Antisense oligonucleotides restore excitability, GABA signalling and sodium current density in a Dravet syndrome model.

Dravet syndrome is an intractable developmental and epileptic encephalopathy caused by de novo variants in SCN1A resulting in haploinsufficiency of the voltage-gated sodium channel Nav1.1. We showed previously that administration of the antisense oligonucleotide STK-001, also called ASO-22, generated using targeted augmentation of nuclear gene output technology to prevent inclusion of the nonsense-mediated decay, or poison, exon 20N in human SCN1A, increased productive Scn1a transcript and Nav1.1 expression and reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy in a mouse model of Dravet syndrome. Here, we investigated the mechanism of action of ASO-84, a surrogate for ASO-22 that also targets splicing of SCN1A exon 20N, in Scn1a+/- Dravet syndrome mouse brain. Scn1a +/- Dravet syndrome and wild-type mice received a single intracerebroventricular injection of antisense oligonucleotide or vehicle at postnatal Day 2. We examined the electrophysiological properties of cortical pyramidal neurons and parvalbumin-positive fast-spiking interneurons in brain slices at postnatal Days 21-25 and measured sodium currents in parvalbumin-positive interneurons acutely dissociated from postnatal Day 21-25 brain slices. We show that, in untreated Dravet syndrome mice, intrinsic cortical pyramidal neuron excitability was unchanged while cortical parvalbumin-positive interneurons showed biphasic excitability with initial hyperexcitability followed by hypoexcitability and depolarization block. Dravet syndrome parvalbumin-positive interneuron sodium current density was decreased compared to wild-type. GABAergic signalling to cortical pyramidal neurons was reduced in Dravet syndrome mice, suggesting decreased GABA release from interneurons. ASO-84 treatment restored action potential firing, sodium current density and GABAergic signalling in Dravet syndrome parvalbumin-positive interneurons. Our work suggests that interneuron excitability is selectively affected by ASO-84. This new work provides critical insights into the mechanism of action of this antisense oligonucleotide and supports the potential of antisense oligonucleotide-mediated upregulation of Nav1.1 as a successful strategy to treat Dravet syndrome.

Hypoxia-induced changes in Ca(2+) mobilization and protein phosphorylation implicated in impaired wound healing.

The process of wound healing must be tightly regulated to achieve successful restoration of injured tissue. Previously, we demonstrated that when corneal epithelium is injured, nucleotides and neuronal factors are released to the extracellular milieu, generating a Ca(2+) wave from the origin of the wound to neighboring cells. In the present study we sought to determine how the communication between epithelial cells in the presence or absence of neuronal wound media is affected by hypoxia. A signal-sorting algorithm was developed to determine the dynamics of Ca(2+) signaling between neuronal and epithelial cells. The cross talk between activated corneal epithelial cells in response to neuronal wound media demonstrated that injury-induced Ca(2+) dynamic patterns were altered in response to decreased O2 levels. These alterations were associated with an overall decrease in ATP and changes in purinergic receptor-mediated Ca(2+) mobilization and localization of N-methyl-d-aspartate receptors. In addition, we used the cornea in an organ culture wound model to examine how hypoxia impedes reepithelialization after injury. There was a change in the recruitment of paxillin to the cell membrane and deposition of fibronectin along the basal lamina, both factors in cell migration. Our results provide evidence that complex Ca(2+)-mediated signaling occurs between sensory neurons and epithelial cells after injury and is critical to wound healing. Information revealed by these studies will contribute to an enhanced understanding of wound repair under compromised conditions and provide insight into ways to effectively stimulate proper epithelial repair.

Antisense oligonucleotides increase Scn1a expression and reduce seizures and SUDEP incidence in a mouse model of Dravet syndrome.

