Theme 07 - Pre-Clinical Therapeutic Strategies

Background and method: Oxidative stress and neuroinflam- mation pave the way leading to neurodegeneration in amyotrophic lateral sclerosis (ALS) (1). Astrogliosis, increased levels of proinflammatory cytokines in the cerebrospinal fluid, and lower GSH content in the motor cortex are associated with ALS progression (1 – 3). Thus, we propose supplementation with N-acetylcysteine (GSH precursor), nicotinamide riboside (NR, a NAD þ promoter) and pterostilbene (PT, a natural antioxidant) could help to slow down the progression of the dis- ease (4,5). Results: NR and PT treatment was efficacious in ALS patients in a pilot human clinical trial (NCT03489200). Following 4 months of treatment (4): a 2.5-point a (cid:2) placebo. Discussion: Combined treatment ameliorated TNF a -induced oxidative stress and motor neuron death in vitro and decreased the microgliosis and astrogliosis associated with ALS progression. Our results support the involvement of oxidative stress, specific Nrf2-dependent antioxidant defenses, motor function in ALS mouse models and greatly extends their lifespan. As AAV9 does not efficiently target nor correct ALS microglia, despite neuron and astrocyte correction, the ALS mice still die of the disease at later time points with extensive microgliosis observed at the end stage. Therefore, it is evident that to provide the best therapeutic outcome for ALS patients, treatments that simultaneously target motor neurons, astrocytes, and microglia are necessary. Objective: To design and evaluate combination treatment approaches targeting neuronal and non-neuronal cells to reach improved therapeutic outcome in the treatment of ALS. Methods: In this currently ongoing study, we test multiple approaches combining our previously developed AAV9 mediated SOD1 downregulation approach in tandem with microglial modulation using ablation or immune modulation. Utilizing this combinatory approach, we indirectly target microglia, thereby dampening the chronic neuroinflammation in ALS mice in add- ition to downregulation of mutant SOD1 in motor neurons and astrocytes. We compare the combination approach against single treatment of SOD1 downregulation, microglia modulations, and untreated controls. Motor function is tested by rotarod performance and grip strength assessments twice per week. Disease onset, duration, and survival are monitored and compared to appropriate controls. Results: Our study shows that SOD1 downregulation in com- bination with small molecule mediated microglia ablation does not have a beneficial synergistic effect, while a novel AAV9 based combination treatment of SOD1 downregulation and a microglial immune modulation transgene lead to significantly longer survival and delayed disease onset of SOD1G93A mice compared to either single treatment. survey caregiver Statistical analyses included frequencies, per- centages, means, medians, and chi-square. Results: The seventy-six participants were caregivers (57%) and people living with neurological conditions (43%). Conditions represented include amyotrophic lateral sclerosis (ALS) (53%), epilepsy (12%), Alzheimer ’ s disease (11%), Parkinson ’ s disease (8%), multiple sclerosis (3%); Huntington ’ s disease (1%); and other (12%). were predominantly white (95%) with bachelor ’ s degrees or higher (75%), and mean age of 54 years. of the the United States (75%). CE ALS/FTD patient-derived cellular models. efficiently the TARDBP expression robustly UNC13A mRNA and protein levels order a robust platform for ASO screening in cell lines with familial and sporadic forms of ALS/FTD. of UNC13A CE targeting ASOs using our in-house software to include criteria for stability, immunogenicity, and hybridization-based off-target prediction. To the in vivo target engagement of the top candidates selected from the cellular model, we a humanized UNC13A mouse model the genomic sequence the CE using an AAV construct and induced the CE inclusion pheno- type by suppressing Tardbp. characterize the functional defi-ciencies in patient-derived neurons that result from cryptic exon inclusion and rescue with ASO treatment, we functional in vitro assays of UNC13A expression, localization, and neurotransmission. suppress UNC13A cryptic exon inclusion in vitro and in vivo, and we anticipate these leads will also rescue functional deficits in patient-derived neurons from multiple forms of familial and sporadic ALS/FTD. Discussion: The genetic evidence suggests that UNC13A levels can modify disease progression in humans. Importantly, the natural history studies also suggest that increasing UNC13A levels could be broadly effective across the ALS and FTLD-TDP populations, including patients with diverse or unknown causal mutations. We have developed novel and potent UNC13A exon skipping ASO candidates for advance-ment into IND-enabling studies. Background: Ferroptosis is a form of iron- and lipid-depend- ent regulated cell death associated with lipid peroxide