Ketogenic diet modifies ribosomal protein dysregulation in KMT2D Kabuki syndrome.

BACKGROUND: Kabuki syndrome (KS) is a genetic disorder caused by DNA mutations in KMT2D, a lysine methyltransferase that methylates histones and other proteins, and therefore modifies chromatin structure and subsequent gene expression. Ketones, derived from the ketogenic diet, are histone deacetylase inhibitors that can 'open' chromatin and encourage gene expression. Preclinical studies have shown that the ketogenic diet rescues hippocampal memory neurogenesis in mice with KS via the epigenetic effects of ketones. METHODS: Single-cell RNA sequencing and mass spectrometry-based proteomics were used to explore molecular mechanisms of disease in individuals with KS (n = 4) versus controls (n = 4). FINDINGS: Pathway enrichment analysis indicated that loss of function mutations in KMT2D are associated with ribosomal protein dysregulation at an RNA and protein level in individuals with KS (FDR <0.05). Cellular proteomics also identified immune dysregulation and increased abundance of other lysine modification and histone binding proteins, representing a potential compensatory mechanism. A 12-year-old boy with KS, suffering from recurrent episodes of cognitive decline, exhibited improved cognitive function and neuropsychological assessment performance after 12 months on the ketogenic diet, with concomitant improvement in transcriptomic ribosomal protein dysregulation. INTERPRETATION: Our data reveals that lysine methyltransferase deficiency is associated with ribosomal protein dysfunction, with secondary immune dysregulation. Diet and the production of bioactive molecules such as ketone bodies serve as a significant environmental factor that can induce epigenetic changes and improve clinical outcomes. Integrating transcriptomic, proteomic, and clinical data can define mechanisms of disease and treatment effects in individuals with neurodevelopmental disorders. FUNDING: This study was supported by the Dale NHMRC Investigator Grant (APP1193648) (R.D), Petre Foundation (R.D), and The Sydney Children's Hospital Foundation/Kids Research Early and Mid-Career Researcher Grant (E.T).

Blood-Based Transcriptomic Biomarkers Are Predictive of Neurodegeneration Rather Than Alzheimer's Disease.

Alzheimer's disease (AD) is a growing global health crisis affecting millions and incurring substantial economic costs. However, clinical diagnosis remains challenging, with misdiagnoses and underdiagnoses being prevalent. There is an increased focus on putative, blood-based biomarkers that may be useful for the diagnosis as well as early detection of AD. In the present study, we used an unbiased combination of machine learning and functional network analyses to identify blood gene biomarker candidates in AD. Using supervised machine learning, we also determined whether these candidates were indeed unique to AD or whether they were indicative of other neurodegenerative diseases, such as Parkinson's disease (PD) and amyotrophic lateral sclerosis (ALS). Our analyses showed that genes involved in spliceosome assembly, RNA binding, transcription, protein synthesis, mitoribosomes, and NADH dehydrogenase were the best-performing genes for identifying AD patients relative to cognitively healthy controls. This transcriptomic signature, however, was not unique to AD, and subsequent machine learning showed that this signature could also predict PD and ALS relative to controls without neurodegenerative disease. Combined, our results suggest that mRNA from whole blood can indeed be used to screen for patients with neurodegeneration but may be less effective in diagnosing the specific neurodegenerative disease.

The neuroprotective effects of estrogen and estrogenic compounds in spinal cord injury.

Spinal cord injury (SCI) occurs when the spinal cord is damaged from either a traumatic event or disease. SCI is characterised by multiple injury phases that affect the transmission of sensory and motor signals and lead to temporary or long-term functional deficits. There are few treatments for SCI. Estrogens and estrogenic compounds, however, may effectively mitigate the effects of SCI and therefore represent viable treatment options. This review systematically examines the pre-clinical literature on estrogen and estrogenic compound neuroprotection after SCI. Several estrogens were examined by the included studies: estrogen, estradiol benzoate, Premarin, isopsoralen, genistein, and selective estrogen receptor modulators. Across these pharmacotherapies, we find significant evidence that estrogens indeed offer protection against myriad pathophysiological effects of SCI and lead to improvements in functional outcomes, including locomotion. A STRING functional network analysis of proteins modulated by estrogen after SCI demonstrated that estrogen simultaneously upregulates known neuroprotective pathways, such as HIF-1, and downregulates pro-inflammatory pathways, including IL-17. These findings highlight the strong therapeutic potential of estrogen and estrogenic compounds after SCI.

