2021-2022
A HUMAN PITT-HOPKINS SYNDROME MODEL FOR THERAPEUTIC PROOF OF PRINCIPLE II
Alysson R. Muotri, PhD
UC San Diego School of Medicine | 2019-2022
$75,000
Research plan: The ultimate goal of aim #1 is to define how nervous system cells are affected by TCF4 haploinsufficiency in human models. We have made good progress on generating these models by reprogramming stem cells from several PTHS patients, generating neural progenitor cells and neurons in 2D culture, and producing cortical organoids in vitro.
Aim #2 Development of strategies for correcting TCF4 expression. Rationale. In this aim, we are testing strategies to manipulate TCF4 expression and correct its mRNA abundance to normal levels. Although the initial experiments are being conducted with in-vitro cultures of patient-derived neural progenitors, neurons, and cerebral organoids, our ultimate goal is to provide proof-of-concept to seed further research on correctional genetic therapies of PTHS to be applied to children patients.
Aim #3 Reversal of PTHS cellular phenotypes by gene expression correction. Rationale. A recurring theme in the study of neurological disease is whether the phenotypic alterations seen in patients at the cellular, tissue and organismic levels are permanent damage caused by the genetic defect, or whether the pathology can be reversed if the gene or its associated molecular pathways are brought back to normal, eventually leading to amelioration or disappearance of the patient’s symptoms. In this aim, we will use the in vitro models of PTHS developed in Aim #1 (stem cells, neural progenitors, neurons, and cerebral organoids derived from patient cells) in combination with the correctional strategies developed in Aim #2 not only to provide proof-of-principle that TCF4 expression and aberrant phenotypes can be corrected in patient cells, but also as a tool to investigate the function of TCF4 and neurological phenotypes in PTHS.
TCF4 GENE ACTIVATION THERAPY FOR PITT-HOPKINS SYNDROME
Principle Investigator (PI): Dan Wang, Ph.D. (Assistant Professor, Horae Gene Therapy Center, University of Massachusetts Medical School)
Co-PI: Guangping Gao, Ph.D. (Professor, Horae Gene Therapy Center, University of Massachusetts Medical School)
Total amount requested: $74,721
2020-2021
Pitt-Hopkins Syndrome (PTHS) patients have mutations or deletions on one of the two TCF4 gene copies, leading to insufficient amount of the gene product, namely the TCF4 protein. This disease mechanism is known as haploinsufficiency. One potential therapeutic strategy for haploinsufficiency is to enhance the protein output (i.e., gene expression) from the normal gene copy to compensate for the mutated one, restoring total protein to the normal level.
Several years ago, the CRISPR activation (CRISPRa) approach was developed to activate targeted gene expression in human cells. In essence, this approach uses an RNA/protein complex to find the target gene and stimulate its expression. In two recent studies, two groups of scientists delivered CRISPRa into mice that suffer from obesity or Dravet Syndrome caused by haploinsufficiency of the Sim1 gene and the Scn1a gene, respectively. They reported that CRISPRa was able to rescue the diseases in mice by activating each gene expression to the normal level. Encouraged by these findings, we propose to test whether CRISPRa can also rescue PTHS by activating the TCF4 gene expression.
We will first optimize the CRISPRa design for TCF4 gene activation in cultured cells including neurons derived from a PTHS mouse model that mimics human disease. The selected CRISPRa reagents will be delivered to the brain of PTHS mice using a viral vector-based method that is similar to what was used in both aforementioned mouse studies. Then TCF4 gene activation in the brain will be assessed by a variety of molecular and cellular assays, and optimized to recapitulate the TCF4 protein level in normal mice.
This 1-year study will serve as a proof-of-concept for the feasibility of using CRISPRa for PTHS. If successful, the knowledge and reagents generated can be immediately applied in a follow-up translational study to examine therapeutic efficacy in PTHS mice, potentially leading to clinical application in PTHS patients.
ANTISENSE OLIGONUCLEOTIDE TREATMENT FOR PITT HOPKINS SYNDROME
Michelina Iacovino PhD
The Lundquist Institute for Biomedical Innovation at Harbor
UCLA Medical Center
$75,000
2021
The main goal of this proposal is to screen dsRNA molecules capable of increasing TCF4 mRNA levels, to restore TCF4 protein levels to normal. Small dsRNA molecules act in many species, as a conserved mechanism to control gene expression (Elbashir, Harborth et al. 2001, Elbashir, Lendeckel et al. 2001, Ipsaro and Joshua-Tor 2015). Classically, this mechanism decreases mRNA levels, by promoting mRNA degradation. Since early 2000, however, several groups have reported that dsRNA can induce gene expression and increase mRNA levels (Li, Okino et al. 2006, Huang, Qin et al. 2010, Portnoy, Huang et al. 2011, Corey 2017). For example, upregulation can occur when the dsRNA targets the gene promoter region (Janowski, Younger et al. 2007) or when the dsRNA targets regulatory transcripts, such as antisense transcripts for the same gene (Voutila, Saetrom et al. 2012, Voutila, Reebye et al. 2017).
