Periodic Reporting for period 1 - UNLEASH (Harnessing the splicing code for targeted control of gene expression)
Reporting period: 2023-06-01 to 2024-11-30
Harnessing the splicing code for targeted control of gene expression
Alternative splicing of messenger RNA precursors plays important roles in tissue-specific gene regulation and biological regulatory mechanisms, where it can radically alter protein expression, cell phenotypes and physiological responses. Altered splicing also contributes to disease mechanisms, ranging from neurodegeneration to cancer. Drugs modulating AS have recently provided the first therapy for Spinal Muscular Atrophy, a common genetic disorder, illustrating the huge potential for treating many other diseases of unmet need, if only we understood the mechanisms controlling splice site selection and how to regulate them with small molecules. Unfortunately, despite decades of research, a comprehensive understanding of the mechanisms that control AS specificity is lacking. This gap in basic knowledge limits future opportunities to harness splicing modulators as tools to study gene function, novel therapeutics or other biotech applications. The UNLEASH Project addresses head-on major technical challenges that have limited progress in the alternative splicing field. Building on extensive preliminary data, we will use a multidisciplinary approach that combines chemical, structural, cellular, systems biology and computational methods to characterize mechanisms of splice site selection and identify targets for tool compounds that will modulate these mechanisms. The outcomes will define key regulatory sequences, splicing factors and molecular interactions involved, thereby illuminating how the splicing machinery efficiently accommodates, yet also discriminates between, a wide range of splice site sequences. Our primary goal is to answer the central question, ‘Is it possible to modulate splicing with high specificity using small molecules?’ Success will transform our basic understanding of human gene expression and unleash major opportunities for Pharma to develop new therapeutics harnessing modulation of splice site selection. In summary, we expect the impact of this project to be a step-change advance in our mechanistic understanding of pre-mRNA splicing and how this can be regulated and harnessed for future therapeutic and biotechnological applications by controlling protein expression.
Alternative splicing of messenger RNA precursors plays important roles in tissue-specific gene regulation and biological regulatory mechanisms, where it can radically alter protein expression, cell phenotypes and physiological responses. Altered splicing also contributes to disease mechanisms, ranging from neurodegeneration to cancer. Drugs modulating AS have recently provided the first therapy for Spinal Muscular Atrophy, a common genetic disorder, illustrating the huge potential for treating many other diseases of unmet need, if only we understood the mechanisms controlling splice site selection and how to regulate them with small molecules. Unfortunately, despite decades of research, a comprehensive understanding of the mechanisms that control AS specificity is lacking. This gap in basic knowledge limits future opportunities to harness splicing modulators as tools to study gene function, novel therapeutics or other biotech applications. The UNLEASH Project addresses head-on major technical challenges that have limited progress in the alternative splicing field. Building on extensive preliminary data, we will use a multidisciplinary approach that combines chemical, structural, cellular, systems biology and computational methods to characterize mechanisms of splice site selection and identify targets for tool compounds that will modulate these mechanisms. The outcomes will define key regulatory sequences, splicing factors and molecular interactions involved, thereby illuminating how the splicing machinery efficiently accommodates, yet also discriminates between, a wide range of splice site sequences. Our primary goal is to answer the central question, ‘Is it possible to modulate splicing with high specificity using small molecules?’ Success will transform our basic understanding of human gene expression and unleash major opportunities for Pharma to develop new therapeutics harnessing modulation of splice site selection. In summary, we expect the impact of this project to be a step-change advance in our mechanistic understanding of pre-mRNA splicing and how this can be regulated and harnessed for future therapeutic and biotechnological applications by controlling protein expression.
Progress made during the first 18 months of the project, along the lines of its main objectives, can be summarised as follows:
- Developing Existing Small Molecule Modulators: 97 compounds have been synthesized or commissioned for SAR analyses as an expansion of chemotypes identified in previous screens of splicing modulators. Transcriptome-wide datasets upon treatment with several previous hits.
- Small Molecule Screening using Chemical Libraries complementary to those already screened: conditions have been set up for a screen using our previous assay with an expanded chemical library of 100.000 compounds.
- Small Molecule Screening using New Assay Formats: major efforts have been made to establish new assay formats assessing early recognition of splice sites by solicing factors.
- Target Identification: Thermal Proteome Profiling and functional network reconstruction identification of targets of small molecule splicing regulators.
- Reconstitution of U1 snRNP from purified components including factors involved in 5’ and 3’ splice site recognition and splice site bridging.
- Mapped binding interfaces by NMR, obtained co-crystal structure of components of splicing complexes and have established various complexes for cryo-EM structural analysis.
- Developed and validated transcriptomic tools to understand the architecture of splicing factor regulatory networks: manuscript published (Rogalska et al 2024) using transcriptome-wide network reconstruction to assess the regulatory potential of core splicing machinery.
- Systematic assessment of cis-acting regulatory sequences: manuscript submitted assessing the optimal mutagenesis design for identification of regulatory sequence elements in alternatively spliced exons.
- Analysis of the biological impact of regulated splicing events: the relevance of alternative splicing events that might explain synthetic lethal effects of splicing inhibitors in cancer cells.
- Developing Existing Small Molecule Modulators: 97 compounds have been synthesized or commissioned for SAR analyses as an expansion of chemotypes identified in previous screens of splicing modulators. Transcriptome-wide datasets upon treatment with several previous hits.
- Small Molecule Screening using Chemical Libraries complementary to those already screened: conditions have been set up for a screen using our previous assay with an expanded chemical library of 100.000 compounds.
- Small Molecule Screening using New Assay Formats: major efforts have been made to establish new assay formats assessing early recognition of splice sites by solicing factors.
- Target Identification: Thermal Proteome Profiling and functional network reconstruction identification of targets of small molecule splicing regulators.
- Reconstitution of U1 snRNP from purified components including factors involved in 5’ and 3’ splice site recognition and splice site bridging.
- Mapped binding interfaces by NMR, obtained co-crystal structure of components of splicing complexes and have established various complexes for cryo-EM structural analysis.
- Developed and validated transcriptomic tools to understand the architecture of splicing factor regulatory networks: manuscript published (Rogalska et al 2024) using transcriptome-wide network reconstruction to assess the regulatory potential of core splicing machinery.
- Systematic assessment of cis-acting regulatory sequences: manuscript submitted assessing the optimal mutagenesis design for identification of regulatory sequence elements in alternatively spliced exons.
- Analysis of the biological impact of regulated splicing events: the relevance of alternative splicing events that might explain synthetic lethal effects of splicing inhibitors in cancer cells.
Rogalska et al. “Transcriptome-wide splicing network reveals specialized regulatory functions of the core spliceosome”. Science, 386: 551-560 (2024) doi: 10.1126/science.adn8105. This publication provides transcriptome-wide datasets of the effects of the knock down of 305 splicing factors/regulators and tools for functional network reconstruction that allow to infer targets and mechanisms of the spliceosome behind splicing perturbations, including those induced by small molecules. It was elected by Forbes as one of the 5 breakthroughs in 2024 that laid a foundation for future tech (https://www.forbesmiddleeast.com/innovation/technology/five-breakthroughs-in-2024-that-laid-a-foundation-for-future-tech(opens in new window)).