Prime editing restores RNA splicing in a minigene carrying a familial dysautonomia variant
The causal variant leads to exon 20 skipping
A cell-based study reports that prime editing restored RNA splicing disrupted by a familial dysautonomia (FD) causal variant. In a variant minigene introduced into HEK293T cells, exon 20 inclusion rose from 19±2% to 60±3% after PE3. A minigene is an experimental construct containing part of a gene that is used to analyze splicing. The findings establish proof of concept only in this model.
FD is a rare autosomal recessive disease characterized by progressive degeneration of sensory and autonomic nerves. According to the paper, the intronic ELP1 variant c.2204+6T>C is causal in 99.8% of patients. The variant causes exon 20 to be skipped during RNA splicing, reducing ELP1 expression. Normal transcript proportions and ELP1 expression are particularly low in nervous tissue.
Substances that modulate splicing have already been studied in cells and animal models. The researchers in this study tested gene-editing strategies that either directly reverse the causal variant or alter a splicing-suppressing site.
Comparing direct correction with alteration of a silencer element
Prime editing (PE) changes a target DNA sequence without a DNA double-strand break or separate donor DNA. PE2 uses an editing protein and a prime editing guide RNA. PE3 adds a guide that induces a single-strand nick on the opposite DNA strand.
The researchers transiently introduced the variant minigene pTB-IKAP and editing components together into HEK293T cells. They extracted RNA 72 hours later and analyzed it by reverse transcription polymerase chain reaction (RT-PCR). Exon 20 inclusion was 19±2% with the variant minigene alone, 48±3% with PE2, and 60±3% with PE3. These values are the mean and standard deviation from three independent experiments. The 19% figure represents the comparison condition in this experiment, not the performance of an existing treatment.
DNA analyses also confirmed correction of the variant with PE3. The researchers performed restriction enzyme analysis and Sanger sequencing, and estimated the correction rate at about 10% using denaturing capillary electrophoresis. This is a DNA-level measurement in the minigene experiment and should be distinguished from the 60% RNA exon inclusion rate. The authors cited assay sensitivity and repeated RNA production from corrected DNA among the reasons for the difference between the two results.
A second strategy altered the splicing silencer ESS2 within exon 20. Introducing a synonymous A>G substitution that does not change the amino acid yielded exon inclusion rates of 30±4% with PE2 and 50±4% with PE3. In this approach, splicing was restored by changing the silencer element rather than directly reversing the causal variant.
Disease-model validation is needed to assess therapeutic potential
The study provides proof of concept for two approaches: direct correction and ESS2 alteration. However, the experiments used only a minigene, which does not fully reflect the genomic context. These results cannot establish effects in patient-derived cells or the nervous system in vivo, restoration of ELP1 protein, or long-term durability.
The paper discusses therapeutic potential based on a prior mouse study in which modest restoration of ELP1 expression alleviated disease. However, expression level and DNA correction rate are distinct measurements. There is no basis for interpreting the approximately 10% correction rate reported here as a threshold that guarantees a cure or treatment benefit for patients. The possibility of editing at sites other than the target was also assessed only by computational prediction. Further studies need to evaluate efficacy in disease models, delivery of the editing tools to the nervous system, and safety through experiments.
Source: Original article in Orphanet Journal of Rare Diseases.
Familial Dysautonomia (FD; OMIM #223900) is characterized by a nearly universal (99.8%) occurrence of FD due to the c.2204+6T>C intronic mutation in the ELP1/IKAP gene, leading to exon 20 skipping and reduced ELP1 protein levels. Although splicing modulation therapies have shown partial efficacy, permanent genetic correction remains unattainable. This study presents the first application of Prime Editing (PE) to rectify the splicing defect responsible for FD. Utilizing a mutant exon-trapping minigene (pTB-IKAP) transiently transfected with PE2 or PE3 components in HEK293T cells, we observed a substantial increase in exon 20 inclusion from 19 ± 2% to 48 ± 3% and further to 60 ± 3% with PE3. Genetic validation through restriction fragment length polymorphism analysis and Sanger sequencing confirmed allele correction, achieving approximately 10% genomic editing efficiency with PE3. Additionally, targeting ESS2 via a silent A>G substitution restored exon inclusion to 50 ± 4%. These findings establish PE3, particularly, as a highly effective strategy for correcting or bypassing the c.2204+6T>C mutation and restoring proper splicing in FD. Considering that modest increases in ELP1 expression (5-10% of wild-type levels) significantly alleviate FD severity in animal models, our results underscore PE3 as a promising curative approach, paving the way for future ex vivo and in vivo therapeutic applications.
In the same variant minigene, this study tested both direct correction of the causal base and alteration of a splicing silencer. Follow-up FD research could apply both strategies to patient-derived cells and disease models to compare whether exon inclusion, ELP1 protein expression, and nervous system function recover together. Measuring DNA correction and RNA exon inclusion separately can help clarify how editing relates to splicing restoration. Developing a therapy will also require validation of nervous system delivery, durability of the effect, and editing at sites other than the target. These findings provide a starting point for that follow-up work; they do not demonstrate symptom improvement in patients.