$60,327
2022
The main goal of this proposal is to screen interference RNA molecules (siRNA or ASO) capable of increasing TCF4 mRNA levels, to restore TCF4 protein levels to normal. We have previously screened double-strand RNA molecules targeting the TCF4 promoter and TCF4 AS-1 and TCF4 AS-2. siRNA does not need packaging to traffic through the cells (it is a small single strand RNA), and therefore is it more amenable to clinical development. We will screen the same DNA regions to develop siRNA capable of inducing TCF4 expression. We will use hNPC to perform the screening, and we will use Pitt Hopkins human brain organoids and oligodendrocytes to test in cell the efficacy to induce TCF4 expression and to revert abnormal biological phenotype reported.
2020
Differential analysis of genome-wide TCF4 binding in neural progenitor cells derived from Pitt Hopkins Syndrome patients and controls
Joseph L. McClay, PhD
Virginia Commonwealth University | 2019-2020
$50,000
Pitt Hopkins Syndrome (PTHS) is caused by mutations at the transcription factor 4 (TCF4) gene. The normal function of TCF4 is to regulate other genes in brain development, but we lack knowledge about how TCF4 mutations disrupt this process. A critical hurdle is that access to the relevant brain cell types is currently impossible from a living human being. Therefore, in the first stage of this project, we will produce the relevant human brain cell types by reprogramming skin cells from PTHS patients using cutting-edge cell technology. Specifically, we will generate neuroprogenitor cells, which are a type of stem cell that develop into to several of the most important brain cell types. Crucially, TCF4 is thought to regulate this developmental process. Therefore, in the second stage of the project, we will study how this regulation is disrupted by TCF4 mutation. We will use a method called chromatin immunoprecipitation sequencing to identify all the regions on neuroprogenitor cell DNA that TCF4 binds and therefore regulates. We will compare TCF4 binding patterns between cells from PTHS patients and controls to identify differences that may underlie pathophysiology in PTHS. Ultimately, our goal is to detect biological processes that are disrupted during brain development due to patient specific mutations in order to identify new potential drug targets to treat PTHS.
EXPLORING MECP2 OVER-EXPRESSION AS A POTENTIAL THERAPEUTIC STRATEGY FOR PITT HOPKINS SYNDROME
Colleen NIswender, PhD, NIswender Lab
Vanderbilt University, 2020
$33,532
Mutations in the MECP2 gene cause Rett Syndrome, a debilitating disorder similar to Pitt Hopkins. Because Rett Syndrome has a much larger diagnosed population than Pitt Hopkins and a longer medical history, research in this disorder is already on the cusp of gene therapy, with clinical trials slated to start later this year. The Niswender lab has preliminary data demonstrating that increased expression of the MeCP2 in Tcf4-deficient mice improves various PTHS symptoms. This grant will further explore the possible benefits of enhanced MeCP2 expression in Pitt Hopkins mouse models. While not as direct of an approach as a straight TCF4 reinstatement, it is anticipated that this approach may capitalize on existing MeCP2-based gene therapy.
Characterization of the expression of TCF4 mRNA and protein isoforms during the development of rodent and human brain
Tonis Timmusk, PhD
Tallinn University of Technology, Estonia | 2019-2020
$50,000
Mutations in the gene called TCF4 gene are associated with Pitt-Hopkins syndrome (PTHS) that features [developmental delay]. The mutation appears in only one of the two gene copies (alleles) of TCF4. Whereas in many other genes the other, unaffected allele, would be able to compensate for the defect, this is not the case in TCF4. This indicates that the protein encoded by the TCF4 gene is essential for the development of the nervous system, and that human development depends significantly on the amount of this protein in the brain and body. Most of the mutations found in PTHS patients are of de novo origin meaning that the mutation is not present in the parents. TCF4 gene has attracted wider interest because polymorphisms (genetic variations that may create
TCF4 predisposition to a disease) in this gene have been linked to schizophrenia.
