CEP290 Exon 36 Skipping for Retinal Dystrophy Truncation
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Solution Overview
Problem
Current therapies for Leber congenital amaurosis (LCA10) caused by CEP290 mutations face challenges due to cargo capacity limitations of AAV vectors and risks of overexpression toxicity, particularly for mutations like c.4723A>T, which truncate the protein and are not effectively addressed by gene augmentation therapy.
Innovation Solution
An antisense oligonucleotide targeting the donor splice site of exon 36 in the CEP290 gene, with sequence SEQ ID NO:1, blocks recognition by the spliceosome to maintain the reading frame, allowing production of near full-length CEP290 protein and bypassing protein truncation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If gene augmentation therapy is used to treat CEP290 mutations, then the therapeutic approach can address protein truncation, but the cargo capacity of AAV vectors is exceeded due to the large CEP290 cDNA size (7.4 Kb)
Solution Approach 1:
The patent applies exon skipping to selectively exclude specific exons (such as exon 36) from the mature mRNA, thereby removing the portion containing the premature termination codon while retaining the functional coding regions. This segmentation approach allows the therapeutic gene to be delivered in a truncated but functional form that fits within AAV cargo capacity constraints.
Solution Approach 2:
The invention extracts and removes the problematic exon containing the mutation from the gene sequence through splice-modulating oligonucleotides. By taking out only the defective portion (exon 36 with the premature stop codon) rather than delivering the entire 7.4 Kb CEP290 gene, the therapy achieves its goal while staying within vector capacity limits.
2Reliability
If gene augmentation therapy is used for CEP290 mutations, then protein truncation can be addressed, but overexpression toxicity risks arise
Solution Approach 1:
Instead of delivering the complete CEP290 gene which would result in full-length protein overexpression and potential toxicity, the patent employs partial action by using exon skipping to produce a selectively truncated protein that lacks only the defective C-terminal portion. This partial restoration of function achieves therapeutic benefit while avoiding the harmful effects of complete gene overexpression.
3Reliability
If antisense oligonucleotide blocks exon 36 recognition by spliceosome, then reading frame is maintained and functional protein is produced, but the complexity of the treatment increases
Solution Approach 1:
The patent introduces an intermediary molecule - the splice-modulating oligonucleotide (SMO) or antisense oligonucleotide - that mediates the correction of the splicing defect. This intermediary blocks the recognition of exon 36 by the spliceosome, thereby preventing inclusion of the premature termination codon and maintaining the reading frame, ultimately leading to production of functional CEP290 protein without directly modifying the gene itself.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The antisense oligonucleotide strategy enhances the production of functional, minimally shortened CEP290 protein, reducing retinal damage and potentially improving visual function by maintaining the open reading frame and promoting the production of a functional protein.
Implementation Method 1
An antisense oligonucleotide targeting the donor splice site of exon 36 in the CEP290 gene, with sequence SEQ ID NO:1, blocks recognition by the spliceosome
Data Source
AI summary
The invention relates to the skipping of the CEP290 exon 36 in an individual suffering from a retinal dystrophy accounted for by a nonsense mutation or a premature termination codon generated by a frameshift mutation in exon 36 or an upstream exon, including the c.4723A>T, c.4771C>T, c.4714G>T, c.4786_4790del, c.4791_4794del, c.4732G>T, c.4625_4626insCATG (35), c.4792_4795del, c.4801C>T, c.4805C>T, or c.4811G>A mutations, to bypass protein truncation and lessen retinal damages. Here, studying fibroblasts from control individuals, and two patients carrying the CEP290 c.4723A>T nonsense mutation, they show low levels of spontaneous skipping of exon 36 arising from both endogenous basal skipping and mutation-induced skipping. The minimally shortened and mutation-free CEP290 mRNA produced by skipping of exon 36 in the fibroblasts of the two patients is translated into a protein isoform that localizes at the centrosome and allows the formation of primary cilia, yet with elongated axonemes. Using an AON consisting of a sequence set forth as SEQ ID NO: 1, complementary to a nucleic acid sequence of CEP290 pre-mRNA, wherein said AON targeting an mRNA encoding the donor splice site (H36D) is capable to alter splicing by blocking the recognition of exon 36 and bypass protein truncation while maintaining the open reading frame, leading to the production of near full-length CEP290 protein, they were able to increase the abundance of the alternatively spliced mRNA and shortened protein and to reduce axonemal length in patient cells.