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MODIFIED U1 RNAs AS SPLICING CORRECTORS IN HUMAN GENETIC DISORDERSKatarzyna Rajkowska, 2018, doctoral dissertation
Abstract: 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.
Keywords: Familial Dysautonomia, IKBKAP, IKAP, splicing, splicing defects, ExSpeU1, U1 snRNA, mouse model, AAV
Published in RUNG: 26.03.2018; Views: 4982; Downloads: 127
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