- Email: [email protected]
ANTISENSE OLIGONUCLEOTIDES AS THERAPEUTIC AGENTS
Symposia: S3-02: Clinical Trials and Frontotemporal Lobar Dementia (FTLD)
who does have dementia "passes" or scores well on the test. Cognition tests in clinical practice are usually used when it is not known who has dementia; the test is used to help find out. False positives and false negatives are of less interest than what fraction of patients who receive a positive test result actually have dementia. However, even when the sensitivity is 90% and sensitivity is 80%, the positive predictive value will be very different in community and primary care settings compared to "specialty" settings. Results: Positive predictive value is driven by the prevalence of the dementia in the population for which a test for cognitive impairment is being applied. In a locked ward, the prevalence is 80% and positive predictive value is 97%. The memory clinic setting will have about 30% of patients with dementia and this results in a positive predictive value of 77%. In a general population or in a primary care clinic, a prevalence of 6% or less will result in a positive predictive value of 34%. Clearly, with greater likelihood of in the community or primary care settings of mislabeling and the risk of over-diagnosis will be greater. Conclusions: For psychometric reasons, mislabeling, including over-diagnosis, is more likely in community and primary care than "specialty" settings. TUESDAY, JULY 15, 2014 SYMPOSIA S3-02 CLINICAL TRIALS AND FRONTOTEMPORAL LOBAR DEMENTIA (FTLD)
tion of the target mRNA. When delivered to the cerebral spinal fluid, the ASOs distribute broadly throughout the central nervous system. In Amyotrophic Lateral Sclerosis, intrathecally delivered ASOs showed excellent safety in a Phase I human clinical trial. Methods: We have designed ASOs that target and selectively reduce either endogenous mouse tau mRNA or human tau mRNA. In mice, the ASOs are delivered to the right lateral ventricle using an indwelling catheter and subcutaneous osmotic pump. Results: In adult non-transgenic mice, ASO reduction of mouse tau mRNA and protein 1) decreased neuronal spike frequency in the hippocampus after administration of picrotoxin (GABA antagonist) and 2) decreased seizure severity after administration of PTZ (pentylenetetrazol, GABA antagonist). In transgenic mice expressing human tau with the P301S mutation, ASOs targeting human tau markedly reduced phosphortau pathology, even when starting treatment in 9 month old mice which, in this model, already show substantial tau pathology. Conclusions: Reducing tau mRNA and protein decreases hyperexcitability and seizures in adult mice. Since abnormal hyperexcitabiltiy may contribute to cognitive dysfunction in Alzheimer’s disease, this effect of tau reduction may be beneficial in an AD therapy. Reducing human mutant tau mRNA and protein reverses some tau pathology in mice. Given recent, positive experience with ASOs in human clinical trials, we are developing tau ASOs as a therapy for Alzheimer’s disease and other tauopathies. S3-02-03
S3-02-01
P201
TARGETING CHAPERONES TO TREAT FTLD
THERAPIES THAT ENHANCE PROGRANULIN EXPRESSION
Chad A. Dickey, University of South Florida, Tampa, Florida, United States. Contact e-mail: [email protected]
Joachim Herz, UT Southwestern, Dallas, Texas, United States. Contact e-mail: [email protected]
Background: The microtubule-associated protein tau becomes neurotoxic in Alzheimer’s disease and frontotemporal dementia. Finding therapeutics to remove this abnormal tau is needed. Small molecule-based drug discovery targeting tau is in its early stages, but major efforts are underway to accelerate this. Currently only one small molecule, a phenothiazine derivative, is in phase III clinical trials. The putative mechanism for this compound is direct inhibition of tau aggregation, but we found that this class of small molecules also blocks chaperones from preserving tau, leading to its clearance. This suggested that common structural properties govern tau aggregation and chaperone binding. Methods: We have used several techniques to evaluate the effects of chaperone inhibitors and their potential synergy with aggregation inhibitors. We have employed cell culture, biochemical, biophysical and ex vivo slice culture strategies to survey the efficacy of approaches that target chaperones alone or in combination with aggregation inhibitors. We have also employed electrophysiology to further explore the effect of this approach on restoring tau-mediated synaptic deficits. Results: We recently discovered the molecular basis for tau interacting with Hsp90, providing the first demonstration of how a client interacts with this important chaperone. Through this and other work, we have determined that the same amino acid sequences in tau are required for both its aggregation and its binding to chaperones. Indeed compounds that inhibit tau aggregation and chaperone function are the most potent for reducing tau and rescuing synaptic deficits caused by tau accumulation. The basis for this mechanism of action is that chaperones have greater access to tau when it is unable to aggregate, creating more chaperone/tau complexes that can be targeted for degradation with inhibitors. We observed this same principle of competition for tau binding when we found that microtubule destabilization also enhanced tau binding to chaperones. Conclusions: While a chaperone/tau interface is potentially "druggable", one of the biggest concerns with chaperone-based therapies is both the on- and off-target toxic liabilities. Perhaps by exploiting our understanding of tau aggregation and tau’s interaction with chaperones, we can lower the doses required for efficacy of either drug, reducing the chances for detrimental side-effects.