production by lipoxygenase enzymes and glutathione depletion. Activation of ferroptosis has been implicated in a growing number of disorders, including Amyotrophic Lateral Sclerosis (ALS). Though the cause of ALS remains unknown, pathogen- icity involves oxidative stress, lipid peroxidation, chronic inflammation and cell death — hallmarks of ferroptosis. Because ferroptosis is regulated by balancing the activities of glutathione peroxidase-4 (GPX4) and 15-lipoxygenase (15-LO), targeting 15-LO provides a therapeutic approach to reducing ferroptosis. Objectives: Using a novel 15-LO inhibitor, PTC857, we set out to demonstrate that 15-LO inhibition prevents ferroptosis and improves cell survival by targeting oxidative stress, lipid peroxidation and inflammation. Based on our in vitro data, we then wanted to evaluate the protective effects of PTC857 on survival in the SOD1 mouse model. In addition to evaluat- ing the efficacy of PTC857 in these models, we sought to identify biomarkers confirming target engagement and mechanism-of-action (MoA). Methods: To assess the effectiveness of 15-LO inhibition on cell survival and neuro-inflammation, human spinal astrocytes were subjected to a pro-ferroptotic challenge (GPX4 inhib- ition by RSL3) in the presence of increasing concentrations of PTC857. To evaluate the effects of PTC857 on survival, 4- week old SOD1 mice were dosed once daily for 16-weeks with representative clinical scores recorded each day ( n ¼ 16 animals/arm). Determination of target engagement and dose selection was performed by measuring the whole blood concentration of the product of 15-LO, 12-hydroxyeicosatetrae- noic acid (12-HETE), in healthy C57B/6 mice following escalating PTC857 doses. Results: PTC857 prevented RSL3-induced ferroptotic cell death of spinal astrocytes, with cellular potency (EC50) values (cid:3) 98nM with no apparent effect on the viability at concentrations (cid:4) 5000nM. In the same study, riluzole conferred no protection up to 5000nM, and PTC857 was 70 times more potent than edaravone (EC50 7000nM) in preventing ferrop- totic death. In the SOD1 mouse study, PTC857 treated animals had improved grip strength, time to morbidity and survival compared to vehicle treated animals. PTC857 exhib- ited a time- and dose-dependent decrease in 12-HETE in mice with a median effective dose (ED50), which elicited a 50% decrease in 12-HETE levels estimated to be 41mg/kg. This change in HETEs can be measured in the clinic. Discussion: In totality, these in vitro and in vivo data pro- vided compelling evidence for targeting 15-LO and ferroptosis as a novel method for treating ALS. Following the completion of the requisite safety studies and regulatory fil- ings, PTC857 completed a Phase 1 testing in healthy adults. PTC857 is currently being tested in a Phase 2 placebo-con-trolled randomized trial to assess efficacy and safety in ALS patients (CARDINALS; NCT05349721). Background: Glucosylceramides are glycosphingolipids that are derived from ceramide by lysosomal (GBA1) or non-lyso- somal (GBA2) glucocerebrosidase. In ALS mice, glucosylcera-mide pathways are perturbed. In particular, an increase in GBA2 activity has been observed in the spinal cord of asymp-tomatic SOD1 mice. Previously, studies in SOD1 mice have shown that inhibition of GBA2 with ambroxol can lead to neuroprotection. However, it is unknown whether ambroxol exerts its neuroprotective effect solely via GBA2, or whether it exerts additional neuroprotection via GBA1. Objectives: Our aim is to use iPSC-derived lower motor neurons (iPSC-LMNs) ( N ¼ 3 Control, N ¼ 3 ALS) to determine whether inhibition of GBA1 or GBA2 can attenuate motor neuron death and TDP-43 mislocalisation. Methods: Inhibitors of GBA1 (OM1) and GBA2 (OM2) were synthesized by the Institut de Chimie Organique et Analytique (ICOA UMR7311, FR). Control and ALS iPSC-LMNs were treated with ambroxol (0.1, 1.0, 10 l M), OM1 (3, 30, 300nM), or OM2 (3, 30, 300nM) at day 35 for 10 days. Immunocytochemistry was performed at the end of the experiment to assess the number of ChAT positive LMNs and degree of TDP-43 mislocalisation. Results: Our preliminary data in control ( N ¼ 1) and ALS ( N ¼ 1) iPSC-LMNs suggest that at baseline, ALS iPSC-LMNs show significant cell death ( (cid:5) 20% compared to control) and 17% loss in neuronal size at day 45 ( p < 0.0001). Treatment motor neuron viability neuron viability ( p < 0.005). At all doses, OM2 but not OM1, was able to improve neuronal size ( p < 0.005). At baseline our C9orf72 ALS iPSC-LMNs exhibit 89% TDP-43 mislocalisation. Upon treatment with ambroxol (0.1 l M) and OM2 (300nM), a significant reduction of TDP-43 mislocalisation was observed ( p < 0.001). Discussion: Our findings to date suggest that high dose ambroxol, while a range of doses of OM1 and OM2 improves motor neuron survival. Interestingly, low dose ambroxol and high dose OM2 attenuates TDP-43 mislocalisation. While our data indicate that the neuroprotective effects of ambroxol are derived fr