TCF4 gene encodes a protein named Transcription Factor 4 (alias ITF2, SEF2 or E2-2). Transcription factors are proteins that regulate expression of genes. There are about 2000 different transcription factors encoded by the human genome. TCF4 is broadly expressed and involved in the development and functioning of many different tissues and cell types. We have previously demonstrated that TCF4 gene codes for numerous TCF4 protein isoforms of different length. All isoforms contain the sequences coded by downstream part of the TCF4 gene while only the longer isoforms contain sequences coded by the upstream part of the gene. Importantly, TCF4 protein isoforms vary in their subcellular distribution and ability to regulate transcription. Considering the diversity of TCF4 isoforms, it is important to find out which isoforms are expressed at the critical period of brain development when gene therapies for PTHS are planned to be developed. The aim of the project is to characterize developmental expression pattern of TCF4 mRNA and protein isoforms in different embryonic and postnatal stages of rodent and human brain development. Knowledge of the developmental expression dynamics of TCF4 isoforms in the brain is instrumental in achieving a better understanding about which TCF4 isoforms and at which developmental stage are needed to be delivered in gene therapy approaches for Pitt Hopkins syndrome.
Reinstatement of Tcf4 function to treat Pitt-Hopkins Syndrome
Principle Investigator (PI): Andrew Kennedy, PhD
Bates College
Awarded from the 2020 MDBR
UPenn Million Dollar Bike Ride Grant
$71,643
The Pitt-Hopkins research community is faced with the pressing question as to whether a gene replacement therapy is worth pursuing. Pitt-Hopkins Syndrome (PTHS), a rare disease characterized by intellectual disability, seizures, and developmental delay, is caused by the haploinsufficiency of the gene transcription factor 4 (Tcf4). Given the fledgling success of gene replacement therapy, mediated by viral vectors, it is critical to know if PTHS is caused by Tcf4-deficiency during development or in the functioning adult CNS. To test this idea, we propose that the selective knockout of Tcf4 in glutamatergic neurons in the adult CNS will elicit similar cognitive deficits as the systemic Tcf4 +/- mice, which models the lifelong Tcf4 haploinsufficiency of PTHS patients. These conditional knockout experiments will elucidate whether Tcf4 is necessary for proper function of excitatory neurons in the hippocampus and forebrain. Additionally, we will test the idea that the selective reinstatement of Tcf4 in glutamatergic neurons is sufficient to rescue cognition in PTHS model mice. Lastly, we propose that Tcf4 reinstatement in oligodendrocytes may function to ameliorate social aversion observed in PTHS mice. This study will elucidate how and when Tcf4-deficiency in mice induces phenotypes consistent with the human condition of PTHS.
2019
A human Pitt Hopkins Syndrome model for therapeutic proof of principle
Alysson R. Muotri, PhD
UC San Diego School of Medicine | 2018-2019
$100,000
We will use patient-derived neurons and cerebral organoids to investigate the cellular/neural phenotypes and molecular mechanisms underlying Pitt-Hopkins Syndrome. Moreover, we will use these tools to test correctional strategies to restore function to TCF4, the single gene causally implicated with the disease in humans. We anticipate that, through the aims outlined below, we will be able to better understand how TCF4 haploinsufficiency leads to the disabling neural phenotypes observed in affected individuals. Most importantly, this proposal will give the first steps toward applying epigenetic and genome editing technology based on the CRISPR/Cas9 system as a method to correct the genetic defects that accompany this severely disabling childhood syndrome. The use of such modern technology might soon be used to therapeutically correct expression of the affected gene and eventually cure the disease in human patients, which we can now envision could be safely and realistically performed in the near future.
TISSUE-SPECIFIC AND TEMPORAL REINSTATEMENT OF TCF4 TO TREAT PITT-HOPKINS SYNDROME, AWARD II
Principle Investigator (PI): Andrew Kennedy, PhD
Bates College
Awarded from the 2019 MDBR
UPenn Million Dollar Bike Ride Grant
$68,709
At present, the Pitt-Hopkins community is faced with the pressing question as to whether a gene replacement therapy is worth pursuing. Pitt-Hopkins Syndrome (PTHS), a rare disease characterized by intellectual disability, seizures, and developmental delay, is caused by the haploinsufficiency of the gene transcription factor 4 (Tcf4). Given the fledgling success and FDA approval of gene replacement therapy, mediated by viral vectors, to treat Spinal Muscular Atrophy, it is critical to know if Tcf4 replacement in post-mitotic neurons in a developed CNS would be sufficient to measurably improve cognition in PTHS model mice. In fact, investigating a gene replacement therapy is a specific goal of the Pitt-Hopkins Research Foundation, an organization of families and supporters that organize research on this ultra-rare disorder. Here, we propose testing whether cell-type specific knockout of Tcf4 gene function is sufficient to induce phenotypes my research lab has characterized in Tcf4 model mice. Moreover, we will test whether cell-type specific and temporal reinstatement of Tcf4 gene function is sufficient to rescue these cognitive measures. Taken together, these experiments will indicate whether a Tcf4 gene replacement therapy is worth pursuing, and what cell populations should be targeted in its construction.
