Efficacy of Vafidemstat in Experimental Autoimmune Encepha-Lomyelitis Highlights the KDM1A/RCOR1/HDAC Epigenetic Axis in Multiple Sclerosis

Lysine specific demethylase 1 (LSD1; also known as KDM1A), is an epigenetic modulator that modifies the histone methylation status. KDM1A forms a part of protein complexes that regulate the expression of genes involved in the onset and progression of diseases such as cancer, central nervous system (CNS) disorders, viral infections, and others. Vafidemstat (ORY-2001) is a clinical stage inhibitor of KDM1A in development for the treatment of neurodegenerative and psychiatric diseases. However, the role of ORY-2001 targeting KDM1A in neuroinflammation remains to be explored. Here, we investigated the effect of ORY-2001 on immune-mediated and virus-induced encephalomyelitis, two experimental models of multiple sclerosis and neuronal damage. Oral ad-ministration of ORY-2001 ameliorated clinical signs, reduced lymphocyte egress and infiltration of immune cells into the spinal cord, and prevented demyelination. Interestingly, ORY-2001 was more effective and/or faster acting than a sphingosine 1-phosphate receptor antagonist in the effector phase of the disease and reduced the inflammatory gene expression signature characteristic ofEAE in the CNS of mice more potently. In addition, ORY-2001 induced gene expression changes con-cordant with a potential neuroprotective function in the brain and spinal cord and reduced neuronal glutamate excitotoxicity-derived damage in explants. These results pointed to ORY-2001 as a promising CNS epigenetic drug able to target neuroinflammatory and neurodegenerative diseases and provided preclinical support for the subsequent design of early-stage clinical trials.This research funded by Oryzon Genomics, S.A. and partially supported by RETOS: (RTC2016-4955-1); EUROSTAR II: EMTherapy (CIIP-20152001/E!9683) and CDTI: EDOTEM (IDI-20180117)

Modulation of KDM1A with vafidemstat rescues memory deficit and behavioral alterations

Transcription disequilibria are characteristic of many neurodegenerative diseases. The activity-evoked transcription of immediate early genes (IEGs), important for neuronal plasticity, memory and behavior, is altered in CNS diseases and governed by epigenetic modulation. KDM1A, a histone 3 lysine 4 demethylase that forms part of transcription regulation complexes, has been implicated in the control of IEG transcription. Here we report the development of vafidemstat (ORY-2001), a brain penetrant inhibitor of KDM1A and MAOB. ORY-2001 efficiently inhibits brain KDM1A at doses suitable for long term treatment, and corrects memory deficit as assessed in the novel object recognition testing in the Senescence Accelerated Mouse Prone 8 (SAMP8) model for accelerated aging and Alzheimer's disease. Comparison with a selective KDM1A or MAOB inhibitor reveals that KDM1A inhibition is key for efficacy. ORY-2001 further corrects behavior alterations including aggression and social interaction deficits in SAMP8 mice and social avoidance in the rat rearing isolation model. ORY-2001 increases the responsiveness of IEGs, induces genes required for cognitive function and reduces a neuroinflammatory signature in SAMP8 mice. Multiple genes modulated by ORY-2001 are differentially expressed in Late Onset Alzheimer's Disease. Most strikingly, the amplifier of inflammation S100A9 is highly expressed in LOAD and in the hippocampus of SAMP8 mice, and down-regulated by ORY-2001. ORY-2001 is currently in multiple Phase IIa studies

Modulation of KDM1A with vafidemstat rescues memory deficit and behavioral alterations

Transcription disequilibria are characteristic of many neurodegenerative diseases. The activity-evoked transcription of immediate early genes (IEGs), important for neuronal plasticity, memory and behavior, is altered in CNS diseases and governed by epigenetic modulation. KDM1A, a histone 3 lysine 4 demethylase that forms part of transcription regulation complexes, has been implicated in the control of IEG transcription. Here we report the development of vafidemstat (ORY-2001), a brain penetrant inhibitor of KDM1A and MAOB. ORY-2001 efficiently inhibits brain KDM1A at doses suitable for long term treatment, and corrects memory deficit as assessed in the novel object recognition testing in the Senescence Accelerated Mouse Prone 8 (SAMP8) model for accelerated aging and Alzheimer's disease. Comparison with a selective KDM1A or MAOB inhibitor reveals that KDM1A inhibition is key for efficacy. ORY-2001 further corrects behavior alterations including aggression and social interaction deficits in SAMP8 mice and social avoidance in the rat rearing isolation model. ORY-2001 increases the responsiveness of IEGs, induces genes required for cognitive function and reduces a neuroinflammatory signature in SAMP8 mice. Multiple genes modulated by ORY-2001 are differentially expressed in Late Onset Alzheimer's Disease. Most strikingly, the amplifier of inflammation S100A9 is highly expressed in LOAD and in the hippocampus of SAMP8 mice, and down-regulated by ORY-2001. ORY-2001 is currently in multiple Phase IIa studies

