Amyotrophic lateral sclerosis (ALS) affects an estimated 30,000 individuals in the United States and 450,000 worldwide. The disease is characterized by loss of motor neurons leading to loss of muscle control and muscle wasting. Patients typically progress from diagnosis to death in 3-5 years. While there are two approved therapies to treat ALS, their effects are limited. The lack of available treatments for ALS patients remains a significant unmet medical need. Unlike numerous other rare diseases, ALS is not monogenic, thus it is unlikely that a single therapy will be able to address the entire patient population. Despite recent advances in sequencing technology, 90% of ALS cases are sporadic making determination of a genetic cause more difficult. Nonetheless, many of the genes originally found in familial cases also appear in the sporadic population including C9orf72, and Optineurin. One of the genes that has recently been shown to be associated with ALS in a dominant fashion is tank binding kinase 1 (TBK1). Using patient derived cells, it has been shown that the mutations lead to a haploinsufficiency and loss of function. Furthermore, studies on TBK1 have shown that it is a key regulator of autophagy and mitophagy and interacts with Optineurin, p62, and Ubiquilin, all of which have also been shown to be associated with ALS. To develop therapies for TBK1 patients, we propose to develop a cellular system with TBK1 haploinsufficiency that can be used to develop an assay to assess the efficacy of potential drug candidates to increase TBK1 function from the remaining allele. We propose to use CRISPR-Cas9 to disrupt a single allele of the TBK1 gene in human induced pluripotent stem cells (hiPSCs) as a phenotypic model of TBK1 patients. Once we have generated a single allele knockout of TBK1, we will assess the phenotype in hiPSC-derived motor neurons in comparison to parental cells. Disease relevant phenotypes will be examined including levels of activated TBK1, autophagy, and mitophagy. Our drug discovery efforts are focused on increasing the levels of activated TBK1 and thus rescuing any deficiency found in the downstream pathways of autophagy and mitophagy. We have identified a molecular target that modulates TBK1 activity and have preliminary data showing that pharmacological modulation of this target leads to an increase in mitophagy in human microglial cells. We are continuing to pursue this target and anticipate that we will have a significant number of compounds to assay once the TBK1 knockout cells are available. The demonstration of activity in a cellular model of ALS is crucial to support our ultimate goal of developing a therapeutic for this patient population. In addition, many ALS associated genes, including TBK1, have also been shown to be associated with Frontotemporal Temporal Dementia (FTD), and autophagy and mitophagy defects are associated with several neurodegenerative diseases, thus this work may have implications for drug discovery beyond ALS.
Public Health Relevance Statement: Project Narrative Despite the fact that amyotrophic lateral sclerosis (ALS) has been known for generations, it is only recently that the genetic underpinnings of this disease have started to be elucidated. It is now clear that ALS can have multiple genetic causes and furthermore that therapies may need to be tailored to specific patient subpopulations to be effective. QurAlis has adopted this approach and is building models and engaged in drug discovery to address each patient population precisely.
NIH Spending Category: ALS; Brain Disorders; Genetics; Neurodegenerative; Neurosciences; Orphan Drug; Rare Diseases; Stem Cell Research; Stem Cell Research - Induced Pluripotent Stem Cell; Stem Cell Research - Induced Pluripotent Stem Cell - Human
Project Terms: Address; Adopted; Affect; Alleles; ALS2 gene; American; Amyotrophic Lateral Sclerosis; Automobile Driving; Autophagocytosis; Autophagosome; base; Binding; Biological; Biological Assay; C9ORF72; Cell Line; Cell model; cell type; Cells; Cessation of life; Chemicals; Complex; CRISPR/Cas technology; Data; Defect; Dementia; Detection; Development; Diagnosis; Disease; Disease model; DNA; Dose; drug candidate; drug discovery; Drug Modelings; Ensure; Familial Amyotrophic Lateral Sclerosis; Functional disorder; Future; Gene Expression; Generations; Genes; Genetic; Genetic Engineering; Goals; Guide RNA; Human; Impairment; improved; Individual; induced pluripotent stem cell; knock-down; Knock-out; Lead; loss of function; Medical; Microglia; miniaturize; model building; Modeling; Molecular Target; Motor Neurons; Muscular Atrophy; Mutation; Nerve Degeneration; Neuraxis; Neurodegenerative Disorders; Neuromuscular Junction; neuronal circuitry; novel; Paralysed; Pathway interactions; patient population; patient subsets; Patients; Pharmaceutical Chemistry; Pharmacology; Phase; Phenotype; Phosphorylation; Phosphotransferases; Population; preservation; programs; Proteins; Rare Diseases; Regulation; Research; response; Risk Factors; RNA; screening; Serine; Site; Small Business Innovation Research Grant; Solid; stem cell technology; System; TANK-binding kinase 1; Technology; Testing; Therapeutic; therapy development; tool; ubiquilin; United States; Validation; Work