Identification of small molecules upregulating Tcf4 in PTHS
Ben Philpot, PhD
University of North Carolina at Chapel Hill | 2019
UPenn Million Dollar Bike Ride Grant
Awarded from the 2018 MDBR
$50,000
PTHS is caused by a lack of transcription factor 4 (TCF4), but the molecular mechanisms of PTHS etiology are largely unknown. TCF4 expression increases during embryonic and early postnatal development, indicating that its transcription can be dynamically regulated. Thus, a potential treatment for PTHS is to reinstate normal TCF4 levels through pharmacologically upregulating the levels of the intact (non-mutated) copy of TCF4. To this end, we will establish the extent to which a novel potential activator compound, which we recently identified through a drug screen, can activate TCF4 isoforms. We will also perform structure-activity relationship studies to identify a lead compound in mouse cortical neurons and fibroblasts. Small molecule activators of TCF4 have the potential to treat PTHS.
GENERATION, VALIDATION, STORAGE, AND DISSEMINATION OF AN INDUCE PLURIPOTENT STEM CELLS (IPSC) COLLECTION DERIVED FROM PITT-HOPKINS SYNDROME (PTHS) PATIENTS
Brady J. Maher Ph.D.; Lead Investigator, Lieber Institute for Brain Development, Asst. Professor, Psychiatry and Behavioral Sciences, Asst. Professor, Dept. of Neuroscience, Asst. Professor Dept. of Biochemistry, Cell and Molecular Biology, Johns Hopkins School of Medicine, 2018-2019
$87,140
The development of new therapies for the treatment of neurodevelopmental disorders, such as PTHS, has been hindered by a large translation gap between animal models and humans. To overcome this translational gap, it is critical to develop human models of PTHS and test whether our current understanding of TCF4 function in animal models translates into human models. Generation of human models is now possible with the advent of reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). iPSCs provide a starting material that is perpetual and are capable of becoming any cell type of interest including cells of the central and peripheral nervous system which are disrupted in PTHS.
For PTHS, iPSCs are uniquely suited as a model system because of the neurodevelopmental nature of the disorder. Here, they propose to provide a perpetual resource for the PHRF by generating, validating and disseminating iPSCs derived from PTHS. In addition, this proposal will directly benefit his group research efforts focused on identifying therapeutic targets for PTHS by giving him immediate access to new patient cell lines. In this regard, his research group is actively studying human neural stem cells and neurons derived from PTHS patients and controls.
Tissue-specific and temporal reinstatement of Tcf4 function to treat Pitt-Hopkins Syndrome
Andrew Kennedy, PhD
Bates College
2019
Awarded from the 2018 MDBR
UPenn Million Dollar Bike Ride Grant
$50,000
The Kennedy Lab was awarded a one-year Orphan Disease Center Million Dollar Bike Ride Pilot Grant to study genetic and epigenetic therapies for Pitt-Hopkins Syndrome. Working in collaboration with Ben Philpot (UNC) and Brady Maher (Lieber Institute), we will test whether normalizing Tcf4 gene function in glutamatergic neurons during pregnancy and/or late adolescence is sufficient to enhance learning and memory in Pitt-Hopkins model mice when they reach adulthood. Additionally, we will test whether reinstatement of Tcf4 function in other cell types throughout the brain can affect cognition. These experiments are key in understanding the feasibility and design of a Tcf4 gene therapy. We are also investigating an epigenetic therapy for Pitt-Hopkins Syndrome that targets the Tet enzymes, which govern how many other genes are regulated in neuronal plasticity. We have demonstrated that Tet enzyme deletion enhances learning and memory in Pitt-Hopkins mice, and we have developed small molecules that target these enzymes. Currently, we are testing whether these drug-like molecules are sufficient to enhance cognition in our model mice.
2018
Protein Replacement Therapy Grant
Michelina Iacovino, PhD Assistant Professor
UCLA | 2017 – 2018
$25,000
Timeline and Milestones
Plasmid preparation: TCF4 reporter and TCF4 overexpressing construct about 4-6 months.
iPS cells generation (including breeding time for TCF4 mice) 3 month (this process will overlap
with Plasmid preparation).
Cell type system for TCF4 overexpression and TCF4 uptake 1-2 months and will follow plasmid
preparation.
Uptake assay and TCF4 protein purification 2-4 month following Cell preparation.