Background: Haploinsufficiency of progranulin causes frontotemporal lobar degeneration, while homozygous loss of progranulin results in a form of neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. Granulins are small highly disulfide-bonded peptides that arise by proteolytic processing from progranulin and resemble saposins, which function as cofactors of lysosomal glycosphingolipid metabolism. These similarities suggest that progranulin haploinsufficiency may lead to progressive lysosomal dysfunction and subsequently neurodegeneration. This mechanism suggests upregulation of the remaining functional progranulin allele as a simple preventive therapeutic strategy. Methods: A BAC construct that drives a luciferase reporter under the control of the authentic human progranulin promoter including upstream and downstream regulatory sequences has been constructed. Results: We have used neuronal cell lines transfected with this reporter to perform high throughput screens of a large chemical compound library and have identified a series of small molecules that increase progranulin promoter activity, mRNA and protein expression. Conclusions: Pharmacological upregulation of progranulin expression may be an achievable way to abrogate the risk for frontotemporal dementia caused by progranulin haploinsufficiency.
S3-02-02
ANTISENSE OLIGONUCLEOTIDES AS THERAPEUTIC AGENTS
Timothy M. Miller, Washington University in St. Louis, St. Louis, Missouri, United States. Contact e-mail: [email protected] Background: Chemically modified antisense oligonucleotides (ASO) bind directly to mRNA and result in sequence specific, RNAseH-mediated reduc-
S3-02-04
MECHANISMS OF TOXICITY AND THERAPEUTIC APPROACHES FOR C9FTD/ALS
Leonard Petrucelli, Mayo Clinic, Jacksonville, Florida, United States. Contact e-mail: [email protected] Background: A significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two diseases commonly seen in comorbidity, carry an expanded non-coding hexanucleotide repeat in the C9orf72 gene, a condition commonly referred to as c9FTD/ALS. We and others have demonstrated that the RNA structure of GGGGCC repeats may cause neurodegeneration via their accumulation into discrete structures in the nucleus, termed RNA foci, and by serving as a template for the synthesis of aggregation-prone "c9RAN proteins" by repeat-associated non-ATG (RAN) translation. Expanded repeat carriers also exhibit reduced C9orf72 mRNA levels, suggesting that C9orf72 loss of function may contribute to neurodegeneration. Methods: Using a variety of approaches, including the generation of novel antibodies, in vitro and in vivo assays, as well as analyses of human tissue, we examine how bidirectional transcription of the expanded C9orf72 repeat and reduced C9orf72 mRNA expression lead to c9FTD/ALS. Results: We discover antisense
who does have dementia "passes" or scores well on the test. Cognition tests in clinical practice are usually used when it is not known who has dementia; the test is used to help find out. False positives and false negatives are of less interest than what fraction of patients who receive a positive test result actually have dementia. However, even when the sensitivity is 90% and sensitivity is 80%, the positive predictive value will be very different in community and primary care settings compared to "specialty" settings. Results: Positive predictive value is driven by the prevalence of the dementia in the population for which a test for cognitive impairment is being applied. In a locked ward, the prevalence is 80% and positive predictive value is 97%. The memory clinic setting will have about 30% of patients with dementia and this results in a positive predictive value of 77%. In a general population or in a primary care clinic, a prevalence of 6% or less will result in a positive predictive value of 34%. Clearly, with greater likelihood of in the community or primary care settings of mislabeling and the risk of over-diagnosis will be greater. Conclusions: For psychometric reasons, mislabeling, including over-diagnosis, is more likely in community and primary care than "specialty" settings. TUESDAY, JULY 15, 2014 SYMPOSIA S3-02 CLINICAL TRIALS AND FRONTOTEMPORAL LOBAR DEMENTIA (FTLD)
tion of the target mRNA. When delivered to the cerebral spinal fluid, the ASOs distribute broadly throughout the central nervous system. In Amyotrophic Lateral Sclerosis, intrathecally delivered ASOs showed excellent safety in a Phase I human clinical trial. Methods: We have designed ASOs that target and selectively reduce either endogenous mouse tau mRNA or human tau mRNA. In mice, the ASOs are delivered to the right lateral ventricle using an indwelling catheter and subcutaneous osmotic pump. Results: In adult non-transgenic mice, ASO reduction of mouse tau mRNA and protein 1) decreased neuronal spike frequency in the hippocampus after administration of picrotoxin (GABA antagonist) and 2) decreased seizure severity after administration of PTZ (pentylenetetrazol, GABA antagonist). In transgenic mice expressing human tau with the P301S mutation, ASOs targeting human tau markedly reduced phosphortau pathology, even when starting treatment in 9 month old mice which, in this model, already show substantial tau pathology. Conclusions: Reducing tau mRNA and protein decreases hyperexcitability and seizures in adult mice. Since abnormal hyperexcitabiltiy may contribute to cognitive dysfunction in Alzheimer’s disease, this effect of tau reduction may be beneficial in an AD therapy. Reducing human mutant tau mRNA and protein reverses some tau pathology in mice. Given recent, positive experience with ASOs in human clinical trials, we are developing tau ASOs as a therapy for Alzheimer’s disease and other tauopathies. S3-02-03