Artificial intelligence-driven meta-analysis of brain gene expression identifies novel gene candidates and a role for mitochondria in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of dementia. There is no treatment and AD models have focused on a small subset of genes identified in familial AD. Microarray studies have identified thousands of dysregulated genes in the brains of patients with AD yet identifying the best gene candidates to both model and treat AD remains a challenge. We performed a meta-analysis of microarray data from the frontal cortex (n = 697) and cerebellum (n = 230) of AD patients and healthy controls. A two-stage artificial intelligence approach, with both unsupervised and supervised machine learning, combined with a functional network analysis was used to identify functionally connected and biologically relevant novel gene candidates in AD. We found that in the frontal cortex, genes involved in mitochondrial energy, ATP, and oxidative phosphorylation, were the most significant dysregulated genes. In the cerebellum, dysregulated genes were involved in mitochondrial cellular biosynthesis (mitochondrial ribosomes). Although there was little overlap between dysregulated genes between the frontal cortex and cerebellum, machine learning models comprised of this overlap. A further functional network analysis of these genes identified that two downregulated genes, ATP5L and ATP5H, which both encode subunits of ATP synthase (mitochondrial complex V) may play a role in AD. Combined, our results suggest that mitochondrial dysfunction, particularly a deficit in energy homeostasis, may play an important role in AD.

Meta-Analysis Identifies BDNF and Novel Common Genes Differently Altered in Cross-Species Models of Rett Syndrome.

Rett syndrome (RTT) is a rare disorder and one of the most abundant causes of intellectual disabilities in females. Single mutations in the gene coding for methyl-CpG-binding protein 2 (MeCP2) are responsible for the disorder. MeCP2 regulates gene expression as a transcriptional regulator as well as through epigenetic imprinting and chromatin condensation. Consequently, numerous biological pathways on multiple levels are influenced. However, the exact molecular pathways from genotype to phenotype are currently not fully elucidated. Treatment of RTT is purely symptomatic as no curative options for RTT have yet to reach the clinic. The paucity of this is mainly due to an incomplete understanding of the underlying pathophysiology of the disorder with no clinically useful common disease drivers, biomarkers, or therapeutic targets being identified. With the premise of identifying universal and robust disease drivers and therapeutic targets, here, we interrogated a range of RTT transcriptomic studies spanning different species, models, and MECP2 mutations. A meta-analysis using RNA sequencing data from brains of RTT mouse models, human post-mortem brain tissue, and patient-derived induced pluripotent stem cell (iPSC) neurons was performed using weighted gene correlation network analysis (WGCNA). This study identified a module of genes common to all datasets with the following ten hub genes driving the expression: ATRX, ADCY7, ADCY9, SOD1, CACNA1A, PLCG1, CCT5, RPS9, BDNF, and MECP2. Here, we discuss the potential benefits of these genes as therapeutic targets.

Gene Editing and Rett Syndrome: Does It Make the Cut?

Rett syndrome (RTT) is a rare neurogenetic disorder caused by pathogenic variants of the Methyl CpG binding protein 2 (MECP2) gene. The RTT is characterized by apparent normal early development followed by regression of communicative and fine motor skills. Comorbidities include epilepsy, severe cognitive impairment, and autonomic and motor dysfunction. Despite almost 60 clinical trials and the promise of a gene therapy, no cure has yet emerged with treatment remaining symptomatic. Advances in understanding RTT has provided insight into the complexity and exquisite control of MECP2 expression, where loss of expression leads to RTT and overexpression leads to MECP2 duplication syndrome. Therapy development requires regulated expression that matches the spatiotemporal endogenous expression of MECP2 in the brain. Gene editing has revolutionized gene therapy and promises an exciting strategy for many incurable monogenic disorders, including RTT, by editing the native locus and retaining endogenous gene expression. Here, we review the literature on the currently available editing technologies and discuss their limitations and applicability to the treatment of RTT.