Sarah Huffman Award for Promising Young Researchers: Novel Therapeutics for Pitt Hopkins Syndrome
Annah Moore, Graduate Student, Pharmacology Department Vanderbilt University under the Mentorship of: Dr. David Sweatt, Chairman, Department of Pharmacology and Dr. Colleen Niswender, Research Associate Professor of Pharmacology
Vanderbilt University | 2018
$62,000.00
This project is focused on the discovery of small molecules designed by the Vanderbilt Center for Neuroscience Drug Discovery as potential therapeutics for Pitt Hopkins Syndrome (PTHS). Preliminary experiments have shown cognitive enhancing properties in Pitt Hopkins model mice after treatment with a drug that acts at the M4 muscarinic acetylcholine receptor. This compound will be tested in additional mouse models for PTHS and additional candidate drugs will also be investigated. Abnormal breathing has been documented in a PTHS mouse line, and further characterization this trait is planned.
Understanding Pitt-Hopkins Disease from a microbiome and metabolome perspective
Daniel McDonald, Ph.D.
American Gut Project, Robert Knight Lab,
University of California San Diego
$65,000
Pitt-Hopkins Syndrome (PTHS) is a rare genetic disorder characterized as a severe form of autism spectrum disorder (ASD). Afflicted individuals exhibit a high incidence of constipation which is often a focal point of longterm care. Relief of constipation has been observed by family members to reduce the rate of seizures, and overall improve the quality of life. Prior research has suggested relationships between the gut microbiome and gastrointestinal distress as well as autism related symptoms in individuals clinical diagnoses of ASD. Here, we provide observations of the pilot microbiome and metabolome investigation into PTHS.
Pilot Study to Identify Small Molecule Activators of TCF4
PI: Philpot, Benjamin D., PhD. | 2017 – 2018
University of North Carolina at Chapel Hill
Million Dollar Bike Ride Pilot Grant (UPenn)
$49,263.00
Pitt-Hopkins syndrome (PTHS) is a complex neurodevelopmental disorder caused by insufficiency of the transcription factor 4 (TCF4). The reduced TCF4 expression is caused by a mutation in one of the two gene copies of TCF4. However, the other TCF4 gene copy remains functional. As one potential treatment for PTHS, we are trying to pharmacologically upregulate the levels of the intact copy of TCF4. To this end, we are using a high throughput drug screen to find small molecules that increase the activity of the intact TCF4 gene. The TCF4 activators that we identify have the potential to treat PTHS by rebalancing TCF4 activity.
Epigenetic Therapy to Treat Pitt-Hopkins Syndrome
Andrew J. Kennedy, PhD | 2017-2018
Department of Chemistry and Biochemistry, Neuroscience Program, Bates College
Million Dollar Bike Ride Pilot Grant (UPenn)
$49,263.00
We aim to use a mouse model to test the hypothesis that an epigenetic therapy can be effective in treating cognitive deficits associated with PTHS.
PTHS Late Onset Gene Therapy Grant
Ben Philpot, PhD, UNC-CH, and Steve Gray, PhD
UT Southwestern | 2018
$123,970
Our labs are currently developing and testing adeno-associated virus (AAV) vectors expressing human TCF4 isoforms (hTCF4) to determine whether early postnatal introduction of TCF4 can normalize phenotypes in Pitt-Hopkins syndrome (PTHS) model mice. Our preliminary data indicate that AAV9/hTCF4(B-) vectors are well tolerated when delivered at the highest concentration to neonates. Encouraged by our preliminary findings suggesting safety of the viral vectors, we will now determine the extent to which adult-onset gene therapy can correct PTHS phenotypes. Further, we will test additional isoforms of TCF4 to evaluate if hTCF4(B-) is the best candidate for gene therapy. These experiments will complement ongoing experiments in which we are optimizing protocols and testing for recovery with neonatal gene therapy.
2017
Identification of Direct Target Genes of TCF4 in Neurons
Tonis Timmusk, PhD, Principal Investigator, Mari Sepp, PhD, Co-Principal Investigator
Tallinn University of Technology, Estonia | UPenn, MDBR, 2017
$50,000
Pitt Hopkins syndrome (PTHS) is a genetic developmental disorder that severely affects cognitive, motor and social development. PTHS is charachterized by distinct facial features, absent speech, absent or delayed walking, low muscle tone, gastrointestinal problems and autistic-like behaviour. The patients may develop breathing problems and/or epilepsy. PTHS has been diagnosed in less than 1000 people in the world. It is caused by mutations in one of the two alleles of a gene called TCF4. Most of the mutations found in PTHS patients are of de novo origin meaning that the mutation is not present in the parents. TCF4 gene has attracted wider interest mainly due to the fact that polymorphisms (genetic variations that may create predisposition to a disease) in this gene have been linked to schizophrenia.
