulation system called Expression Attenuation via Construct Tuning (EXACT), which uses a self-contained, microRNA-based feed-forward loop that not only ensures more consistent transgene expression but also protects against excessive expression. Through cell-based screening assays, we demonstrated the ability of the EXACT circuit to modulate the expression of full-length human MeCP2. Compared with a conventional construct, an EXACT-MECP2 construct exhibited a narrower range of cellular protein abundance. Furthermore, the degree of regulation by the EXACT circuit increased with higher transgene doses in vitro and in wild-type mice and mice modeling RTT. On the basis of cellular and in vivo testing, we identified an optimal configuration for the adeno-associated virus serotype 9 (AAV9) construct for self-regulated MECP2 gene therapy, designated NGN-401. Delivery of NGN-401 to neonatal male Mecp2-/y hemizygous mice via intracerebroventricular injection resulted in prolonged survival and amelioration of RTT-like phenotypes compared with vehicle-treated animals. NGN-401 was also well tolerated by female Mecp2+/- mice and healthy juvenile nonhuman primates, in contrast with a conventional construct, which caused toxicity. The results from these studies underpin a first-in-human pediatric trial of NGN-401 in RTT (ClinicalTrials.gov, NCT05898620).

Cre/Lox technology is a powerful tool in the mouse genetics tool-box as it enables tissue-specific and inducible mutagenesis of specific gene loci. Correct interpretation of phenotypes depends upon knowledge of the Cre expression pattern in the chosen mouse driver line to ensure that appropriate cell types are targeted. For studies of the brain and neurological disease a pan-neuronal promoter that reliably drives efficient neuron-specific transgene expression would be valuable. Here we compare a widely used “pan-neuronal” mouse Cre driver line, Syn1-cre, with a little-known alternative, Snap25-IRES2-cre. Our results show that the Syn1-cre line broadly expresses in the brain but is indetectable in more than half of all neurons and weakly active in testes. In contrast the Snap25-IRES2-cre line expressed Cre in a high proportion of neurons (~85%) and was indetectable in all non-brain tissues that were analysed, including testes. Our findings suggest that for many purposes Snap25-IRES2-cre is superior to Syn1-cre as a potential pan-neuronal cre driver.

DNA methylation is implicated in neuronal biology via the protein MeCP2, the mutation of which causes Rett syndrome. MeCP2 recruits the NCOR1/2 co-repressor complexes to methylated cytosine in the CG dinucleotide, but also to sites of non-CG methylation, which are abundant in neurons. To test the biological significance of the dual-binding specificity of MeCP2, we replaced its DNA binding domain with an orthologous domain from MBD2, which can only bind mCG motifs. Knockin mice expressing the domain-swap protein displayed severe Rett-syndrome-like phenotypes, indicating that normal brain function requires the interaction of MeCP2 with sites of non-CG methylation, specifically mCAC. The results support the notion that the delayed onset of Rett syndrome is due to the simultaneous post-natal accumulation of mCAC and its reader MeCP2. Intriguingly, genes dysregulated in both Mecp2 null and domain-swap mice are implicated in other neurological disorders, potentially highlighting targets of relevance to the Rett syndrome phenotype.

Mammalian genomes contain long domains with distinct average compositions of A/T versus G/C base pairs. In a screen for proteins that might interpret base composition by binding to AT-rich motifs, we identified the stem cell factor SALL4, which contains multiple zinc fingers. Mutation of the domain responsible for AT binding drastically reduced SALL4 genome occupancy and prematurely upregulated genes in proportion to their AT content. Inactivation of this single AT-binding zinc-finger cluster mimicked defects seen in Sall4 null cells, including precocious differentiation of embryonic stem cells (ESCs) and embryonic lethality in mice. In contrast, deletion of two other zinc-finger clusters was phenotypically neutral. Our data indicate that loss of pluripotency is triggered by downregulation of SALL4, leading to de-repression of a set of AT-rich genes that promotes neuronal differentiation. We conclude that base composition is not merely a passive byproduct of genome evolution and constitutes a signal that aids control of cell fate.

