Engineering human-derived programmable RNA effectors to retune gene expression

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Project Summary Genetic deletion (haploinsufficiency) or duplication events are often associated with a variety of diseases, including: cancers, cardiovascular and metabolic disease, and neuropathies. Because many genes are sensitive to both over- and under-expression, with imbalances in gene product in either direction leading to disease, tight regulatory control is a therapeutic necessity. RNA-targeting technologies, however, provide a mechanism to appropriately and reversibly modulate gene expression at the transcript level, resulting in tunable, cell-specific remediation of disease-causing shifts in gene dosage. Our recently developed CRISPR- Cas- inspired RNA targeting system (CIRTS), integrates small, human-derived proteins and guide RNA into an easily programmed and packaged technology for tunable gene expression manipulation at the transcript level. Notably, CIRTS imparts a major clinical advantage over that of functionally-related, but bacteria-derived CRISPR/Cas systems: minimized immunogenic risk stemming from the use of human protein parts. This proposal seeks to use protein engineering and cell-based screens to create highly efficient, optimized CIRTS- based technologies – both for gene activation and deactivation – and assess their generality across a panel of disease-relevant targets, and to test the potential of the technology in vivo. Initial screening will focus on the gene PMP22, an exemplar gene for which both over- and under-activation result in distinct, but related, genetic disorders. This provides a fertile testbed for optimization of both CIRTS degraders (Aim 1) and activators (Aim 2). In Aim 1, the human proteome will be mined to identify and characterize functional domains that can be integrated into CIRTS to programmably degrade PMP22 RNA. How target RNA landing sites correlate with the functional outcomes of each successful design, as well as generality against another target, SNCA, will be simultaneously pursued. Aim 2 of the proposal focuses on developing CIRTS activators, mining the human proteome for novel functional domains and using novel reporter systems to efficiently screen and characterize them, and finally, assessing lead designs in two other target genes, SCN1a (brain) and JAG1 (liver). Aim 3 will assess selectivity and specificity constraints of the system, and the generality of the CIRTS technology across a range of biological contexts. Here, lead CIRTS architectures will be benchmarked against state-of-the-art Cas13-based systems, comparing immunogenicity in vivo and testing the efficacy of CIRTS-based regulatory systems in “real world” contexts, namely mouse models of PMP22-based gene dosing disorders. Critically, data from all three aims feed off of and inform one another, resulting in a tripartite design-build-test optimization cycle. Completion of this project will generate a multi-modal, programmable CIRTS toolkit for the manipulation of gene expression at the RNA level. In addition to providing proof-of-principle validation of this system’s potential in one exemplar clinical application, this work will also produce a unique suite RNA-targeting effectors with broad utility across a wide variety of biomedical research and synthetic biology fields.