Improving clinical trial readiness to accelerate development of new therapeutics for Rett syndrome.

Rett syndrome is associated with severe functional impairments and many comorbidities, each in urgent need of treatments. Mutations in the MECP2 gene were identified as causing Rett syndrome in 1999. Over the past 20 years there has been an abundance of preclinical research with some studies leading to human clinical trials. Despite this, few viable therapeutic options have emerged from this investment of effort. Reasons for this lack of success as they relate both to preclinical research and the clinical trial landscape are discussed. Considering what needs to be done to promote further success in the field, we take a positive and constructive approach and introduce the concept of clinical trial readiness and its necessary ingredients for Rett syndrome. These include: listening to the needs of families; support from advocacy groups; optimising use of existing clinic infrastructures and available natural history data; and, finally, the validation of existing outcome measures and/or the development and validation of new measures. We conclude by reiterating the need for a collaborative and coordinated approach amongst the many different stakeholder groups and the need to engage in new types of trial design which could be much more efficient, less costly and much less burdensome on families.

Emerging evidence of Toll-like receptors as a putative pathway linking maternal inflammation and neurodevelopmental disorders in human offspring: A systematic review.

Inflammation is increasingly recognised to play a major role in gene-environment interactions in neurodevelopmental disorders (NDDs). The effects of aberrant immune responses to environmental stimuli in the mother and in the child can affect neuroimmune signalling that is central to brain development. Toll-like receptors (TLR) are the best known innate immune pattern and danger recognition sensors to various environmental threats. In animal models, maternal immune activation (MIA), secondary to inflammatory factors including maternal gestational infection, obesity, diabetes, and stress activate the TLR pathway in maternal blood, placenta, and fetal brain, which correlate with offspring neurobehavioral abnormalities. Given the central role of TLR activation in animal MIA models, we systematically reviewed the human evidence for TLR activation and response to stimulation across the maternal-fetal interface. Firstly, we included 59 TLR studies performed in peripheral blood of adults in general population (outside of pregnancy) with six chronic inflammatory factors which have epidemiological evidence for increased risk of offspring NDDs, namely, obesity, diabetes mellitus, depression, low socio-economic status, autoimmune diseases, and asthma. Secondly, eight TLR studies done in human pregnancies with chronic inflammatory factors, involving maternal blood, placenta, and cord blood, were reviewed. Lastly, ten TLR studies performed in peripheral blood of individuals with NDDs were included. Despite these studies, there were no studies which examined TLR function in both the pregnant mother and their offspring. Increased TLR2 and TLR4 mRNA and/or protein levels in peripheral blood were common in obesity, diabetes mellitus, depression, autoimmune thyroid disease, and rheumatoid arthritis. To a lesser degree, TLR 3, 7, 8, and 9 activation were found in peripheral blood of humans with autoimmune diseases and depression. In pregnancy, increased TLR4 mRNA levels were found in the peripheral blood of women with diabetes mellitus and systemic lupus erythematosus. Placental TLR activation was found in mothers with obesity or diabetes. Postnatally, dysregulated TLR response to stimulation was found in peripheral blood of individuals with NDDs. This systematic review found emerging evidence that TLR activation may represent a mechanistic link between maternal inflammation and offspring NDD, however the literature is incomplete and longitudinal outcome studies are lacking. Identification of pathogenic mechanisms in MIA could create preventive and therapeutic opportunities to mitigate NDD prevalence and severity.

Common targetable inflammatory pathways in brain transcriptome of autism spectrum disorders and Tourette syndrome.