Generation and characterisation of Friedreich ataxia YG8R mouse fibroblast and neural stem cell models

This article has been made available through the Brunel Open Access Publishing Fund.Background: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease caused by GAA repeat expansion in the first intron of the FXN gene, which encodes frataxin, an essential mitochondrial protein. To further characterise the molecular abnormalities associated with FRDA pathogenesis and to hasten drug screening, the development and use of animal and cellular models is considered essential. Studies of lower organisms have already contributed to understanding FRDA disease pathology, but mammalian cells are more related to FRDA patient cells in physiological terms. Methodology/Principal Findings: We have generated fibroblast cells and neural stem cells (NSCs) from control Y47R mice (9 GAA repeats) and GAA repeat expansion YG8R mice (190+120 GAA repeats). We then differentiated the NSCs in to neurons, oligodendrocytes and astrocytes as confirmed by immunocytochemical analysis of cell specific markers. The three YG8R mouse cell types (fibroblasts, NSCs and differentiated NSCs) exhibit GAA repeat stability, together with reduced expression of frataxin and reduced aconitase activity compared to control Y47R cells. Furthermore, YG8R cells also show increased sensitivity to oxidative stress and downregulation of Pgc-1α and antioxidant gene expression levels, especially Sod2. We also analysed various DNA mismatch repair (MMR) gene expression levels and found that YG8R cells displayed significant reduction in expression of several MMR genes, which may contribute to the GAA repeat stability. Conclusions/Significance: We describe the first fibroblast and NSC models from YG8R FRDA mice and we confirm that the NSCs can be differentiated into neurons and glia. These novel FRDA mouse cell models, which exhibit a FRDA-like cellular and molecular phenotype, will be valuable resources to further study FRDA molecular pathogenesis. They will also provide very useful tools for preclinical testing of frataxin-increasing compounds for FRDA drug therapy, for gene therapy, and as a source of cells for cell therapy testing in FRDA mice. © 2014 Sandi et al

Ciliary Beating Recovery in Deficient Human Airway Epithelial Cells after Lentivirus Ex Vivo Gene Therapy

Primary Ciliary Dyskinesia is a heterogeneous genetic disease that is characterized by cilia dysfunction of the epithelial cells lining the respiratory tracts, resulting in recurrent respiratory tract infections. Despite lifelong physiological therapy and antibiotics, the lungs of affected patients are progressively destroyed, leading to respiratory insufficiency. Recessive mutations in Dynein Axonemal Intermediate chain type 1 (DNAI1) gene have been described in 10% of cases of Primary Ciliary Dyskinesia. Our goal was to restore normal ciliary beating in DNAI1–deficient human airway epithelial cells. A lentiviral vector based on Simian Immunodeficiency Virus pseudotyped with Vesicular Stomatitis Virus Glycoprotein was used to transduce cultured human airway epithelial cells with a cDNA of DNAI1 driven by the Elongation Factor 1 promoter. Transcription and translation of the transduced gene were tested by RT–PCR and western blot, respectively. Human airway epithelial cells that were DNAI1–deficient due to compound heterozygous mutations, and consequently had immotile cilia and no outer dynein arm, were transduced by the lentivirus. Cilia beating was recorded and electron microscopy of the cilia was performed. Transcription and translation of the transduced DNAI1 gene were detected in human cells treated with the lentivirus. In addition, immotile cilia recovered a normal beat and outer dynein arms reappeared. We demonstrated that it is possible to obtain a normalization of ciliary beat frequency of deficient human airway epithelial cells by using a lentivirus to transduce cells with the therapeutic gene. This preliminary step constitutes a conceptual proof that is indispensable in the perspective of Primary Ciliary Dyskinesia's in vivo gene therapy. This is the first time that recovery of cilia beating is demonstrated in this disease

