Document text
Principal Investigator: Jacob Litke
Organization: CHIMERNA THERAPEUTICS INC.
Fiscal Year: 2022
Award: $256,158
Funding agency: National Institute of Neurological Disorders and Stroke
SUMMARY
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by inclusion bodies
containing the TDP-43 protein in the cytoplasm of motor neurons. These inclusion bodies are caused by TDP-
43 aggregation, which is caused by pathways or mutations that cause TDP-43 to localize to the cytoplasm.
TDP-43 is an RNA-binding protein and its ability to bind mRNA is a critical step in the process that ultimately
leads to TDP-43 aggregates. When TDP-43 binds cellular RNAs, it can “phase separate” and become
enriched in stress granules, where TDP-43 slowly forms insoluble aggregates. A widely accepted therapeutic
goal is to prevent TDP-43 from undergoing phase separation into stress granules, since this is a prerequisite
for TDP-43 aggregation. To block TDP-43 phase separation, we are using RNA aptamers that bind TDP-43’s
RNA-binding domain and thus act as a “decoy” to block TDP-43 from binding to cellular mRNA. However,
RNA aptamers are rapidly degraded in the cytosol. Chimerna scientists have developed an innovative
expression system that allows RNA aptamers to rapidly be converted into “circular” RNAs in cells, thus making
them resistant to cellular exonucleases. This dramatically increases RNA aptamer stability, leading to
expression levels that are at least 100 times higher than any previous expression system. Using this system,
we found that circular TDP-43-binding aptamers can block phase separation of TDP-43 using an optogenetic
TDP-43 phase-separation assay. Since the circular RNAs are cytosolic, they selectively block the cytosolic
form of TDP-43 without affecting the normal function of TDP-43 in the nucleus. The critical question is whether
TDP-43-binding aptamers will prevent TDP-43 aggregation, toxicity, and neurodegeneration in disease-
relevant models. To test this, the specific aims of this proposal are: (1) To evolve TDP-43 aptamers for
higher affinity and lowest IC50 for blocking phase separation. We will use directed evolution to evolve
high-affinity TDP-43 aptamers. We will use our optogenetic platform for inducing TDP-43 phase separation in
cells to calculate IC50 values for the top evolved aptamers. These experiments will reveal the aptamers that
have the greatest potential to disrupt TDP-43 phase separation. (2) To test the ability of circular TDP-43
aptamers to block phase separation, aggregation, and toxicity of TDP-43 in human iPS neurons. Here,
we will use iPS-derived ALS motor neurons that exhibit TDP-43 aggregates, markers of aggregate toxicity, and
increased susceptibility to oxidative stress-mediated cell death. We will determine if TDP-43 aptamers can
block these key parameters of TDP-43 aggregate toxicity, thus providing critical data about the validity of our
aptamer approach using disease-relevant model. Overall, these experiments will test a new concept for
blocking TDP-43 aggregation using highly stable circular TDP-43 aptamers, that will be tested using an
optogenetic phase separation assay and ALS iPS-derived motor neurons.
Terms: <ALS patients><Affect><Affinity><Amyotrophic Lateral Sclerosis><Amyotrophic Lateral Sclerosis Motor Neuron Disease><Amyotrophic Lateral Sclerosis patients><Assay><Binding><Binding Proteins><Bioassay><Biologic Assays><Biological Assay><Body Tissues><Catalytic RNA><Cell Body><Cell Death><Cell Nucleus><Cells><Cellular Inclusions><Circular Fold><Clinical Trials><Cytoplasm><Cytoplasmic Protein><Cytosol><DNA Alteration><DNA Sequence Alteration><DNA mutation><Data><Degenerative Neurologic Diseases><Degenerative Neurologic Disorders><Directed Molecular Evolution><Disease><Disorder><Dose><Evolution><Exhibits><Exonuclease><FDA approved><Gehrig's Disease><Gene Transcription><Genetic Alteration><Genetic Change><Genetic Transcription><Genetic defect><Genetic mutation><Goals><Human><Inclusion Bodies><Kerckring's valve><Legal patent><Ligand Binding Protein><Ligand Binding Protein Gene><Link><Lou Gehrig Disease><Mediating><Messenger RNA><Modeling><Modern Man><Molecular Interaction><Motor Cell><Motor Neurons><Mutation><Nerve Cells><Nerve Degeneration><Nerve Unit><Nervous System Degenerative Diseases><Neural Cell><Neural Degenerative Diseases><Neural degenerative Disorders><Neurocyte><Neurodegenerative Diseases><Neurodegenerative Disorders><Neurologic Degenerative Conditions><Neuron Degeneration><Neurons><Non-Polyadenylated RNA><Nucleus><Oligo><Oligonucleotides><Oxidative Stress><Patents><Pathway interactions><Patients><Phase><Polyribonucleotide Synthetase><Predisposition><Prevention><Problem Solving><Process><Protein Binding><Putative RNA-Binding Region><RNA><RNA Binding><RNA Binding Domain><RNA Expression><RNA Gene Products><RNA Instability><RNA Ligase (ATP)><RNA Recognition Motif><RNA Sequences><RNA Splicing><RNA based therapeutics><RNA bound><RNA ligase><RNA-Binding Proteins><RNP Domain><RNP Motif><RNP-1 Signature><Resistance><Ribonucleic Acid><Ribozymes><Scientist><Sequence Alteration><Small RNA><Splicing><Stress><Structure><Susceptibility><System><TAR DNA-binding protein 43><TDP-43><TDP-43 aggregate><TDP-43 aggregation><TDP43><TDP43 aggregate><TDP43 aggregation><Technology><Testing><Therapeutic><Time><Tissues><Tornadoes><Toxic effect><Toxicities><Transcription><Transcription Repressor><Transcriptional Repressor><Universities><Viral><appropriate dose><aptamer><base><bound protein><circular RNA><clinical development><closed circular RNA><degenerative diseases of motor and sensory neurons><degenerative neurological diseases><directed evolution><experiment><experimental research><experimental study><genetic repressor><genome mutation><genomic alteration><iPS><iPSC><iPSCs><induced pluripotent stem cell><inducible pluripotent stem cell><inhibitor><innovate><innovation><innovative><insoluble aggregate><lipid based nanoparticle><lipid nanoparticle><mRNA><motoneuron><nano particle delivery><nanoparticle delivered><nanoparticle delivery><necrocytosis><nerve cell death><nerve cell loss><neural degeneration><neurodegeneration><neurodegenerative><neurodegenerative illness><neurological degeneration><neuron cell death><neuron cell loss><neuron death><neuron loss><neuronal><neuronal cell death><neuronal cell loss><neuronal death><neuronal degeneration><neuronal loss><new approaches><new drug treatments><new drugs><new therapeutics><new therapy><next generation therapeutics><novel><novel approaches><novel drug treatments><novel drugs><novel strategies><novel strategy><novel therapeutics><novel therapy><oligos><optimal drug dosage><optimal drug dose><optogenetics><pathway><pre-clinical development><preclinical development><prevent><preventing><protein TDP-43><protein TDP43><protein aggregate><protein aggregation><recruit><resistant><stem cell based model><stem cell derived model><stem cell model><stress granule><therapeutic RNA>