Dravet syndrome (DS) is an intractable developmental and epileptic encephalopathy caused largely by de novo variants in the SCN1A gene, resulting in haploinsufficiency of the voltage-gated sodium channel α subunit NaV1.1. Here, we used Targeted Augmentation of Nuclear Gene Output (TANGO) technology, which modulates naturally occurring, nonproductive splicing events to increase target gene and protein expression and ameliorate disease phenotype in a mouse model. We identified antisense oligonucleotides (ASOs) that specifically increase the expression of productive Scn1a transcript in human cell lines, as well as in mouse brain. We show that a single intracerebroventricular dose of a lead ASO at postnatal day 2 or 14 reduced the incidence of electrographic seizures and sudden unexpected death in epilepsy (SUDEP) in the F1:129S-Scn1a +/- × C57BL/6J mouse model of DS. Increased expression of productive Scn1a transcript and NaV1.1 protein was confirmed in brains of treated mice. Our results suggest that TANGO may provide a unique, gene-specific approach for the treatment of DS.

Antisense oligonucleotide modulation of non-productive alternative splicing upregulates gene expression.

While most monogenic diseases are caused by loss or reduction of protein function, the need for technologies that can selectively increase levels of protein in native tissues remains. Here we demonstrate that antisense-mediated modulation of pre-mRNA splicing can increase endogenous expression of full-length protein by preventing naturally occurring non-productive alternative splicing and promoting generation of productive mRNA. Bioinformatics analysis of RNA sequencing data identifies non-productive splicing events in 7,757 protein-coding human genes, of which 1,246 are disease-associated. Antisense oligonucleotides targeting multiple types of non-productive splicing events lead to increases in productive mRNA and protein in a dose-dependent manner in vitro. Moreover, intracerebroventricular injection of two antisense oligonucleotides in wild-type mice leads to a dose-dependent increase in productive mRNA and protein in the brain. The targeting of natural non-productive alternative splicing to upregulate expression from wild-type or hypomorphic alleles provides a unique approach to treating genetic diseases.

CXCL10 stabilizes T cell-brain endothelial cell adhesion leading to the induction of cerebral malaria.

Malaria remains one of the world's most significant human infectious diseases and cerebral malaria (CM) is its most deadly complication. CM pathogenesis remains incompletely understood, hindering the development of therapeutics to prevent this lethal complication. Elevated levels of the chemokine CXCL10 are a biomarker for CM, and CXCL10 and its receptor CXCR3 are required for experimental CM (ECM) in mice, but their role has remained unclear. Using multiphoton intravital microscopy, CXCR3 receptor- and ligand-deficient mice and bone marrow chimeric mice, we demonstrate a key role for endothelial cell-produced CXCL10 in inducing the firm adhesion of T cells and preventing their cell detachment from the brain vasculature. Using a CXCL9 and CXCL10 dual-CXCR3-ligand reporter mouse, we found that CXCL10 was strongly induced in the brain endothelium as early as 4 days after infection, while CXCL9 and CXCL10 expression was found in inflammatory monocytes and monocyte-derived DCs within the blood vasculature on day 8. The induction of both CXCL9 and CXCL10 was completely dependent on IFN-γ receptor signaling. These data demonstrate that IFN-γ-induced, endothelium-derived CXCL10 plays a critical role in mediating the T cell-endothelial cell adhesive events that initiate the inflammatory cascade that injures the endothelium and induces the development of ECM.

Chemoattractant-mediated leukocyte trafficking enables HIV dissemination from the genital mucosa.

HIV vaginal transmission accounts for the majority of newly acquired heterosexual infections. However, the mechanism by which HIV spreads from the initial site of viral entry at the mucosal surface of the female genital tract to establish a systemic infection of lymphoid and peripheral tissues is not known. Once the virus exits the mucosa it rapidly spreads to all tissues, leading to CD4+ T cell depletion and the establishment of a viral reservoir that cannot be eliminated with current treatments. Understanding the molecular and cellular requirements for viral dissemination from the genital tract is therefore of great importance, as it could reveal new strategies to lengthen the window of opportunity to target the virus at its entry site in the mucosa where it is the most vulnerable and thus prevent systemic infection. Using HIV vaginal infection of humanized mice as a model of heterosexual transmission, we demonstrate that blocking the ability of leukocytes to respond to chemoattractants prevented HIV from leaving the female genital tract. Furthermore, blocking lymphocyte egress from lymph nodes prevented viremia and infection of the gut. Leukocyte trafficking therefore plays a major role in viral dissemination, and targeting the chemoattractant molecules involved can prevent the establishment of a systemic infection.