TCF4 gene encodes a protein named Transcription Factor 4 (alias ITF2, SEF2 or E2-2). Transcription factors are proteins that regulate expression of genes. There are about 2000 different transcription factors encoded by the human genome. TCF4 is broadly expressed and involved in the development and functioning of many different tissues and cell types. Evidence is accumulating that in the nervous system TCF4 plays an important role in proliferation, differentiation and migration of neurons, as well as brain plasticity – a process that enables the brain to rewire itself in response to the stimuli from learning and experience.
The goal of the current project is to identify genes that are regulated by TCF4 in the brain neurons. We will generate adeno-associated virus based vectors for overexpression and knockdown of TCF4 protein, identify TCF4-regulated genes and determine the binding sites of TCF4 in its target genes. The knowledge of TCF4 target genes is instrumental in deciphering the role of TCF4 in biological processes that contribute to the pathology of Pitt Hopkins syndrome. It also allows us to test whether increasing the activity of the remaining TCF4 protein (produced from the intact allele in PTHS patients) by pharmacological modulation is feasible for PTHS treatment. Focusing on target genes that are related to brain plasticity, a process that is ongoing lifelong, enables us to obtain insights into adult functions of TCF4 that in turn may represent suitable targets for therapeutic intervention of Pitt-Hopkins syndrome.
Pharmacological rescue and screening in a Pitt Hopkins model
Daniel R. Marenda Ph.D., Principal Investigator
Drexel University | UPenn, MDBR, 2017
$50,000
Screening drugs in cells and animals is essential to identifying therapeutic compounds and targets for disease. Though screening drugs in cells is fast, these drugs often fail when tested in animal models of a disease. However, screening in animal models (such as mice and rats) is far too expensive and time consuming to make large scale drug screening feasible in most cases. This proposal will utilize a novel animal model for Pitt Hopkins by using the fruit fly Drosophila melanogaster to screen for potential therapeutic compounds for the treatment of this disorder. Drosophila offer a powerful, well tested, cheap, and quick way to test for new drugs in an established animal model.
Using an established Drosophila model for Pitt Hopkins, we performed a small drug screen and identified three drugs that have been previously identified as having potential for therapeutic use in Pitt Hopkins, suggesting that this model is capable of identifying molecules with the potential for further exploration. We are expanding upon this approach, and will use this animal model to screen for additional compounds in a large library of drugs. Our hope is to identify new compounds in this animal model which can be further explored in mammalian models of Pitt Hopkins, and eventually be furthered explored in humans.
Ann D. Bornstein Gene Therapy Grant
PTHS Gene Therapy Grant
Steve Gray, PhD, Principal Investigator, and Ben Philpot, PhD, Co-Principal Investigator
University of North Carolina at Chapel Hill | 2016 – 2017
$68,156
The laboratories of Dr. Ben Philpot and Dr. Steven Gray at the University of North Carolina at Chapel Hill are collaborating on a project to investigate the feasibility of a gene therapy approach for Pitt-Hopkins syndrome (PTHS). This collaborative study combines Dr. Philpot’s expertise in autism and neuroscience with Dr. Gray’s expertise in translational gene therapy for neurological disorders. The project will follow a platform gene transfer approach using AAV vectors taken by Dr. Gray to initiate a human Phase I trial for Giant Axonal Neuropathy. The approach uses an engineered virus, AAV, to carry a functional copy of the gene disrupted in PTHS into the body and distribute it across the nervous system. In this fashion, a single dose of this gene therapy could permanently restore the gene to cells across the nervous system, treating the disease at its source. This initial pilot study is meant to assess the potential of this as a treatment approach for PTHS, and identify any roadblocks that may exist.
A Model of Pitt-Hopkins Syndrome and Efficacy Testing of HDAC Inhibitors
Jackson Lab, HDACi: Vorinostat Control and Other HDACi | 2017
FIRST INSTALLMENT: $27,117
Validation of behavioral testing in B6;129-Tcf4tm1Zhu/J mice.
A Drug Repurposing Project
to identify potential drug candidates for the treatment of Pitt Hopkins Syndrome
Timm Guillams, PhD, Healx, Cambridge, UK
2017 – 2018
$45,000
Phase 1: Bicuration: A biocuration phase precedes the full drug repurposing work to provide a comprehensive data analysis and landscape of PHTS and its current treatment options. This first step is essential to ensure drug repurposing predictions with a higher likelihood of success.
Phase II: Drug Repurposing & Healx Methods, Drug-Gene Expression Matching, Prediction of Repurposed Indications with Similarity Matrices, Similarity Network Analysis based upon Predicted Protein Targets, Literature Mining, Drug Ranking
Phase III: Expert Pharmacology Review
Single Concentration Re-purposing Screen
Aaron Gerlach, Ph.D, Icagen Inc., and Sean Ekins, PhD
Ion Channel Inhibition Grant | 2017
$35,000.00
Starting the deeper dive of Five top compounds from Initial screen. Larger drug repurposing proposal in review.
