Suicide Gene Intron Design for Tumor-Specific Abnormal Splicing
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Solution Overview
Problem
Existing treatments for myelodysplastic syndrome (MDS), myeloid leukemia, and uveal melanoma are ineffective due to abnormal splicing induced by mutations in spliceosome constituents like SF3B1, SRSF2, and ZRSR2, leading to oncogenic transformation and abnormal protein expression.
Innovation Solution
A recombinant DNA encoding a suicide gene with an intron sequence that undergoes abnormal splicing only in tumor cells, expressing proteins like HSV-TK or iCasp9 to inhibit cell proliferation, while maintaining normal splicing in healthy cells, using expression vectors and promoters to control transcription.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If a conventional gene therapy approach is used to treat tumor cells, then normal cells may be affected by the treatment, but selective targeting of tumor cells without affecting normal cells is difficult to achieve
Solution Approach 1:
The patent applies local quality by designing an intron sequence with specific abnormal splicing sites that are only recognized and processed by mutant spliceosomes in tumor cells. The intron contains specific sequence motifs (such as 5' splice site, 3' splice site, and branch point sequences) that differ from normal introns, creating a localized molecular signature that confers selectivity. This allows the suicide gene to be expressed only in cells with the specific splicing mutation, achieving local quality in terms of cellular specificity.
Solution Approach 2:
The patent utilizes parameter changes by exploiting the altered splicing parameters (sequence recognition, splice site selection) in mutant spliceosomes. The intron sequence is designed with modified splicing parameters that match the altered recognition patterns of mutant spliceosomes (e.g., SF3B1, SRSF2, ZRSR2 mutations). This parameter mismatch ensures that only tumor cells with the specific splicing defect can process the intron, while normal cells with wild-type spliceosomes cannot, thereby achieving selective targeting.
2Productivity
If the suicide gene is always expressed to kill tumor cells, then normal cells expressing the gene would also die, but the gene must be conditionally expressed only in tumor cells
Solution Approach 1:
The patent applies preliminary action by pre-installing the intron sequence within the suicide gene construct before introduction into cells. This intron acts as a conditional switch that is already in place but remains inactive in normal cells. Only when the construct enters tumor cells with the specific splicing mutation does the intron get abnormally spliced and trigger gene expression. This preliminary positioning of the splicing-dependent element ensures that the toxic effect is prepared but only activated under the correct conditions.
Solution Approach 2:
The intron sequence serves as an intermediary element that mediates between the suicide gene and the mutant spliceosome. It translates the presence of the splicing mutation into gene expression: the mutant spliceosome recognizes and processes the abnormal intron, which then allows removal of the intron sequence and enables translation of the suicide gene. This intermediary mechanism decouples gene expression from direct promoter control, instead coupling it to the splicing status, thereby providing conditional expression.
3Reliability
If the intron sequence is designed for abnormal splicing in tumor cells, then normal splicing in healthy cells may be disrupted, but the intron must be processed correctly in normal cells to avoid toxicity
Solution Approach 1:
The patent applies asymmetry by designing the intron sequence with asymmetric splicing recognition features that create different outcomes in normal versus tumor cells. The intron contains specific sequence asymmetries (such as atypical 5' or 3' splice sites, unusual branch points) that are poorly recognized by wild-type spliceosomes but well-recognized by mutant spliceosomes. This asymmetric design ensures directional specificity: normal cells fail to process the intron (leaving it retained and blocking translation), while tumor cells successfully process it (enabling gene expression).
Data Source
AI summary
An object of the invention is to provide a novel pharmaceutical composition. The pharmaceutical composition of the disclosure contains a DNA encoding a suicide gene having at least one intron sequence. The intron sequence has a donor sequence or an acceptor sequence to be used in a tumor cell with abnormal splicing not in a normal cell. In a transcript of the DNA, the suicide gene is expressed when the intron is abnormally spliced and the suicide gene is not expressed when the intron is not abnormally spliced.