Stable S/MAR-based episomal vectors are regulated at the chromatin level

Episomal vectors assembled from defined genetic components are a promising alternative to traditional gene therapy vectors that integrate in the host genome and may cause insertional mutations. The vector pEPI-eGFP is stably retained in the episomal state in cultured mammalian cells at low copy number for many generations without integration into the host genome. Although pEPI-eGFP is a fully engineered vector, little is known about how it interacts with the host genome and about the molecular mechanisms that are responsible for its transcriptional activity. We have analyzed the expression of the episomal reporter gene eGFP under conditions that affect the chromatin state of the genome. We have also constructed pEPI derivatives carrying a tandem array of lac operator sequences, which allows in vivo visualization and manipulation of the chromatin state of the episome. We show that changes in chromatin state of both the host and pEPI-eGFP induces changes in episomal gene activity and influences the episome’s nuclear distributions. We conclude that episomal genes are subject to control systems of the host, similarly to their counterparts in the host genome

A portable BRCA1-HAC (human artificial chromosome) module for analysis of BRCA1 tumor suppressor function

BRCA1 is involved in many disparate cellular functions, including DNA damage repair, cell-cycle checkpoint activation, gene transcriptional regulation, DNA replication, centrosome function and others. The majority of evidence strongly favors the maintenance of genomic integrity as a principal tumor suppressor activity of BRCA1. At the same time some functional aspects of BRCA1 are not fully understood. Here, a HAC (human artificial chromosome) module with a regulated centromere was constructed for delivery and expression of the 90 kb genomic copy of the BRCA1 gene into BRCA1-deficient human cells. A battery of functional tests was carried out to demonstrate functionality of the exogenous BRCA1. In separate experiments, we investigated the role of BRCA1 in maintenance of heterochromatin integrity within a human functional kinetochore. We demonstrated that BRCA1 deficiency results in a specific activation of transcription of higher-order alpha-satellite repeats (HORs) assembled into heterochromatin domains flanking the kinetochore. At the same time no detectable elevation of transcription was observed within HORs assembled into centrochromatin domains. Thus, we demonstrated a link between BRCA1 deficiency and kinetochore dysfunction and extended previous observations that BRCA1 is required to silence transcription in heterochromatin in specific genomic loci. This supports the hypothesis that epigenetic alterations of the kinetochore initiated in the absence of BRCA1 may contribute to cellular transformation

Rottura prematura delle membrane ed infezioni batteriche vulvovaginali: aspetti ultrastrutturali, microbiologici, diagnostici e terapeutici.

Analisi delle modificazioni nella struttura delle membrane amniotiche in seguito alla colonizzazione microbica. Possibilità di prevenzione delle alterazioni flogistiche vulvovaginali e terapia antibiotica della PRO

Advances in high-capacity extrachromosomal vector technology: episomal maintenance, vector delivery, and transgene expression.

Recent developments in extrachromosomal vector technology have offered new ways of designing safer, physiologically regulated vectors for gene therapy. Extrachromosomal, or episomal, persistence in the nucleus of transduced cells offers a safer alternative to integrating vectors which have become the subject of safety concerns following serious adverse events in recent clinical trials. Extrachromosomal vectors do not cause physical disruption in the host genome, making these vectors safe and suitable tools for several gene therapy targets, including stem cells. Moreover, the high insert capacity of extrachromosomal vectors allows expression of a therapeutic transgene from the context of its genomic DNA sequence, providing an elegant way to express normal splice variants and achieve physiologically regulated levels of expression. Here, we describe past and recent advances in the development of several different extrachromosomal systems, discuss their retention mechanisms, and evaluate their use as expression vectors to deliver and express genomic DNA loci. We also discuss a variety of delivery systems, viral and nonviral, which have been used to deliver episomal vectors to target cells in vitro and in vivo. Finally, we explore the potential for the delivery and expression of extrachromosomal transgenes in stem cells. The long-term persistence of extrachromosomal vectors combined with the potential for stem cell proliferation and differentiation into a wide range of cell types offers an exciting prospect for therapeutic interventions

Replication-competent herpes simplex vectors: design and applications

Replication-competent vectors are derived from attenuated viruses whose genes, that are nonessential for replication in cultured cells in vitro, are either mutated or deleted. The removal of one or more nonessential genes may reduce pathogenicity without requiring a cell line to complement growth. Herpes simplex viruses (HSV) are potential vectors for several applications in human healthcare. These include delivery and expression of human genes to cells of the nervous systems, selective destruction of cancer cells, prophylaxis against infection with HSV or other infectious diseases, and targeted infection to specific tissues or organs. This review highlights the progress in creating attenuated genetically engineered HSV vectors