Aim of study: The aim of the study is to test the inhibitor activity of the 1280 compound Prestwick library against recombinantly expressed human KCNQ1 and Nav1.8 channels.
2016
Awarded $75,000, PHRF, 2016: Benjamin D. Philpot, Ph.D., Principal Investigator; Alexander D. Kloth, Ph.D., Co-Principal Investigator; Courtney L. Thaxton, Ph.D., Co-Principal Investigator, The University of North Carolina at Chapel Hill
Characterization and Generation of PTHS Model Mice for Rational Therapeutic Discovery
Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder characterized by intellectual disability, absent speech, seizures, ataxia, and breathing anomalies. In support for future therapeutic development for PTHS, we will pursue two independent aims: (1) to uncover the neural impairments that are common across multiple PTHS mouse models, and (2) to develop new tools to analyze TCF4 expression in neuronal subtypes throughout development and adulthood. In the first aim, we will follow up on our finding that long-term changes in synaptic function related to experience are enhanced in multiple PTHS-related mouse models. We hypothesize that this deficit is related to altered function of a glutamate receptor, the NMDA receptor, and we will rigorously test this hypothesis using electrophysiology, biochemistry and pharmacological methods in multiple PTHS-related mouse models. In the second aim, we will develop a unique mouse model toward determining effective drug targets that affect TCF4 expression levels, as well as be able to alter TCF4 activity in a spatiotemporal manner. This novel binary “reporter-reinstatement” mouse will not only allow for a stream-lined and genetically precise approach to drug discovery for PTHS, but also will allow us to determine the most efficacious time in which to reinstate TCF4 function to alleviate the pathophysiologies associated with PTHS. In all, the proposed project pursues incisive approaches that will provide guidance to the development of PTHS therapeutics.
Awarded $75,000, PHRF, 2016: Andrew John Kennedy, Ph.D., Principal Investigator, Bates College; J. David Sweatt, Ph.D., Co-Principal Investigator, Vanderbilt University
Kindal Kivisto Award for Promising Young Researchers:
Investigating Therapies for Pitt-Hopkins Syndrome
The central strategy of our research program consists of two goals: the near-term goal to identify FDA approved drugs as potential translatable therapies for Pitt-Hopkins Syndrome (PTHS) and the long-term goal to develop novel neuroepigenetic therapies that fundamentally reverse the effects of PTHS. Over the past three years, we have characterized a genetically engineered heterozygous deletion mouse model of PTHS (Tcf4 +/-), validated the histone deacetylase enzyme Hdac2 as a target to treat the cognitive deficits associated with PTHS, and undertaken a drug screening program. This grant will investigate the efficacy of Fingolimod (trade name Gilenya), as well as other FDA approved therapeutics that target Hdac2, to improve learning, problem solving, and associative memory in PTHS mice. These experiments will focus on identifying a plausible drug candidate that can be translated to a clinical setting and effectively improve cognition in PTHS patients. Additionally, more advanced epigenetic therapies will be developed to address the genetic cause of PTHS. Every person has two functioning copies of Tcf4 with the exception of individuals with PTHS, who have a mutation or deletion that yields only one functioning copy. Epigenetic therapies, which alter the epigenetic states at specific genes within the genome, are being designed to allow PTHS models to use their one functioning copy of Tcf4 twice as much, hopefully restoring full Tcf4 function and reversing the cognitive deficits associated with Pitt-Hopkins. Taken together, these approaches investigate already-available FDA approved drugs and cutting edge genetic techniques to identify potential therapies that improve cognition in the near-term and attempt to address and compensate for the underlying cause of Pitt-Hopkins Syndrome.
Awarded $75,000, PHRF, 2016: Benjamin D. Philpot, Ph.D., Principal Investigator; Alexander D. Kloth, Ph.D., Co-Principal Investigator; Courtney L. Thaxton, Ph.D., Co-Principal Investigator, The University of North Carolina at Chapel Hill
Investigating Therapies for Pitt-Hopkins Syndrome
Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder characterized by intellectual disability, absent speech, seizures, ataxia, and breathing anomalies. In support for future therapeutic development for PTHS, we will pursue two independent aims: (1) to uncover the neural impairments that are common across multiple PTHS mouse models, and (2) to develop new tools to analyze TCF4 expression in neuronal subtypes throughout development and adulthood. In the first aim, we will follow up on our finding that long-term changes in synaptic function related to experience are enhanced in multiple PTHS-related mouse models. We hypothesize that this deficit is related to altered function of a glutamate receptor, the NMDA receptor, and we will rigorously test this hypothesis using electrophysiology, biochemistry and pharmacological methods in multiple PTHS-related mouse models. In the second aim, we will develop a unique mouse model toward determining effective drug targets that affect TCF4 expression levels, as well as be able to alter TCF4 activity in a spatiotemporal manner. This novel binary “reporter-reinstatement” mouse will not only allow for a stream-lined and genetically precise approach to drug discovery for PTHS, but also will allow us to determine the most efficacious time in which to reinstate TCF4 function to alleviate the pathophysiologies associated with PTHS. In all, the proposed project pursues incisive approaches that will provide guidance to the development of PTHS therapeutics.
