Defining short RNA therapeutics that combat TDP-43 proteinopathy connected to AD and ADRDs.

PROJECT SUMMARY There are no effective therapeutics for several devastating neurodegenerative disorders, including: amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), Alzheimer's disease (AD), limbic- predominant age-related TDP-43 encephalopathy (LATE), and chronic traumatic encephalopathy (CTE). A unifying pathological feature of ~50% AD cases, ~45% FTD cases, ~97% ALS cases, all LATE cases, and ~85% of CTE cases is the aberrant phase separation of TDP-43, an essential RNA-binding protein with a prion-like domain, into cytoplasmic inclusions in degenerating neurons. Abundant evidence suggests that the aberrant phase transition of TDP-43 in the cytoplasm is a key pathological event that is difficult for neurons to reverse. Methods to prevent and reverse the aberrant phase transitions of TDP-43 and restore functional TDP- 43 to the nucleus in the degenerating neurons of ALS/FTD, AD, LATE, and CTE patients are likely to be therapeutic for these disorders. Indeed, such an agent would simultaneously eliminate any toxic gain-of- function of aberrant TDP-43 conformers in the cytoplasm and any toxic loss-of-function caused by depletion of TDP-43 from the nucleus. We have established that short, specific RNAs provide a novel mechanism to antagonize neurotoxic phase transitions of TDP-43. These short RNAs can engage TDP-43, prevent aberrant phase separation, reverse the formation of existing TDP-43 aggregates, restore nuclear localization of TDP-43, and protect human neurons against TDP-43 toxicity. Importantly, these short RNAs are similar in size to FDA- approved antisense oligonucleotides that can be delivered successfully to the CNS of patients to treat neurodegenerative disorders. However, despite their protective effects, advancing these short RNAs to in vivo studies will require sequence and chemical modifications to mimic existing FDA-approved therapeutic oligonucleotides and limit `off-target' effects. Here, we propose to optimize the sequence, chemistry, and length of our lead oligonucleotides and assess their efficacy in model systems. Thus, we will pursue three aims: (1) Define optimized, modified RNA oligonucleotides that prevent and reverse aberrant TDP-43 phase separation at the pure protein level; (2) Define optimized, modified RNA oligonucleotides that prevent and reverse aberrant TDP-43 phase separation and toxicity in human neuronal models of TDP-43 proteinopathy; and (3) Define optimized, modified RNA oligonucleotides that mitigate aberrant TDP-43 phenotypes in patient-derived neurons and in mouse models of TDP-43 proteinopathy. Our studies hold the potential to yield the first therapeutic oligonucleotides that reverse TDP-43 aggregation and mitigate neurodegeneration in patient- derived neurons and the mammalian brain. We envision a therapeutic strategy whereby specific short oligonucleotides reverse TDP-43 aggregation in AD, ALS/FTD, LATE, and CTE and restore functional TDP-43 to the nucleus.