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Opis: The experimental work of this thesis was performed at the International Centre for Genetic Engineering and Biotechnology (ICGEB) in the Human Molecular Genetics Group, under the scientific direction of Prof. Franco Pagani. The project was developed during the academic years 2014-2017. Modified U1 RNAs, also named Exon Specific U1s (ExSpeU1s) represent a novel class of small RNA-based molecules that correct exons splicing defects. To evaluate their therapeutic potential focused on Familial Dysautonomia (FD), a rare autosomal recessive disorder characterized by progressive degeneration of the sensory and autonomic nervous system. More than 99% of patients are homozygous for the T to C transition in position 6 of the IKBKAP intron 20 (c.2204+6T>C). This substitution modifies the exon 20 5’ splice site (5’ss) inducing exon skipping in a tissue-specific manner and reducing the total amount of IKAP protein. The molecular mechanisms underlying the IKBKAP mis-splicing are not completely clear and there are no effective treatments. In this thesis, I investigated the therapeutic potential of ExSpeU1s and the role of cis- and trans-acting factors that regulates IKBKAP splicing. Using a splicing functional assay, I identified ExSpeU1s that bind to intron 20 sequences and rescue the exon 20 skipping defect. Interestingly, their rescue activity was modulated by several splicing factors and requires a critical exonic splicing enhancer element. Transfection experiment showed the involvement of both enhancing (TIA1, PTBP1 and PTB4) and inhibitory (SRSF3, hnRNPA1, FOX and FUS) splicing factors in IKBKAP splicing. To better evaluate the ExSpeU1s therapeutic efficacy, I transduced FD patient’s fibroblasts with a lentiviral vector expressing the most active ExSpeU1. This resulted in a complete rescue of the exon skipping defect and improvement in IKAP protein expression. Most importantly, intraperitoneal delivery of ExSpeU1s by AAV9 into a transgenic mouse model, that recapitulates the tissue-specific mis-splicing seen in FD patients, corrected the aberrant splicing patterns in several tissues increasing the amount of the corresponding IKAP protein. All together, these results identify novel regulatory splicing factors involved in the IKBKAP exon 20 regulation and provide the proof of principle that ExSpeU1s delivered in vivo by AAV vectors represent a novel therapeutic strategy for FD.
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Povzetek najdenega: ...small RNA-based molecules that correct exons splicing defects. To evaluate their therapeutic potential focused on Familial...
Ključne besede: Familial Dysautonomia, IKBKAP, IKAP, splicing, splicing defects, ExSpeU1, U1 snRNA, mouse model, AAV
Objavljeno: 26.03.2018; Ogledov: 1815; Prenosov: 52
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