OTC intron 4 variations mediate pathogenic splicing patterns caused by the c.386G>A mutation in humans and spfash mice, and govern susceptibility to RNA-based therapies

Abstract

Background Aberrant splicing is a common outcome in the presence of exonic or intronic variants that might hamper the intricate network of interactions defining an exon in a specific gene context. Therefore, the evaluation of the functional, and potentially pathological, role of nucleotide changes remains one of the major challenges in the modern genomic era. This aspect has also to be taken into account during the pre-clinical evaluation of innovative therapeutic approaches in animal models of human diseases. This is of particular relevance when developing therapeutics acting on splicing, an intriguing and expanding research area for several disorders. Here, we addressed species-specific splicing mechanisms triggered by the OTC c.386G>A mutation, relatively frequent in humans, leading to Ornithine TransCarbamylase Deficiency (OTCD) in patients and spfash mice, and its differential susceptibility to RNA therapeutics based on engineered U1snRNA. Methods Creation and co-expression of engineered U1snRNAs with human and mouse minigenes, either wild-type or harbouring different nucleotide changes, in human (HepG2) and mouse (Hepa1-6) hepatoma cells followed by analysis of splicing pattern. RNA pulldown studies to evaluate binding of specific splicing factors. Results Comparative nucleotide analysis suggested a role for the intronic +10-11 nucleotides, and pull-down assays showed that they confer preferential binding to the TIA1 splicing factor in the mouse context, where TIA1 overexpression further increases correct splicing. Consistently, the splicing profile of the human minigene with mouse +10-11 nucleotides overlapped that of mouse minigene, and restored responsiveness to TIA1 overexpression and to compensatory U1snRNA. Swapping the human +10-11 nucleotides into the mouse context had opposite effects. Moreover, the interplay between the authentic and the adjacent cryptic 5′ss in the human OTC dictates pathogenic mechanisms of several OTCD-causing 5′ss mutations, and only the c.386+5G>A change, abrogating the cryptic 5′ss, was rescuable by engineered U1snRNA. Conclusions Subtle intronic variations explain species-specific OTC splicing patterns driven by the c.386G>A mutation, and the responsiveness to engineered U1snRNAs, which suggests careful elucidation of molecular mechanisms before proposing translation of tailored therapeutics from animal models to humans.

Development of Engineered-U1 snRNA Therapies: Current Status

“…However, the existence of species-specific splicing profiles can constitute a challenge to the development and validation of these therapies. This is elegantly presented in two recent studies developed by the same group [62,63], both targeting a splicing variant (c.386G>A) in the OTC gene, the gene mutated in Ornithine transcarbamylase deficiency (OTCD). The presence of the c.386G>A variant in the last base of exon 4 leads to aberrant splicing but with a substantially different impact between humans and mice.…”

“…The same was observed in vivo upon delivery via an AAV8 of the same ExSpeU1 to a mouse model carrying the c.386G>A variant (spf ash mouse). In a second study [63], the same group dissected the molecular mechanisms mediating the differences in the pathogenic splicing patterns between mice and humans in the presence of the c.386G>A variant and how it can influence susceptibility to RNA-based therapies. Actually, they tested in vitro several engineered U1 snRNAs for the human sequence and none of those U1 snRNAs improved the selection of the authentic mutated 5 ss, because while decreasing exon skipping, they further promoted the usage of the adjacent cryptic 5 ss.…”

Counteracting the Common Shwachman–Diamond Syndrome-Causing SBDS c.258+2T>C Mutation by RNA Therapeutics and Base/Prime Editing

“…Among RNA therapeutics acting on pre-mRNA splicing, modified U1snRNA and antisense oligonucleotides (AON) represent some of the most attractive, with numerous experimental evidence in cellular and animal models [ 4 , 5 , 6 , 7 , 8 , 9 , 10 ] and also in humans [ 11 , 12 ]. In particular, either the compensatory or the exon-specific (ExSpe) U1 variants of U1snRNA, the RNA component of the spliceosomal U1 ribonucleoprotein (U1snRNP) mediating 5′ss recognition in the earliest splicing steps [ 13 ], were shown to rescue splicing mutations both in cellular and animal models of human diseases [ 14 , 15 , 16 , 17 , 18 , 19 ].…”