Neurodevelopmental disorders (NDDs), including autism-spectrum disorders (ASD) and Tourette syndrome (TS) are common brain conditions which often co-exist, and have no approved treatments targeting disease mechanisms. Accumulating literature implicates the immune system in NDDs, and transcriptomics of post-mortem brain tissue has revealed an inflammatory signal. We interrogated two RNA-sequencing datasets of ASD and TS and identified differentially expressed genes, to explore commonly enriched pathways through GO, KEGG, and Reactome. The DEGs [False Discovery Rate (FDR) <0.05] in the ASD dataset (n = 248) and the TS dataset (n = 156) enriched pathways involving inflammation, cytokines, signal transduction and cell signalling. Of the DEGs from the ASD and TS analyses, 23 were shared, all of which were up-regulated: interaction networks of the common protein-coding genes using STRING revealed 5 central up-regulated hub genes: CCL2, ICAM1, HMOX1, MYC, and SOCS3. Applying KEGG and Reactome analysis to the 23 common genes identified pathways involving the innate immune response such as interleukin and interferon signalling pathways. These findings bring new evidence of shared immune signalling in ASD and TS brain transcriptome, to support the overlapping symptoms that individuals with these complex disorders experience.

Breaking Boundaries in the Brain-Advances in Editing Tools for Neurogenetic Disorders.

Monogenic neurological disorders are devastating, affecting hundreds of millions of people globally and present a substantial burden to individuals, carers, and healthcare systems. These disorders are predominantly caused by inherited or de novo variants that result in impairments to nervous system development, neurodegeneration, or impaired neuronal function. No cure exists for these disorders with many being refractory to medication. However, since monogenic neurological disorders have a single causal factor, they are also excellent targets for innovative, therapies such as gene therapy. Despite this promise, gene transfer therapies are limited in that they are only suitable for neurogenetic disorders that fit within the technological reach of these therapies. The limitations include the size of the coding region of the gene, the regulatory control of expression (dosage sensitivity), the mode of expression (e.g., dominant negative) and access to target cells. Gene editing therapies are an alternative strategy to gene transfer therapy as they have the potential of overcoming some of these hurdles, enabling the retention of physiological expression of the gene and offers precision medicine-based therapies where individual variants can be repaired. This review focusses on the existing gene editing technologies for neurogenetic disorders and how these propose to overcome the challenges common to neurogenetic disorders with gene transfer therapies as well as their own challenges.

WGCNA Identifies Translational and Proteasome-Ubiquitin Dysfunction in Rett Syndrome.

Rett Syndrome (RTT) is an X linked neurodevelopmental disorder caused by mutations in the methyl-CpG-binding protein 2 (MECP2) gene, resulting in severe cognitive and physical disabilities. Despite an apparent normal prenatal and postnatal development period, symptoms usually present around 6 to 18 months of age. Little is known about the consequences of MeCP2 deficiency at a molecular and cellular level before the onset of symptoms in neural cells, and subtle changes at this highly sensitive developmental stage may begin earlier than symptomatic manifestation. Recent transcriptomic studies of patient induced pluripotent stem cells (iPSC)-differentiated neurons and brain organoids harbouring pathogenic mutations in MECP2, have unravelled new insights into the cellular and molecular changes caused by these mutations. Here we interrogated transcriptomic modifications in RTT patients using publicly available RNA-sequencing datasets of patient iPSCs harbouring pathogenic mutations and healthy control iPSCs by Weighted Gene Correlation Network Analysis (WGCNA). Preservation analysis identified core gene pathways involved in translation, ribosomal function, and ubiquitination perturbed in some MECP2 mutant iPSC lines. Furthermore, differential gene expression of the parental fibroblasts and iPSC-derived neurons revealed alterations in genes in the ubiquitination pathway and neurotransmission in fibroblasts and differentiated neurons respectively. These findings might suggest that global translational dysregulation and proteasome ubiquitin function in Rett syndrome begins in progenitor cells prior to lineage commitment and differentiation into neural cells.

Anti-Semaphorin 4D Rescues Motor, Cognitive, and Respiratory Phenotypes in a Rett Syndrome Mouse Model.

