Casarosa, S. | Search bioRxiv

bioRxiv2021

Exon skipping via chimeric antisense U1 snRNAs to correct Retinitis Pigmentosa GTPase-Regulator (RPGR) splice defect.

Inherited retinal dystrophies are caused by mutations in more than 250 genes, each of them carrying several types of mutations that can lead to different clinical phenotypes. Mutations in Retinitis Pigmentosa GTPase-Regulator (RPGR) cause X-linked Retinitis pigmentosa (RP). A nucleotide substitution in intron 9 of RPGR causes the increase of an alternatively spliced isoform of the mature mRNA, bearing exon 9a (E9a). This introduces a stop codon, leading to truncation of the protein. Aiming at restoring impaired gene expression, we developed an antisense RNA-based therapeutic approach for the skipping of RPGR E9a. We designed a set of specific U1 antisense snRNAs (U1_asRNAs) and tested their efficacy in vitro, upon transient co-transfection with RPGR minigene reporter systems in HEK-293T and PC-12 cell lines. We thus identified three chimeric U1_asRNAs that efficiently mediate E9a skipping, correcting the genetic defect. Unexpectedly, the U1-5antisense construct, which exhibited the highest exon-skipping efficiency in PC-12 cells, induced E9a inclusion in HEK-293T cells, indicating caution in the choice of preclinical model systems when testing RNA splicing-correcting therapies. Our data provide a proof of principle for the application of U1_snRNA exon skipping-based approach to correct splicing defects in RPGR.

molecular biology↗

bioRxiv2021

Expression and protein sequence analyses of zebrafish impg2a and impg2b, two proteoglycans of the interphotoreceptor matrix

Photoreceptor outer segments projecting from the surface of the neural retina toward the retinal pigment epithelium (RPE) are surrounded by a carbohydrate-rich matrix, the interphotoreceptor matrix (IPM) [1,2]. This extracellular compartment is necessary for physiological retinal function. However, specific roles for molecules characterizing the IPM have not been clearly defined [3]. Recent studies have found the presence of nonsense mutations in the interphotoreceptor matrix proteoglycan 2 (IMPG2) gene in patients affected by autosomal recessive Retinitis Pigmentosa (arRP) [4,5] and autosomal dominant and recessive vitelliform macular dystrophy (VMD) [6,7]. The gene encodes for a proteoglycan synthesized by photoreceptors and secreted in the IPM. However, little is known about the function and structure of this protein. We used the teleost zebrafish (D.rerio) as a model to study IMPG2 expression both during development and in adulthood, as its retina is very similar in humans [8]. In zebrafish, there are two IMPG2 proteins, IMPG2a and IMPG2b. We generated a phylogenetic tree based on IMPG2 protein sequence similarity among different vertebrate species, showing a significant similarity despite the evolutionary distance between humans and teleosts. In fact, human IMPG2 and D.rerio IMPG2a and IMPG2b share conserved SEA and EGF-like domains. Homology models of these domains were obtained by using the iTasser server. Finally, expression analyses of impg2a and impg2b during development and in the adult fish showed expression of both mRNAs starting from 3 days post fertilization (dpf) in the outer nuclear layer of zebrafish retina that continues throughout adulthood. This data lays the groundwork for the generation of novel and most needed animal models for the study of IMPG2-related inherited retinal dystrophies.

developmental biology↗