Splice sensors for cancer drug discovery - SUMMARY:
RNA splicing plays a central role in the generation of proteome diversity and in gene regulation. Splicing impacts
vital cellular processes, such as cell-fate and differentiation, acquisition of tissue-identity, and organ
development. Thus, defects in splicing has been linked to many diseases, including spinal muscular atrophy,
Duchenne muscular dystrophy, Parkinson’s disease, and several types of cancer. RNA splicing is even more
relevant in cancer due to the higher incidence of mis-splicing events than in normal cells. Thus, it is not surprising
that aberrant splicing has been linked to important hallmarks of cancer such as proliferation, proliferation,
apoptosis evasion, metastasis, and angiogenesis, and in the development of cellular resistance against cancer
therapeutics. Despite the significance of splicing in cancer and other diseases, drug discovery efforts targeting
them are far and few. A critical bottleneck for such efforts is the lack of robust high-throughput assay tools to
monitor endogenous spliced RNA in the cell. Even though there are excellent tools such as RT-qPCR and RNA-
seq to study RNA splicing, they are not readily adaptable for high-throughput screening (HTS) due to their
complex and time-consuming methodology. While splice-mini-gene method offers the advantage of higher-
throughput, it lacks in the ability to monitor the endogenous target RNA due its artificial design. Thus, there is an
unmet need for simple and robust HTS-ready assay tools to monitor RNA splicing. Addressing this need, we
proposed the development of an easy-to-use, HTS-ready splice sensor platform that can specifically detect a
spliced RNA isoform of interest. In the Phase I, we used pyruvate kinase isoforms M1 (PKM1) and M2 (PKM2)
as model targets and developed prototype sensors that emit fluorescence signal when they bind to their
respective target RNA isoforms. PKM isoform switching is one of the ways cancer cells reprogram their glycolytic
pathways to meet cellular demands of energy and biosynthetic intermediates for growth and proliferation. With
the goals of commercializing the splice sensor platform for drug discovery, in the Phase II, we will perform a pilot
HTS for chemical modulators of PKM splicing and validate the splice sensor as a HTS-ready platform. To
demonstrate the versatility of the assay platform to target any RNA isoform of interest, we will port the modular
splice sensor platform to detect two new RNA isoforms linked to cancer. To operationalize the splice sensor
development process in the Aim 3, we will streamline standard operating procedures and create benchmarks for
the development, production, and kitting of the splice sensor assay. Lastly, we will create a next-generation
splice sensor-origami (SSO) system that will comprise of a Spinach reader and a user-customizable companion
probe set to target any spliced RNA isoform of interest. The SSO will rely on toe-hold mediated strand
displacement to achieve specificity and activation of Spinach fluorescence. Thus, at the end of Phase II, we will
commercialize the splice sensors developed as assay kits for direct sales and work with biopharma companies
to create custom splice sensors for internal drug discovery needs.
