RNA Trans-Splicing Molecule for HERV Targeting
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Solution Overview
Problem
Current RNA trans-splicing technologies lack specificity and efficiency in targeting human endogenous retrovirus (HERV) pre-mRNA for selective killing of cancer cells, leading to potential off-target effects and reduced efficacy.
Innovation Solution
Development of an RNA trans-splicing molecule (RTM) with a binding region specific for HERV pre-mRNA, a trans-splicing domain, and a coding sequence for a suicide protein, such as HSV thymidine kinase, which mediates trans-splicing to express the suicide protein in HERV-expressing cells, enabling targeted cell death upon activation by a cytotoxic compound like ganciclovir.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current RNA trans-splicing technologies are used to target HERV pre-mRNA, then cell death is achieved, but specificity is reduced leading to off-target effects
Solution Approach 1:
The RTM is divided into distinct functional modules: a binding region (20-50 nucleotides) that specifically recognizes HERV pre-mRNA sequences, a trans-splicing domain that catalyzes the splicing reaction, and a coding sequence for the suicide protein. This segmentation allows the binding region to be optimized for high specificity to HERV sequences while the trans-splicing domain performs the catalytic function, reducing off-target effects.
Solution Approach 2:
The binding region of the RTM is designed with specific nucleotide sequences (20-50 nucleotides) that complementarily bind to unique HERV pre-mRNA sequences. This local optimization of binding affinity and specificity ensures that trans-splicing occurs only at the intended target site on HERV pre-mRNA, preventing off-target effects while maintaining efficient cell death induction.
2Productivity
If RNA trans-splicing is used to deliver suicide gene, then cancer cell killing is achieved, but efficiency is reduced
Solution Approach 1:
The RTM is designed to undergo trans-splicing with HERV pre-mRNA as a preliminary step before translation of the suicide protein. The binding region first specifically binds to HERV pre-mRNA, then the trans-splicing domain catalyzes the joining of the RTM coding sequence to the HERV pre-mRNA, creating a chimeric mRNA that is then translated. This preliminary trans-splicing action ensures efficient and targeted delivery of the suicide gene to cancer cells expressing HERV.
3Ease of manufacture
If trans-splicing domain is included in RTM, then suicide protein expression is achieved, but device complexity increases
Solution Approach 1:
The trans-splicing domain in the RTM serves multiple functions: it catalyzes the trans-splicing reaction between the RTM and HERV pre-mRNA, ensures proper positioning of the coding sequence for translation, and facilitates the creation of the chimeric mRNA molecule. By incorporating this multi-functional domain, the RTM achieves suicide protein expression without requiring multiple separate molecular components, thereby managing complexity while maintaining functionality.
Data Source
AI summary
This invention relates to an RNA trans-splicing molecule (RTM) that targets human endogenous retrovirus (HERV) pre-mRNA. The RTM comprises (i) a binding region specific for a HERV pre-mRNA, (ii) a trans-splicing splice domain and (ill) a coding sequence for a suicide protein. The binding region of the RTM binds to HERV pre-mRNA in a cell, such that the coding sequence is trans-spliced through the trans- splicing domain with the HERV pre-mRNA, resulting in a chimeric mRNA causing the suicide protein to be expressed in the cell. RTMs of the invention may be useful in selectively killing cells that express HERV genes, for example cancer cells. RTMs, encoding nucleic acids, methods of treatment and associated methods and uses are provided.