Epigenetic editing for autosomal dominant neurological disorders.

Epigenetics refers to the molecules and mechanisms that modify gene expression states without changing the nucleotide context. These modifications are what encode the cell state during differentiation or epigenetic memory in mitosis. Epigenetic modifications can alter gene expression by changing the chromatin architecture by altering the affinity for DNA to wrap around histone octamers, forming nucleosomes. The higher affinity the DNA has for the histones, the tighter it will wrap and therefore induce a heterochromatin state, silencing gene expression. Several groups have shown the ability to harness the cell's natural epigenetic modification pathways to engineer proteins that can induce changes in epigenetics and consequently regulate gene expression. Therefore, epigenetic modification can be used to target and treat disorders through the modification of endogenous gene expression. The use of epigenetic modifications may prove an effective path towards regulating gene expression to potentially correct or cure genetic disorders.

Role of MEF2C in the Endothelial Cells Derived from Human Induced Pluripotent Stem Cells.

Human induced pluripotent stem cells (hiPSCs) not only provide an abundant source of vascular cells for potential therapeutic applications in vascular disease but also constitute an excellent model for understanding the mechanisms that regulate the differentiation and the functionality of vascular cells. Here, we reported that myocyte enhancer factor 2C (MEF2C) transcription factor, but not any other members of the MEF2 family, was robustly upregulated during the differentiation of vascular progenitors and endothelial cells (ECs) from hiPSCs. Vascular endothelial growth factors (VEGF) strongly induced MEF2C expression in endothelial lineage cells. The specific upregulation of MEF2C during the commitment of endothelial lineage was dependent on the extracellular signal regulated kinase (ERK). Moreover, knockdown of MEF2C with shRNA in hiPSCs did not affect the differentiation of ECs from these hiPSCs, but greatly reduced the migration and tube formation capacity of the hiPSC-derived ECs. Through a chromatin immunoprecipitation-sequencing, genome-wide RNA-sequencing, quantitative RT-PCR, and immunostaining analyses of the hiPSC-derived endothelial lineage cells with MEF2C inhibition or knockdown compared to control hiPSC-derived ECs, we identified TNF-related apoptosis inducing ligand (TRAIL) and transmembrane protein 100 (TMEM100) as novel targets of MEF2C. This study demonstrates an important role for MEF2C in regulating human EC functions and highlights MEF2C and its downstream effectors as potential targets to treat vascular malfunction-associated diseases.

Transcriptional reprogramming restores UBE3A brain-wide and rescues behavioral phenotypes in an Angelman syndrome mouse model.

Angelman syndrome (AS) is a neurogenetic disorder caused by the loss of ubiquitin ligase E3A (UBE3A) gene expression in the brain. The UBE3A gene is paternally imprinted in brain neurons. Clinical features of AS are primarily due to the loss of maternally expressed UBE3A in the brain. A healthy copy of paternal UBE3A is present in the brain but is silenced by a long non-coding antisense transcript (UBE3A-ATS). Here, we demonstrate that an artificial transcription factor (ATF-S1K) can silence Ube3a-ATS in an adult mouse model of Angelman syndrome (AS) and restore endogenous physiological expression of paternal Ube3a. A single injection of adeno-associated virus (AAV) expressing ATF-S1K (AAV-S1K) into the tail vein enabled whole-brain transduction and restored UBE3A protein in neurons to ∼25% of wild-type protein. The ATF-S1K treatment was highly specific to the target site with no detectable inflammatory response 5 weeks after AAV-S1K administration. AAV-S1K treatment of AS mice showed behavioral rescue in exploratory locomotion, a task involving gross and fine motor abilities, similar to low ambulation and velocity in AS patients. The specificity and tolerability of a single injection of AAV-S1K therapy for AS demonstrate the use of ATFs as a promising translational approach for AS.

Polyamine Metabolites as Biomarkers in Head and Neck Cancer Biofluids.

Background: Novel, non-invasive diagnostic biomarkers that facilitate early intervention in head and neck cancer are urgently needed. Polyamine metabolites have been observed to be elevated in numerous cancer types and correlated with poor prognosis. The aim of this study was to assess the concentration of polyamines in the saliva and urine from head and neck cancer (HNC) patients, compared to healthy controls. Methods: Targeted metabolomic analysis was performed on saliva and urine from 39 HNC patient samples and compared to 89 healthy controls using a quantitative, targeted liquid chromatography mass spectrometry approach. Results: The metabolites N1-acetylspermine (ASP), N8-acetylspermidine (ASD) and N1,N12-diacetylspermine (DAS) were detected at significantly different concentrations in the urine of HNC patients as compared to healthy controls. Only ASP was detected at elevated levels in HNC saliva as compared to healthy controls. Conclusion: These data suggest that assessment of polyamine-based metabolite biomarkers within the saliva and urine warrants further investigation as a potential diagnostic in HNC patients.