S3-02-01
P201
TARGETING CHAPERONES TO TREAT FTLD
THERAPIES THAT ENHANCE PROGRANULIN EXPRESSION
Chad A. Dickey, University of South Florida, Tampa, Florida, United States. Contact e-mail: [email protected]
Joachim Herz, UT Southwestern, Dallas, Texas, United States. Contact e-mail: [email protected]
Background: The microtubule-associated protein tau becomes neurotoxic in Alzheimer’s disease and frontotemporal dementia. Finding therapeutics to remove this abnormal tau is needed. Small molecule-based drug discovery targeting tau is in its early stages, but major efforts are underway to accelerate this. Currently only one small molecule, a phenothiazine derivative, is in phase III clinical trials. The putative mechanism for this compound is direct inhibition of tau aggregation, but we found that this class of small molecules also blocks chaperones from preserving tau, leading to its clearance. This suggested that common structural properties govern tau aggregation and chaperone binding. Methods: We have used several techniques to evaluate the effects of chaperone inhibitors and their potential synergy with aggregation inhibitors. We have employed cell culture, biochemical, biophysical and ex vivo slice culture strategies to survey the efficacy of approaches that target chaperones alone or in combination with aggregation inhibitors. We have also employed electrophysiology to further explore the effect of this approach on restoring tau-mediated synaptic deficits. Results: We recently discovered the molecular basis for tau interacting with Hsp90, providing the first demonstration of how a client interacts with this important chaperone. Through this and other work, we have determined that the same amino acid sequences in tau are required for both its aggregation and its binding to chaperones. Indeed compounds that inhibit tau aggregation and chaperone function are the most potent for reducing tau and rescuing synaptic deficits caused by tau accumulation. The basis for this mechanism of action is that chaperones have greater access to tau when it is unable to aggregate, creating more chaperone/tau complexes that can be targeted for degradation with inhibitors. We observed this same principle of competition for tau binding when we found that microtubule destabilization also enhanced tau binding to chaperones. Conclusions: While a chaperone/tau interface is potentially "druggable", one of the biggest concerns with chaperone-based therapies is both the on- and off-target toxic liabilities. Perhaps by exploiting our understanding of tau aggregation and tau’s interaction with chaperones, we can lower the doses required for efficacy of either drug, reducing the chances for detrimental side-effects.
Background: Haploinsufficiency of progranulin causes frontotemporal lobar degeneration, while homozygous loss of progranulin results in a form of neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. Granulins are small highly disulfide-bonded peptides that arise by proteolytic processing from progranulin and resemble saposins, which function as cofactors of lysosomal glycosphingolipid metabolism. These similarities suggest that progranulin haploinsufficiency may lead to progressive lysosomal dysfunction and subsequently neurodegeneration. This mechanism suggests upregulation of the remaining functional progranulin allele as a simple preventive therapeutic strategy. Methods: A BAC construct that drives a luciferase reporter under the control of the authentic human progranulin promoter including upstream and downstream regulatory sequences has been constructed. Results: We have used neuronal cell lines transfected with this reporter to perform high throughput screens of a large chemical compound library and have identified a series of small molecules that increase progranulin promoter activity, mRNA and protein expression. Conclusions: Pharmacological upregulation of progranulin expression may be an achievable way to abrogate the risk for frontotemporal dementia caused by progranulin haploinsufficiency.
S3-02-02
ANTISENSE OLIGONUCLEOTIDES AS THERAPEUTIC AGENTS
Timothy M. Miller, Washington University in St. Louis, St. Louis, Missouri, United States. Contact e-mail: [email protected] Background: Chemically modified antisense oligonucleotides (ASO) bind directly to mRNA and result in sequence specific, RNAseH-mediated reduc-
S3-02-04
MECHANISMS OF TOXICITY AND THERAPEUTIC APPROACHES FOR C9FTD/ALS
Leonard Petrucelli, Mayo Clinic, Jacksonville, Florida, United States. Contact e-mail: [email protected] Background: A significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), two diseases commonly seen in comorbidity, carry an expanded non-coding hexanucleotide repeat in the C9orf72 gene, a condition commonly referred to as c9FTD/ALS. We and others have demonstrated that the RNA structure of GGGGCC repeats may cause neurodegeneration via their accumulation into discrete structures in the nucleus, termed RNA foci, and by serving as a template for the synthesis of aggregation-prone "c9RAN proteins" by repeat-associated non-ATG (RAN) translation. Expanded repeat carriers also exhibit reduced C9orf72 mRNA levels, suggesting that C9orf72 loss of function may contribute to neurodegeneration. Methods: Using a variety of approaches, including the generation of novel antibodies, in vitro and in vivo assays, as well as analyses of human tissue, we examine how bidirectional transcription of the expanded C9orf72 repeat and reduced C9orf72 mRNA expression lead to c9FTD/ALS. Results: We discover antisense