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Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy.
Reetta A E Eriksson1,2, Tiina Nieminen1, Lionel Galibert1
1Kuopio Center for Gene and Cell Therapy, Kuopio, Finland.
Frontiers in Medicine
|December 30, 2022
Summary
Researchers developed a novel gene therapy method using tetracycline-inducible riboswitches for precise temporal control of therapeutic gene expression. This system offers dose-dependent, reversible gene regulation within hours, enhancing gene therapy applications.
Area of Science:
- Molecular Biology
- Gene Therapy
- RNA Therapeutics
Background:
- Temporal regulation of gene expression is crucial for effective gene therapy.
- Riboswitches are RNA elements that can control gene expression in response to small molecules.
- Existing riboswitches require optimization for robust and controllable transgene expression.
Purpose of the Study:
- To compare different tetracycline and toyocamycin-inducible ON-riboswitches for drug-responsive transgene expression.
- To optimize riboswitch-controlled gene expression cassettes for enhanced therapeutic applications.
- To evaluate the impact of expression cassette elements on riboswitch functionality.
Main Methods:
- Screening of multiple tetracycline-dependent and toyocamycin-dependent ON-riboswitches.
- Application of the best-performing riboswitch (K19) to various transgenes using plasmid and adeno-associated virus (AAV) vectors.
- Optimization of gene expression cassettes using multiple riboswitches and linker sequences.
- Assessment of gene expression induction, dose-dependency, reversibility, and kinetics.
- Evaluation of promoter independence and the effect of WPRE elements.
Main Results:
- The tetracycline-dependent K19 riboswitch demonstrated superior control over transgene expression.
- Gene expression was induced 6- to 10-fold, showing dose-dependency and reversibility within hours.
- Dual or triple riboswitches, particularly with a linker sequence, reduced leakiness and improved dynamic range.
- Riboswitch function was independent of the promoter, but WPRE elements negatively impacted functionality.
- An optimized dual riboswitch construct at the 3' end of the transgene with a linker was developed.
Conclusions:
- The K19 riboswitch provides effective temporal control for gene therapy applications.
- Optimized riboswitch expression cassettes significantly enhance dynamic range and reduce leakiness.
- Understanding riboswitch function and expression cassette design is critical for advancing gene therapy.
- This study provides a foundation for developing advanced riboswitch-based gene regulation systems.