Touchscreen cognitive deficits, hyperexcitability and hyperactivity in males and females using two models of Cdkl5 deficiency.

Many neurodevelopmental disorders (NDDs) are the result of mutations on the X chromosome. One severe NDD resulting from mutations on the X chromosome is CDKL5 deficiency disorder (CDD). CDD is an epigenetic, X-linked NDD characterized by intellectual disability (ID), pervasive seizures and severe sleep disruption, including recurring hospitalizations. CDD occurs at a 4:1 ratio, with a female bias. CDD is driven by the loss of cyclin-dependent kinase-like 5 (CDKL5), a serine/threonine kinase that is essential for typical brain development, synapse formation and signal transmission. Previous studies focused on male subjects from animal models, likely to avoid the complexity of X mosaicism. For the first time, we report translationally relevant behavioral phenotypes in young adult (8-20 weeks) females and males with robust signal size, including impairments in learning and memory, substantial hyperactivity and increased susceptibility to seizures/reduced seizure thresholds, in both sexes, and in two models of CDD preclinical mice, one with a general loss-of-function mutation and one that is a patient-derived mutation.

Cell-Based Delivery Approaches for DNA-Binding Domains to the Central Nervous System.

Advancements in programmable DNA-Binding Proteins (DBDs) that target the genome, such as zinc fingers, transcription activator-like effectors, and Cas9, have broadened drug target design beyond traditional protein substrates. Effective delivery methodologies remain a major barrier in targeting the central nervous system. Currently, adeno-associated virus is the most wellvalidated delivery system for the delivery of DBDs towards the central nervous with multiple, ongoing clinical trials. While effective in transducing neuronal cells, viral delivery systems for DBDs remain problematic due to inherent viral packaging limits or immune responses that hinder translational potential. Direct administration of DBDs or encapsulation in lipid nanoparticles may provide alternative means towards delivering gene therapies into the central nervous system. This review will evaluate the strengths and limitations of current DBD delivery strategies in vivo. Furthermore, this review will discuss the use of adult stem cells as a putative delivery vehicle for DBDs and the potential advantages that these systems have over previous methodologies.

An in vivo Cell-Based Delivery Platform for Zinc Finger Artificial Transcription Factors in Pre-clinical Animal Models.

Zinc finger (ZF), transcription activator-like effectors (TALE), and CRISPR/Cas9 therapies to regulate gene expression are becoming viable strategies to treat genetic disorders, although effective in vivo delivery systems for these proteins remain a major translational hurdle. We describe the use of a mesenchymal stem/stromal cell (MSC)-based delivery system for the secretion of a ZF protein (ZF-MSC) in transgenic mouse models and young rhesus monkeys. Secreted ZF protein from mouse ZF-MSC was detectable within the hippocampus 1 week following intracranial or cisterna magna (CM) injection. Secreted ZF activated the imprinted paternal Ube3a in a transgenic reporter mouse and ameliorated motor deficits in a Ube3a deletion Angelman Syndrome (AS) mouse. Intrathecally administered autologous rhesus MSCs were well-tolerated for 3 weeks following administration and secreted ZF protein was detectable within the cerebrospinal fluid (CSF), midbrain, and spinal cord. This approach is less invasive when compared to direct intracranial injection which requires a surgical procedure.

Artificial escape from XCI by DNA methylation editing of the CDKL5 gene.

A significant number of X-linked genes escape from X chromosome inactivation and are associated with a distinct epigenetic signature. One epigenetic modification that strongly correlates with X-escape is reduced DNA methylation in promoter regions. Here, we created an artificial escape by editing DNA methylation on the promoter of CDKL5, a gene causative for an infantile epilepsy, from the silenced X-chromosomal allele in human neuronal-like cells. We identify that a fusion of the catalytic domain of TET1 to dCas9 targeted to the CDKL5 promoter using three guide RNAs causes significant reactivation of the inactive allele in combination with removal of methyl groups from CpG dinucleotides. Strikingly, we demonstrate that co-expression of TET1 and a VP64 transactivator have a synergistic effect on the reactivation of the inactive allele to levels >60% of the active allele. We further used a multi-omics assessment to determine potential off-targets on the transcriptome and methylome. We find that synergistic delivery of dCas9 effectors is highly selective for the target site. Our findings further elucidate a causal role for reduced DNA methylation associated with escape from X chromosome inactivation. Understanding the epigenetics associated with escape from X chromosome inactivation has potential for those suffering from X-linked disorders.

