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using lipid nanoparticle delivery or encapsulation within dendrimer complexes with a small positive charge 59. Recent studies reported positive results of intranasal delivery of miRNAs on seizure development in experimental models of epilepsy 51, 60. Currently, biotechnology companies are working on the development of RNA-based therapeutics for the treatment of epilepsy, which brings the clinical applicability one step closer 61, 62.
Extracellular miRNAs and circular RNAs
In addition to tissue expression throughout the body in various cell types, miRNAs have also been detected in various biological fluids. These extracellular miRNAs can be assessed in blood, plasma, and serum and altered expression patterns were found in different pathologies 63. Circulating miRNAs may form a novel way of intercellular com- munication 64. It was recently reported that miRNAs can be excreted by glia to modulate synaptic genes in neurons in response to inflammation, indicating a novel pathogenic mechanism where miRNAs contribute to modulation of excitatory signalling in disease 65. miRNAs may either be actively secreted either via exosomes, or via coupling with RNA- binding proteins 66. Exosomes are able to cross the blood-brain barrier and may function as useful disease biomarkers in epilepsy 67, 68. For example, studies in experimental models of epilepsy have identified clusters of plasma miRNAs that were differentially expressed, some even before seizure onset, indicating a potential biomarker of epileptogenesis 69-71. Additionally, deregulated expression of several miRNAs was confirmed in serum of epi- lepsy patients 68. For example, miR146a and miR106b were recently shown to be both upregulated in serum from patients with general or focal epilepsy and the expression was correlated with seizure severity, indicating that these miRNAs can be potentially used for epilepsy diagnosis and/or to guide treatment 72. Currently, we are performing a large-scale biomarker study in a group of 101 TSC patients in a European Commission 7th Framework Programme funded consortium (EPISTOP). Of these young patients, clinical records are collected on a regular basis and serum is obtained at different time points: at a few weeks of age, and at 6 and 24 months of age. Additionally, serum samples are also collected once EEG abnormalities are observed and at clinical manifestation of seizures. Using a custom miRNA PCR panel based on preliminary sequencing results of serum of a small, selected cohort of patients, we aim to identify deregulated miRNAs and will correlate expression data to clinical information like development of seizures, autism spectrum characteristics and response to anti-epileptic drugs, in order to identify poten- tial biomarkers. Furthermore, a new interesting field of non-coding small RNAs is circu- lar RNAs (circRNAs), that are produced by back-splicing of precursor mRNAs and can modulate gene expression by regulating miRNAs. Novel sequencing approaches allowed the detection of over thousands circRNAs in the brain, where they can act as miRNA sponges by competing with mRNAs for miRNA binding sites 73. circRNAs are detectable in blood and may be used as disease biomarkers 74, 75. Currently, we are investigating the expression of circRNAs in response to inflammatory stimuli in human astrocytes derived from patients with TSC or vanishing white matter, a disorder which is, in contrast to TSC, known for its impaired maturation of astrocytes and low levels of gliosis 76, 77. We aim to identify circRNAs related to inflammatory mechanisms and their function, in order to find new therapeutic strategies for restoring altered gene expression in these patholo- gies. This would allow the development of a novel, additional therapeutic approach using
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