PROJECT SUMMARY Patients with retinitis pigmentosa (RP) due a mutation in inosine monophosphate dehydrogenase 1 (IMPDH1) expe- rience an early onset and debilitating retinal degeneration that leads to blindness. IMPDH1, a critical enzyme in the de novo synthesis of guanine nucleotides, places it at the nexus of cellular homeostasis. While guanine nucleo- tides are universally indispensable, mutations in IMPDH1 primarily manifest in retinal degeneration, emphasizing the unique vulnerability of the retina to IMPDH1 dysregulation. While IMPDH1's central role in nucleotide biosyn- thesis is well established, the nuanced regulatory mechanisms dictating its activity in the human retina are un- known. Preliminary investigations have revealed a link between IMPDH1 and MOK kinase (MAPK/MAK/MRK Overlapping Kinase), a member of the RCK (ros-cross hybridizing kinases) family known for its role in cilium length regulation. Given the established involvement of RCK kinases in cilium length regulation, it is reasonable to postulate that MOK kinase may play a crucial role in modulating photoreceptor outer segments. Our hypoth- esis is that IMPDH1 is negatively regulated by MOK kinase and that mutations associated with RP disrupt the interaction between these enzymes leading to decreased feedback inhibition and accumulation of toxic levels of guanine nucleotides. Specific Aim 1 will test the hypothesis that MOK kinase increases IMPDH1 sensitivity to GTP feedback inhibition in human photoreceptor cells and that this sensitivity is lost with the IMPDH1 D311N mutation. We will use patient-derived retinal organoids to model the human retina to determine the effect of MOK kinase on IMPDH1 enzyme kinetics. We will compare IMPDH1 enzyme activity in MOK-knock out (KO) retinal organoids expressing wild type (WT) IMPDH1 or the common RP mutation D311N. Specific Aim 2 will test the hypothesis that guanine nucleotide pools increase IMPDH1 interactions with MOK kinase and that mutations in IMPDH1