The iNs and Outs of Direct Reprogramming to Induced Neurons.

Understanding of cell-type specific transcription factors has promoted progress in methods for cellular reprogramming, such as directly reprogramming somatic cells to induced neurons (iN). Methods for direct reprogramming require neuronal-fate determining gene activation via neuron-specific microRNAs, chemical modulation of key neuronal signaling pathways or overexpression via viral vectors, with some reprogramming strategies requiring a combination of these methods to induce the neuronal-cell fate. These methods have been employed in a multitude of cell types, including fibroblasts, hepatocytes, peripheral blood mononuclear, and T cells. The ability to create iN from skin biopsies and blood samples coupled with recent advancements in artificially inducing age- and disease-associated phenotypes are accelerating the development of disease models for late-onset neurodegenerative disorders. Here, we review how activation of the neuronal transcriptome alters the epigenetic landscape of the donor cell to facilitate reprogramming to neurons. We also discuss the advantages of using DNA binding domains such as CRISPR/dCas9 to overcome epigenetic barriers to induce neuronal-cell fate by activating endogenous neuronal cell-fate determining genes.

Ezh2-dCas9 and KRAB-dCas9 enable engineering of epigenetic memory in a context-dependent manner.

BACKGROUND: Rewriting of the epigenome has risen as a promising alternative to gene editing for precision medicine. In nature, epigenetic silencing can result in complete attenuation of target gene expression over multiple mitotic divisions. However, persistent repression has been difficult to achieve in a predictable manner using targeted systems. RESULTS: Here, we report that persistent epigenetic memory required both a DNA methyltransferase (DNMT3A-dCas9) and a histone methyltransferase (Ezh2-dCas9 or KRAB-dCas9). We demonstrate that the histone methyltransferase requirement can be locus specific. Co-targeting Ezh2-dCas9, but not KRAB-dCas9, with DNMT3A-dCas9 and DNMT3L induced long-term HER2 repression over at least 50 days (approximately 57 cell divisions) and triggered an epigenetic switch to a heterochromatic environment. An increase in H3K27 trimethylation and DNA methylation was stably maintained and accompanied by a sustained loss of H3K27 acetylation. Interestingly, substitution of Ezh2-dCas9 with KRAB-dCas9 enabled long-term repression at some target genes (e.g., SNURF) but not at HER2, at which H3K9me3 and DNA methylation were transiently acquired and subsequently lost. Off-target DNA hypermethylation occurred at many individual CpG sites but rarely at multiple CpGs in a single promoter, consistent with no detectable effect on transcription at the off-target loci tested. Conversely, robust hypermethylation was observed at HER2. We further demonstrated that Ezh2-dCas9 required full-length DNMT3L for maximal activity and that co-targeting DNMT3L was sufficient for persistent repression by Ezh2-dCas9 or KRAB-dCas9. CONCLUSIONS: These data demonstrate that targeting different combinations of histone and DNA methyltransferases is required to achieve maximal repression at different loci. Fine-tuning of targeting tools is a necessity to engineer epigenetic memory at any given locus in any given cell type.

Primed mesenchymal stem cells package exosomes with metabolites associated with immunomodulation.

Mesenchymal stem cell (MSC) based therapies are currently being evaluated as a putative therapeutic in numerous human clinical trials. Recent reports have established that exosomes mediate much of the therapeutic properties of MSCs. Exosomes are nanovesicles which mediate intercellular communication, transmitting signals between cells which regulate a diverse range of biological processes. MSC-derived exosomes are packaged with numerous types of proteins and RNAs, however, their metabolomic and lipidomic profiles to date have not been well characterized. We previously reported that MSCs, in response to priming culture conditions that mimic the in vivo microenvironmental niche, substantially modulate cellular signaling and significantly increase the secretion of exosomes. Here we report that MSCs exposed to such priming conditions undergo glycolytic reprogramming, which homogenizes MSCs' metabolomic profile. In addition, we establish that exosomes derive from primed MSCs are packaged with numerous metabolites that have been directly associated with immunomodulation, including M2 macrophage polarization and regulatory T lymphocyte induction.

Exosomes Derived from Human Primed Mesenchymal Stem Cells Induce Mitosis and Potentiate Growth Factor Secretion.

