Development of anti-inflammatory RNA therapeutics

Protein kinase R (PKR) is a double-stranded RNA (dsRNA)-activated serine/threonine kinase integral to the cellular stress response. Upon binding to dsRNA, PKR undergoes autophosphorylation, leading to the phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), resulting in the inhibition of global protein synthesis and the activation of pro-inflammatory pathways . While this mechanism is crucial for antiviral defense, aberrant PKR activation has been implicated in various neurological disorders. In monogenic neurological disorders such as DYT-PRKRA (dystonia 16), mutations in the PKR activator PACT lead to dysregulated PKR activation, resulting in enhanced neuronal apoptosis . Similarly, in neurodegenerative diseases like Alzheimer's and Parkinson's, elevated PKR activity has been observed, contributing to neuronal loss and cognitive deficits . These findings suggest that PKR-mediated pathways play a detrimental role in neuronal health, making PKR a potential therapeutic target.​ Interestingly, endogenous RNA species, including circular RNAs and Alu elements, have been shown to bind and inhibit PKR in resting cells, preventing unwarranted activation . This natural inhibitory mechanism offers a blueprint for developing synthetic RNA oligonucleotides that can mimic these endogenous inhibitors to therapeutically modulate PKR activity in neurological conditions.​ Project Aims: 1. To screen synthetic RNA oligonucleotides that can specifically bind to and inhibit PKR. 2. To determine the structural requirements of RNA molecules required for specific inhibition of PKR 3. To test PKR inhibiting oligonucleotides in induced pluripotent stem cell models of neurological conditions Methodology: -high throughput screening -induced pluripotent stem cell and organoid culture -CRISPR/Cas9 gene editing -high resolution imaging of live cells -analysis of RNA structure using SHAPE-MaP and Insilco modelling -high resolution imaging of live cells Significance This project will explore the development of RNA-based therapeutics that target PKR, a kinase implicated in the pathology of several neurological disorders. Building on recent insights into the natural regulation of PKR by endogenous RNAs, we aim to design synthetic oligonucleotides that can modulate its activity. This approach may provide a targeted strategy to reduce neuronal stress in conditions where PKR is aberrantly activated and could contribute to the development of new therapeutic options for diseases which have limited or no disease modifying therapies.