įollowing transfection of the vector into HEK293T cells, AONs are co-transfected, and the splicing correction can be assessed at RNA level. Technology is a suitable approach that has been shown to produce reliable results. For the early investigation of possible causative variantĪffecting pre-mRNA splicing in retinal genes and subsequent assessment of AON potency, midigene Generating a reliable artificial splicing system is of major importance when it comes to the development of a potential therapeutic They are used in the field of IRDs, as well as in other geneticĭiseases as the purpose of AONs is not limited to splice-switch function only. AONs are chemically modified RNA molecules that have the ability of modulating splicing by binding to pre-mRNA and interfering with the spliceosome. AON-based therapies represent a very effective approach to target mis-splicing mutations. ![]() Molecules such as antisense oligonucleotides In addition, validated midigenes harboring splicing variants represent a system for reliable and relatively quick identification of potential therapeutic The suitability of midigenes for some cell lines reduced the complexity of the study of pathologic deep-intronic variants (among other types of mutations) and their effect on splicing. , as the entire 128-kb gene has been successfully spanned in a set of midigenes representing an alternative to the impossibility of cloning such a large genomic region in a single vector. These “artificial” genomic vectors were shown to be very valuable when it comes to ABCA4 An even longer genomic content allows for inclusion of long-range cis-acting elements to more accurately reflect the dynamics of splicing. In general, splice vectors or midigenes refer to a specific type of vector containing a large genomic region that allows to study the splicing processes between the included exons. Engineering and use of multi-exon splice vectors have been proven to be extremely effective in gaining insight into IRDs and, more specifically, STDG1. In parallel, great efforts have been made to develop a more cost-effective and less time-consuming strategy to reach the same goal, by trying to mimic the pathological situation in a reliable and controlled manner. Variants of interest, as these represent the right cell type(s) with the proper geneticĬontext. Currently, the ideal model to assess and correct mis-splicing mutations in STGD1 are iPSC-derived retina-like cells (such as photoreceptor precursor cells (PPCs) or retinal organoids) from a patient harboring the genetic In vitro functional assays can provide insight into the underlying mechanisms behind aberrant splicing and identify the mutations that interfere with this process. the second variantĬannot be identified in the coding regions of adenosine triphosphate (ATP) binding cassette type A4 ( ABCA4ĭatabase, mis-splicing mutations have been estimated to represent 8.6% of the total mutations underlying inherited diseases (23,868/275,716). In the field of inherited retinal diseases (IRDs), a common autosomal recessive condition known as Stargardt disease (STDG1) lacks the bi-allelic molecular diagnosis in 30% of cases, i.e. This is especially relevant for intronic variants, as many of them have an unknown functional significance. ![]() , it is crucial to be able to correctly identify and distinguish disease-causing variants from single nucleotide polymorphisms (SNPs). Variants from coding regions to the entire genome. Technologies such as next generation sequencing (NGS) expanded the discovery of genetic Here, we describe how to engineer splicing vectors, validate the reliability and reproducibility of alternative cellular systems, assess pre-mRNA splicing defects involved in IRD, and finally correct those by using antisense oligonucleotide-based strategies. the retinal tissue, cells of which cannot be easily obtained and/or cultured. For some genetic disorders, including inherited retinal diseases (IRDs), reproducing splicing dynamics in vitro is a challenge due to the specific environment provided by, e.g. These changes often lead to the formation of aberrant transcripts that can induce nonsense-mediated decay, and a subsequent lack of functional protein. A significant proportion of mutations underlying genetic disorders affect pre-mRNA splicing, generally causing partial or total skipping of exons, and/or inclusion of pseudoexons.
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