John Ravits, MD - US grants

DESCRIPTION (provided by applicant): Expanded hexanucleotide repeats in a non-coding region of C9orf72 are the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontal temporal degeneration (FTD) (C9-ALS/FTD). Many recent lines of evidence strongly suggest that pathogenesis is related to RNA toxicity, similar to a subset of other repeat expansion diseases. There are two, non-mutually exclusive mechanisms by which RNA- mediated toxicity is thought to occur: one is sequestration of nuclear RNA binding proteins, the hallmarks of which are RNA nuclear foci; and the other is translation of unconventional, repeat-associated non-ATG translation (RAN translation), the hallmarks of which are dipeptide repeat proteins (DPRs). With either of these mechanisms, antisense oligonucleotides (ASOs) that cause sequence-selective transcript degradation in the nucleus can be engineered to target the transcripts that are toxic and thus potentially provide therapy that is fundamental in pathogenesis. ASOs have already proven to be safe in humans in a clinical trial for SOD1 mutation-mediated ALS, are now in trial for spinal muscular atrophy, and are expected to enter trials next year for myotonic muscular dystrophy and Huntington's disease. Our ultimate long-term goal is ASO therapy development for C9-ALS/FTD patients. Recently, we and others have shown that expanded repeats in C9orf72 are bidirectionally transcribed in C9-ALS/FTD: the signature features of RNA nuclear foci and DPRs are generated from both sense and antisense strands. Thus, the critical next steps and the primary objectives for this proposal are to determine the relative contributions of each strand to pathogenesis and, in turn, to establish which targets and ASOs are the most critical for testing in patients. The research team and collaborators have in hand the preliminary data, the patient derived materials, the cellular and animal models, and the critical tools to determine this. In Aim 1, we will calibrate ASOs targeting sense and antisense transcripts measuring transcript levels, foci and RAN-translated products in cell culture models and transgenic mice expressing human C9orf72 with ~450 hexanucleotide repeats. In Aim 2, we will define the RNA signature in the transgenic mice using RNA-seq of the disease-relevant cell type, spinal motor neurons, which will be isolated by laser capture microdissection. In Aim 3, we will quantitatively compare efficacy of ASOs targeting each strand to correct the RNA signature in the transgenic mice. In Aim 4, we will define dose and duration effects of ASOs in transgenic mice. Upon completion of the projects, we will have determined whether one or both strands carrying the expansion are most critical for patient therapy and define a strategy for clinical trials.

Prion diseases are relentlessly progressive neurodegenerative disorders with death often within six months of the onset of neurologic symptoms. Pathologic features include widespread extracellular prion aggregates, spongiform degeneration, synaptic and neuronal loss, and severe astrogliosis and microgliosis. The structural determinants of the prion protein (PrP) and endogenous co-factors that drive aggregation, govern prion assembly, and impact aggregate spread through the central nervous system are unclear. A major goal of this application is to define when and how the endogenous co-factor, heparan sulfate (HS), promotes fibril assembly in the parenchyma and blood vessels and slows PrP clearance through the interstitial fluid using in vitro and in vivo model systems. We have previously pursued a range of approaches using cell-based prion conversion assays and newly generated transgenic and knock-in mouse models in collaboration with structural biologists to define the mechanisms that underlie PrP self-assembly and species barriers to prion conversion. We discovered using knock-in mouse models that N-linked glycans on PrP reduce spongiform degeneration, hinder plaque formation, and repel HS binding. Further, we found that plaque-forming prions were composed of poorly glycosylated, GPI-anchorless PrP bound to highly sulfated HS, underscoring the pivotal role of PrP post- translational modifications in driving the aggregate conformation and disease phenotype. We also found that reducing HS chain length decreases parenchymal plaque formation and prolongs survival. Finally, we identified highly amyloidogenic segments in the PrP sequence that control cross species prion conversion, as the number and location of glutamine and asparagine residues in PrP raise or lower the prion transmission barrier. In this renewal, we aim to determine the PrP-HS interactions that promote prion aggregate assembly and accelerate disease. We build on our long-standing observation that structural features of PrP, together with host glycosaminoglycans, drive efficient prion conversion. First, we will genetically manipulate neuronal, astrocytic, and endothelial HS chains and determine the impact on prion cell targets and survival using mouse models. Second, we will define how endogenous HS regulates PrP clearance through the interstitial fluid using conditional HS mouse models and radiolabeled PrP. Third, we will test the efficacy of antisense oligonucleotides (ASOs) targeting HS biosynthetic enzymes or Prnp mRNA in the early and mid-stages of prion plaque development in a prion disease model. We expect these mechanistic studies will (i) define how an endogenous co-factor, HS, accelerates and modifies prion disease, and (ii) determine whether reducing PrP interactions with this potential therapeutic target blocks prion spread.