A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases

Schneider, Nina; Steinberg, Ricky; Ben-David, Amit; Valensi, Johanna; David-Kadoch, Galit; Rosenwasser, Zohar; Banin, Eyal; Levanon, Erez Y.; Sharon, Dror; Ben-Aroya, Shay

doi:10.1016/j.omtn.2024.102130

Cited by 4 publications

(3 citation statements)

References 51 publications

(62 reference statements)

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“…While NMD does not seem to affect KIZ transcripts in fibroblasts (and potentially in the retina), however, the effect of c.226C>T on splicing might limit the number of transcripts for readthrough drugs. RNA editing, which is another mutation-specific therapy that is based on the adenosine deaminases acting on RNA (ADAR) enzyme, is usually performed at the pre-mRNA stage and is therefore less affected by NMD, however editing levels reported thus far are for this mutation are relatively low and need to be improved before considered as a potential therapy [33]. As such, the most suitable therapeutical approach for KIZ is currently gene augmentation therapy, which has not yet been developed for this particular gene.…”

Section: Discussionmentioning

confidence: 99%

Genetic and Clinical Analyses of the KIZ-c.226C>T Variant Resulting in a Dual Mutational Mechanism

Sundaresan,

Rivera,

Obolensky

et al. 2024

Preprint

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Retinitis pigmentosa (RP) is a heterogeneous inherited retinal disorder. Mutations in KIZ cause autosomal recessive (AR) RP. We aimed to characterize the genotype, expression pattern, and phenotype in a large cohort of KIZ cases. Sanger and whole exome sequencing were used to identify KIZ variants. Medical records were reviewed and analyzed. Thirty one patients with biallelic KIZ mutations were identified: 28 homozygous for c.226C>T (p.R76*), 2 compound heterozygous for p.R76* and c.3G>A (p.M1?), and one homozygous for c.247C>T (p.R83*). c.226C>T is a founder mutation among patients of Jewish descent. Clinical parameters were less severe in KIZ compared to DHDDS and FAM161A cases. RT-PCR analysis in fibroblast cells revealed the presence of four different transcripts in both WT and mutant samples with a lower percentage of the WT transcript in patients. Sequence analysis identified an exonic sequence enhancer (ESE) that includes the c.226 position which is affected by the mutation. KIZ mutations are an uncommon cause of IRD worldwide, but are not rare among Ashkenazi Jews. Our data indicate that p.R76* affect an ESE which in turn results in pronounced skipping of exon 3. Therefore, RNA-based therapies might show low efficacy since the mutant transcripts is spliced.

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Section: Discussionmentioning

confidence: 99%

Genetic and Clinical Analyses of the KIZ-c.226C>T Variant Resulting in a Dual Mutational Mechanism

Sundaresan,

Rivera,

Obolensky

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…While NMD does not seem to affect KIZ transcripts in fibroblasts (and potentially in the retina), the effect of c.226C>T on splicing might limit the number of transcripts for readthrough drugs. RNA editing, which is another mutation-specific therapy that is based on the adenosine deaminases acting on RNA (ADAR) enzyme [ 43 ], is usually performed at the pre-mRNA stage [ 44 ] and is therefore less affected by NMD; however, editing levels reported thus far for this mutation are relatively low and need to be improved before being considered as a potential therapy [ 45 ]. As such, the most suitable therapeutical approach for KIZ is currently gene augmentation therapy [ 46 , 47 , 48 ], which has not yet been developed for this particular gene.…”

Section: Discussionmentioning

confidence: 99%

Genetic and Clinical Analyses of the KIZ-c.226C>T Variant Resulting in a Dual Mutational Mechanism

Sundaresan,

Rivera,

Obolensky

et al. 2024

Genes

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Retinitis pigmentosa (RP) is a heterogeneous inherited retinal disorder. Mutations in KIZ cause autosomal recessive (AR) RP. We aimed to characterize the genotype, expression pattern, and phenotype in a large cohort of KIZ cases. Sanger and whole exome sequencing were used to identify the KIZ variants. Medical records were reviewed and analyzed. Thirty-one patients with biallelic KIZ mutations were identified: 28 homozygous for c.226C>T (p.R76*), 2 compound heterozygous for p.R76* and c.3G>A (p.M1?), and one homozygous for c.247C>T (p.R83*). c.226C>T is a founder mutation among patients of Jewish descent. The clinical parameters were less severe in KIZ compared to DHDDS and FAM161A cases. RT-PCR analysis in fibroblast cells revealed the presence of four different transcripts in both WT and mutant samples with a lower percentage of the WT transcript in patients. Sequence analysis identified an exonic sequence enhancer (ESE) that includes the c.226 position which is affected by the mutation. KIZ mutations are an uncommon cause of IRD worldwide but are not rare among Ashkenazi Jews. Our data indicate that p.R76* affect an ESE which in turn results in the pronounced skipping of exon 3. Therefore, RNA-based therapies might show low efficacy since the mutant transcripts are spliced.

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“…For instance, Diaz Quiroz et al developed an in vitro assay to select optimized guide RNAs to target an adenosine residue within a PTC [ 88 ]. On the other hand, Schneider et al presented a yeast system to identify the best ADAR targets that are more suitable for A-to-I editing [ 89 ]. The authors also discovered the most efficient guide RNAs to edit retinal disease targets carrying nonsense mutations using this assay.…”

Section: Nonsense Suppression Strategiesmentioning

confidence: 99%

Therapeutic Nonsense Suppression Modalities: From Small Molecules to Nucleic Acid-Based Approaches

Morais,

Zhang,

2024

Biomedicines

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Nonsense mutations are genetic mutations that create premature termination codons (PTCs), leading to truncated, defective proteins in diseases such as cystic fibrosis, neurofibromatosis type 1, Dravet syndrome, Hurler syndrome, Beta thalassemia, inherited bone marrow failure syndromes, Duchenne muscular dystrophy, and even cancer. These mutations can also trigger a cellular surveillance mechanism known as nonsense-mediated mRNA decay (NMD) that degrades the PTC-containing mRNA. The activation of NMD can attenuate the consequences of truncated, defective, and potentially toxic proteins in the cell. Since approximately 20% of all single-point mutations are disease-causing nonsense mutations, it is not surprising that this field has received significant attention, resulting in a remarkable advancement in recent years. In fact, since our last review on this topic, new examples of nonsense suppression approaches have been reported, namely new ways of promoting the translational readthrough of PTCs or inhibiting the NMD pathway. With this review, we update the state-of-the-art technologies in nonsense suppression, focusing on novel modalities with therapeutic potential, such as small molecules (readthrough agents, NMD inhibitors, and molecular glue degraders); antisense oligonucleotides; tRNA suppressors; ADAR-mediated RNA editing; targeted pseudouridylation; and gene/base editing. While these various modalities have significantly advanced in their development stage since our last review, each has advantages (e.g., ease of delivery and specificity) and disadvantages (manufacturing complexity and off-target effect potential), which we discuss here.

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A pipeline for identifying guide RNA sequences that promote RNA editing of nonsense mutations that cause inherited retinal diseases

Cited by 4 publications

References 51 publications

Genetic and Clinical Analyses of the KIZ-c.226C>T Variant Resulting in a Dual Mutational Mechanism

Genetic and Clinical Analyses of the KIZ-c.226C>T Variant Resulting in a Dual Mutational Mechanism

Genetic and Clinical Analyses of the KIZ-c.226C>T Variant Resulting in a Dual Mutational Mechanism

Therapeutic Nonsense Suppression Modalities: From Small Molecules to Nucleic Acid-Based Approaches

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