Rett syndrome is a neurodevelopmental disorder caused by mutations of the methyl-CpG binding protein 2 gene. Abnormal physiological functions of glial cells contribute to pathogenesis of Rett syndrome. Semaphorin 4D (SEMA4D) regulates processes central to neuroinflammation and neurodegeneration including cytoskeletal structures required for process extension, communication, and migration of glial cells. Blocking SEMA4D-induced gliosis may preserve normal glial and neuronal function and rescue neurological dysfunction in Rett syndrome. We evaluated the pre-clinical therapeutic efficacy of an anti-SEMA4D monoclonal antibody in the Rett syndrome Mecp2T158A transgenic mouse model and investigated the contribution of glial cells as a proposed mechanism of action in treated mice and in primary glial cultures isolated from Mecp2T158A/y mutant mice. SEMA4D is upregulated in neurons while glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1-positive cells are upregulated in Mecp2T158A/y mice. Anti-SEMA4D treatment ameliorates Rett syndrome-specific symptoms and improves behavioural functions in both pre-symptomatic and symptomatic cohorts of hemizygous Mecp2T158A/y male mice. Anti-SEMA4D also reduces astrocyte and microglia activation in vivo. In vitro experiments demonstrate an abnormal cytoskeletal structure in mutant astrocytes in the presence of SEMA4D, while anti-SEMA4D antibody treatment blocks SEMA4D-Plexin B1 signaling and mitigates these abnormalities. These results suggest that anti-SEMA4D immunotherapy may be an effective treatment option to alleviate symptoms and improve cognitive and motor function in Rett syndrome.

Pre-clinical Investigation of Rett Syndrome Using Human Stem Cell-Based Disease Models.

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, mostly caused by mutations in MECP2. The disorder mainly affects girls and it is associated with severe cognitive and physical disabilities. Modeling RTT in neural and glial cell cultures and brain organoids derived from patient- or mutation-specific human induced pluripotent stem cells (iPSCs) has advanced our understanding of the pathogenesis of RTT, such as disease-causing mechanisms, disease progression, and cellular and molecular pathology enabling the identification of actionable therapeutic targets. Brain organoid models that recapitulate much of the tissue architecture and the complexity of cell types in the developing brain, offer further unprecedented opportunity for elucidating human neural development, without resorting to conventional animal models and the limited resource of human neural tissues. This review focuses on the new knowledge of RTT that has been gleaned from the iPSC-based models as well as limitations of the models and strategies to refine organoid technology in the quest for clinically relevant disease models for RTT and the broader spectrum of neurodevelopmental disorders.

Neurological Disorders Associated with WWOX Germline Mutations-A Comprehensive Overview.

The transcriptional regulator WW domain-containing oxidoreductase (WWOX) is a key player in a number of cellular and biological processes including tumor suppression. Recent evidence has emerged associating WWOX with non-cancer disorders. Patients harboring pathogenic germline bi-allelic WWOX variants have been described with the rare devastating neurological syndromes autosomal recessive spinocerebellar ataxia 12 (SCAR12) (6 patients) and WWOX-related epileptic encephalopathy (DEE28 or WOREE syndrome) (56 patients). Individuals with these syndromes present with a highly heterogenous clinical spectrum, the most common clinical symptoms being severe epileptic encephalopathy and profound global developmental delay. Knowledge of the underlying pathophysiology of these syndromes, the range of variants of the WWOX gene and its genotype-phenotype correlations is limited, hampering therapeutic efforts. Therefore, there is a critical need to identify and consolidate all the reported variants in WWOX to distinguish between disease-causing alleles and their associated severity, and benign variants, with the aim of improving diagnosis and increasing therapeutic efforts. Here, we provide a comprehensive review of the literature on WWOX, and analyze the pathogenic variants from published and unpublished reports by collecting entries from the ClinVar, DECIPHER, VarSome, and PubMed databases to generate the largest dataset of WWOX pathogenic variants. We estimate the correlation between variant type and patient phenotype, and delineate the impact of each variant, and used GnomAD to cross reference these variants found in the general population. From these searches, we generated the largest published cohort of WWOX individuals. We conclude with a discussion on potential personalized medicine approaches to tackle the devastating disorders associated with WWOX mutations.