Mesenchymal stem cells (MSCs) facilitate functional recovery in numerous animal models of inflammatory and ischemic tissue-related diseases with a growing body of research suggesting that exosomes mediate many of these therapeutic effects. It remains unclear, however, which types of proteins are packaged into exosomes compared with the cells from which they are derived. In this study, using comprehensive proteomic analysis, we demonstrated that human primed MSCs secrete exosomes (pMEX) that are packaged with markedly higher fractions of specific protein subclasses compared with their cells of origin, indicating regulation of their contents. Notably, we found that pMEX are also packaged with substantially elevated levels of extracellular-associated proteins. Fibronectin was the most abundant protein detected, and data established that fibronectin mediates the mitogenic properties of pMEX. In addition, treatment of SHSY5Y cells with pMEX induced the secretion of growth factors known to possess mitogenic and neurotrophic properties. Taken together, our comprehensive analysis indicates that pMEX are packaged with specific protein subtypes, which may provide a molecular basis for their distinct functional properties.

Beyond autophagy: a novel role for autism-linked Wdfy3 in brain mitophagy.

WD repeat and FYVE domain-containing 3 (WDFY3; also known as Autophagy-Linked FYVE or Alfy) is an identified intellectual disability, developmental delay and autism risk gene. This gene encodes for a scaffolding protein that is expressed in both the developing and adult central nervous system and required for autophagy and aggrephagy with yet unexplored roles in mitophagy. Given that mitochondrial trafficking, dynamics and remodeling have key roles in synaptic plasticity, we tested the role of Wdfy3 on brain bioenergetics by using Wdfy3+/lacZ mice, the only known Wdfy3 mutant animal model with overt neurodevelopmental anomalies that survive to adulthood. We found that Wdfy3 is required for sustaining brain bioenergetics and morphology via mitophagy. Decreased mitochondrial quality control by conventional mitophagy was partly compensated for by the increased formation of mitochondria-derived vesicles (MDV) targeted to lysosomal degradation (micromitophagy). These observations, extended through proteomic analysis of mitochondria-enriched cortical fractions, showed significant enrichment for pathways associated with mitophagy, mitochondrial transport and axon guidance via semaphorin, Robo, L1cam and Eph-ephrin signaling. Collectively, our findings support a critical role for Wdfy3 in mitochondrial homeostasis with implications for neuron differentiation, neurodevelopment and age-dependent neurodegeneration.

dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression.

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A 'direct tethering' strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a 'recruitment' strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.

Plasma metabolic profile delineates roles for neurodegeneration, pro-inflammatory damage and mitochondrial dysfunction in the FMR1 premutation.

Carriers of premutation CGG expansions in the fragile X mental retardation 1 (FMR1) gene are at higher risk of developing a late-onset neurodegenerative disorder named Fragile X-associated tremor ataxia syndrome (FXTAS). Given that mitochondrial dysfunction has been identified in fibroblasts, PBMC and brain samples from carriers as well as in animal models of the premutation and that mitochondria are at the center of intermediary metabolism, the aim of the present study was to provide a complete view of the metabolic pattern by uncovering plasma metabolic perturbations in premutation carriers. To this end, metabolic profiles were evaluated in plasma from 23 premutation individuals and 16 age- and sex-matched controls. Among the affected pathways, mitochondrial dysfunction was associated with a Warburg-like shift with increases in lactate levels and altered Krebs' intermediates, neurotransmitters, markers of neurodegeneration and increases in oxidative stress-mediated damage to biomolecules. The number of CGG repeats correlated with a subset of plasma metabolites, which are implicated not only in mitochondrial disorders but also in other neurological diseases, such as Parkinson's, Alzheimer's and Huntington's diseases. For the first time, the identified pathways shed light on disease mechanisms contributing to morbidity of the premutation, with the potential of assessing metabolites in longitudinal studies as indicators of morbidity or disease progression, especially at the early preclinical stages.

Plasma Biomarkers for Monitoring Brain Pathophysiology in FMR1 Premutation Carriers.

Premutation carriers have a 55-200 CGG expansion in the fragile X mental retardation 1 (FMR1) gene. Currently, 1.5 million individuals are affected in the United States, and carriers are at risk of developing the late-onset neurodegenerative disorder Fragile X-associated tremor ataxia syndrome (FXTAS). Limited efforts have been made to develop new methods for improved early patient monitoring, treatment response, and disease progression. To this end, plasma metabolomic phenotyping was obtained for 23 premutation carriers and 16 age- and sex-matched controls. Three biomarkers, phenylethylamine normalized by either aconitate or isocitrate and oleamide normalized by isocitrate, exhibited excellent model performance. The lower phenylethylamine and oleamide plasma levels in carriers may indicate, respectively, incipient nigrostriatal degeneration and higher incidence of substance abuse, anxiety and sleep disturbances. Higher levels of citrate, isocitrate, aconitate, and lactate may reflect deficits in both bioenergetics and neurotransmitter metabolism (Glu, GABA). This study lays important groundwork by defining the potential utility of plasma metabolic profiling to monitor brain pathophysiology in carriers before and during the progression of FXTAS, treatment efficacy and evaluation of side effects.