Most human genes are associated with promoters embedded in non-methylated, G + C-rich CpG islands (CGIs). Not all CGIs are found at annotated promoters, however, raising the possibility that many serve as promoters for transcripts that do not code for proteins. To test this hypothesis, we searched for novel transcripts in embryonic stem cells (ESCs) that originate within orphan CGIs. Among several candidates, we detected a transcript that included three members of the let-7 micro-RNA family: Let-7a-1, let-7f-1, and let-7d. Deletion of the CGI prevented expression of the precursor RNA and depleted the included miRNAs. Mice homozygous for this mutation were sub-viable and showed growth and other defects. The results suggest that despite the identity of their seed sequences, members of the let-7 miRNA family exert distinct functions that cannot be complemented by other members.

Most missense mutations causing Rett syndrome (RTT) affect domains of MeCP2 that have been shown to either bind methylated DNA or interact with a transcriptional co-repressor complex. Several mutations, however, including the C-terminal truncations that account for ∼10% of cases, fall outside these characterized domains. We studied the molecular consequences of four of these 'non-canonical' mutations in cultured neurons and mice to see if they reveal additional essential domains without affecting known properties of MeCP2. The results show that the mutations partially or strongly deplete the protein and also in some cases interfere with co-repressor recruitment. These mutations therefore impact the activity of known functional domains and do not invoke new molecular causes of RTT. The finding that a stable C-terminal truncation does not compromise MeCP2 function raises the possibility that small molecules which stabilize these mutant proteins may be of therapeutic value.

MeCP2 is a nuclear protein that is mutated in the severe neurological disorder Rett syndrome (RTT). The ability to target β-galactosidase to the nucleus was previously used to identify a conserved nuclear localization signal (NLS) in MeCP2 that interacts with the nuclear import factors KPNA3 and KPNA4. Here, we report that nuclear localization of MeCP2 does not depend on its NLS. Instead, our data reveal that an intact methyl-CpG binding domain (MBD) is sufficient for nuclear localization, suggesting that MeCP2 can be retained in the nucleus by its affinity for DNA. Consistent with these findings, we demonstrate that disease progression in a mouse model of RTT is unaffected by an inactivating mutation in the NLS of MeCP2. Taken together, our work reveals an unexpected redundancy between functional domains of MeCP2 in targeting this protein to the nucleus, potentially explaining why NLS-inactivating mutations are rarely associated with disease.

Duplication of the X-linked MECP2 gene causes a severe neurological syndrome whose molecular basis is poorly understood. To determine the contribution of known functional domains to overexpression toxicity, we engineered a mouse model that expresses wild-type or mutated MeCP2 from the Mapt (Tau) locus in addition to the endogenous protein. Animals that expressed approximately four times the wild-type level of MeCP2 failed to survive to weaning. Strikingly, a single amino acid substitution that prevents MeCP2 from binding to the TBL1X(R1) subunit of nuclear receptor corepressor 1/2 (NCoR1/2) complexes, when expressed at equivalent high levels, was phenotypically indistinguishable from wild type, suggesting that excessive corepressor recruitment underlies toxicity. In contrast, mutations affecting the DNA-binding domain were toxic when overexpressed. As the NCoR1/2 corepressors are thought to act through histone deacetylation by histone deacetylase 3 (HDAC3), we asked whether mutations in NCoR1 and NCoR2 that drastically reduced their ability to activate this enzyme would relieve the MeCP2 overexpression phenotype. Surprisingly, severity was unaffected, indicating that the catalytic activity of HDAC3 is not the mediator of toxicity. Our findings shed light on the molecular mechanisms underlying MECP2 duplication syndrome and call for a re-evaluation of the precise biological role played by corepressor recruitment.