Maternal autoimmunity and inflammation are associated with childhood tics and obsessive-compulsive disorder: Transcriptomic data show common enriched innate immune pathways.

Although genetic variation is a major risk factor of neurodevelopmental disorders, environmental factors during pregnancy and early life are also important in disease expression. Animal models demonstrate that maternal inflammation causes fetal neuroinflammation and neurodevelopmental deficits, and brain transcriptomics of neurodevelopmental disorders in humans show upregulated differentially expressed genes are enriched in immune pathways. We prospectively recruited 200 sequentially referred children with tic disorders/obsessive-compulsive disorder (OCD), 100 autoimmune neurological controls, and 100 age-matched healthy controls. A structured interview captured the maternal and family history of autoimmune disease and other pro-inflammatory states. Maternal blood and published Tourette brain transcriptomes were analysed for overlapping enriched pathways. Mothers of children with tics/OCD had a higher rate of autoimmune disease compared with mothers of children with autoimmune neurological conditions (p = 0.054), and mothers of healthy controls (p = 0.0004). Autoimmunity was similarly elevated in first- and second-degree maternal relatives of children with tics/OCD (p < 0.0001 and p = 0.014 respectively). Other pro-inflammatory states were also more common in mothers of children with tics/OCD than controls (p < 0.0001). Upregulated differentially expressed genes in maternal autoimmune disease and Tourette brain transcriptomes were commonly enriched in innate immune processes. Pro-inflammatory states, including autoimmune disease, are more common in the mothers and families of children with tics/OCD. Exploratory transcriptome analysis indicates innate immune signalling may link maternal inflammation and childhood tics/OCD. Targeting inflammation may represent preventative strategies in pregnancy and treatment opportunities for children with neurodevelopmental disorders.

Maternal acute and chronic inflammation in pregnancy is associated with common neurodevelopmental disorders: a systematic review.

Inflammation is increasingly recognized as a cause or consequence of common problems of humanity including obesity, stress, depression, pollution and disease states such as autoimmunity, asthma, and infection. Maternal immune activation (MIA), triggered by both acute and systemic chronic inflammation, is hypothesized to be one of the mechanisms implicated in the pathogenesis of neurodevelopmental disorders (NDD). Although there is substantial preclinical evidence to support the MIA hypothesis, the human evidence is disparate. We performed a systematic review on human studies examining associations between maternal inflammatory states and offspring NDDs (autism spectrum disorder- ASD, attention deficit hyperactivity disorder-ADHD, Tourette syndrome-TS). 32 meta-analyses and 26 additional individual studies were identified. Maternal states associated with ASD include obesity, gestational diabetes mellitus, pre-eclampsia, pollution, stress, depression, autoimmune diseases, and infection. Maternal states associated with ADHD include obesity, pre-eclampsia, smoking, low socioeconomic status (SES), stress, autoimmune disease, and asthma. Maternal states associated with TS include low SES, depression, and autoimmune diseases. Diverse maternal inflammatory states in pregnancy are associated with common offspring NDDs. Given the increased prevalence of NDDs, there is urgent need to explore relative and cumulative maternal risk factors and disease mechanisms. Defining preventable risk factors in high-risk pregnancies could mitigate the expression and severity of NDDs.

Expansion of the phenotypic spectrum of de novo missense variants in kinesin family member 1A (KIF1A).

Defects in the motor domain of kinesin family member 1A (KIF1A), a neuron-specific ATP-dependent anterograde axonal transporter of synaptic cargo, are well-recognized to cause a spectrum of neurological conditions, commonly known as KIF1A-associated neurological disorders (KAND). Here, we report one mutation-negative female with classic Rett syndrome (RTT) harboring a de novo heterozygous novel variant [NP_001230937.1:p.(Asp248Glu)] in the highly conserved motor domain of KIF1A. In addition, three individuals with severe neurodevelopmental disorder along with clinical features overlapping with KAND are also reported carrying de novo heterozygous novel [NP_001230937.1:p.(Cys92Arg) and p.(Pro305Leu)] or previously reported [NP_001230937.1:p.(Thr99Met)] variants in KIF1A. In silico tools predicted these variants to be likely pathogenic, and 3D molecular modeling predicted defective ATP hydrolysis and/or microtubule binding. Using the neurite tip accumulation assay, we demonstrated that all novel KIF1A variants significantly reduced the ability of the motor domain of KIF1A to accumulate along the neurite lengths of differentiated SH-SY5Y cells. In vitro microtubule gliding assays showed significantly reduced velocities for the variant p.(Asp248Glu) and reduced microtubule binding for the p.(Cys92Arg) and p.(Pro305Leu) variants, suggesting a decreased ability of KIF1A to move along microtubules. Thus, this study further expanded the phenotypic characteristics of KAND individuals with pathogenic variants in the KIF1A motor domain to include clinical features commonly seen in RTT individuals.

High-Throughput In Vitro, Ex Vivo, and In Vivo Screen of Adeno-Associated Virus Vectors Based on Physical and Functional Transduction.

Adeno-associated virus (AAV) vectors are quickly becoming the vectors of choice for therapeutic gene delivery. To date, hundreds of natural isolates and bioengineered variants have been reported. While factors such as high production titer and low immunoreactivity are important to consider, the ability to deliver the genetic payload (physical transduction) and to drive high transgene expression (functional transduction) remains the most important feature when selecting AAV variants for clinical applications. Reporter expression assays are the most commonly used methods for determining vector fitness. However, such approaches are time consuming and become impractical when evaluating a large number of variants. Limited access to primary human tissues or challenging model systems further complicates vector testing. To address this problem, convenient high-throughput methods based on next-generation sequencing (NGS) are being developed. To this end, we built an AAV Testing Kit that allows inherent flexibility in regard to number and type of AAV variants included, and is compatible with in vitro, ex vivo, and in vivo applications. The Testing Kit presented here consists of a mix of 30 known AAVs where each variant encodes a CMV-eGFP cassette and a unique barcode in the 3'-untranslated region of the eGFP gene, allowing NGS-barcode analysis at both the DNA and RNA/cDNA levels. To validate the AAV Testing Kit, individually packaged barcoded variants were mixed at an equal ratio and used to transduce cells/tissues of interest. DNA and RNA/cDNA were extracted and subsequently analyzed by NGS to determine the physical/functional transduction efficiencies. We were able to assess the transduction efficiencies of immortalized cells, primary cells, and induced pluripotent stem cells in vitro, as well as in vivo transduction in naïve mice and a xenograft liver model. Importantly, while our data validated previously reported transduction characteristics of individual capsids, we also identified novel previously unknown tropisms for some AAV variants.

Genome-wide transcriptomic and proteomic studies of Rett syndrome mouse models identify common signaling pathways and cellular functions as potential therapeutic targets.

The discovery that Rett syndrome is caused by mutations in the MECP2 gene has provided a major breakthrough in our understanding of the disorder. However, despite this, there is still limited understanding of the underlying pathophysiology of the disorder hampering the development of curative treatments. Over the years, a number of animal models have been developed contributing to our knowledge of the role of MECP2 in development and improving our understanding of how subtle expression levels affect brain morphology and function. Transcriptomic and proteomic studies of animal models are useful in identifying perturbations in functional pathways and providing avenues for novel areas of research into disease. This review focuses on published transcriptomic and proteomic studies of mouse models of Rett syndrome with the aim of providing a summary of all the studies, the reported dysregulated genes and functional pathways that are found to be perturbed. The 36 articles identified highlighted a number of dysfunctional pathways as well as perturbed biological networks and cellular functions including synaptic dysfunction and neuronal transmission, inflammation, and mitochondrial dysfunction. These data reveal biological insights that contribute to the disease process which may be targeted to investigate curative treatments.