Awarded $50,0000, UPenn, MDBR, 2015-2016: Brady Maher, Ph.D., Principal Investigator; Huei-Ying Chen Ph.D.; Stephanie Cerceo-Page, Ph.D.; Lieber Institute for Brain Development, Johns Hopkins School of Medicine
Exploring the Impact of a TCF4 Mutation on the Physiology of Inhibitory Neurons of the Prefrontal Cortex
PTHS is a neurodevelopmental disorder due to mutation or deletion of one copy of the TCF4 gene. TCF4 is a transcription factor that can regulate the expression of many downstream genes and therefore regulates the genetic programs necessary for normal brain development. We measured the expression of TCF4 mRNA across the lifespan in humans and rodents and observed a peak in TCF4 expression occurs during the formation of the cerebral cortex, a region of the brain important to higher cognitive functions including learning and memory. Using a mouse model of PTHS that has a mutation in one copy of the TCF4 gene, we observed that TCF expression is blunted during the developmental peak in expression compared to control animals, and we believe this indicates a causal time period for the development of PTHS. Unfortunately, this critical period occurs in utero and prior to when diagnosis is currently made, thus complicating our ability design treatment strategies during this causal phase of the disorder. Therefore, our research group is focused on understanding the underlying pathophysiology that produces symptomatology in PTHS so that we can normalize this pathophysiology in children and adults. Using our animal models of PTHS, we have identified a sodium channel that is normally expressed in the peripheral nervous system, but is ectopically expressed in the central nervous system when TCF4 is mutated. Experiments are currently underway to determine if blocking this Na channel with drugs can lead to improvement on behavioral tests in our PTHS mouse model. In our current proposal, we would like to follow up a preliminary result that suggests inhibitory transmission onto excitatory neurons in the cortex is decreased in the PTHS mouse compared to control littermates. In addition, using RNA sequencing of the PTHS mouse model we observed that many genes that are specific to inhibitory neurons show decreased expression compared to control animals, and we identified a specific population of inhibitory neurons (cortistatin positive) that normally show high levels of TCF4 expression. These cortistatin positive interneurons are known to release a neuropeptide called cortistatin that has been shown to inhibit the generation of seizures and regulate sleep states. Given the prevalence of seizures and sleep disturbances in PTHS, we believe this population of inhibitory neurons may underlie clinical aspects of the disorder. Therefore, we propose to breed the PTHS mouse with another mouse that allows us to visualize cortistatin positive interneurons and we will use electrophysiology and microscopic imaging to determine if these cells are disrupted in the PTHS mouse model compared to control littermates. If deficits are observed in this population we will determine the cellular and molecular mechanism using pharmacological rescue and/or molecular phenocopy. Identified molecular mechanisms will then be deemed potential therapeutic targets and these targets will be tested for their ability to normalization of behavioral deficits in the PTHS mouse.
Tilly Hadlow Young Investigator Award: Joseph Alaimo, Ph.D., Principal Investigator; Sarah Elsea, Ph.D., Co-Investigator/Mentor, Baylor College of Medicine, 2016
Delineating Therapeutic Targets using Global Metabolic Profiling in Pitt Hopkins Syndrome
Defining the cellular defects due to alterations in TCF4 function is paramount in order to determine the proper molecular and biochemical targets for therapeutic intervention in Pitt-Hopkins syndrome. To identify and characterize the biochemical and molecular dysfunction due to altered TCF4 function, we plan to take a clinical and translational approach by recruiting a cohort of individuals with PTHS in collaboration with the Pitt-Hopkins Research Foundation and current PTHS clinics, phenotypically and molecularly characterizing the cohort, and employing state-of-the-art metabolomics screening to identify pharmacologically targetable molecular and biochemical pathways. Our unique approach will utilize a special type of biochemical genetic test called global metabolomics assisted pathway screening (Global MAPS). Global MAPS is currently the most comprehensive small molecule screen available in the clinical setting and is only available through Baylor College of Medicine’s Biochemical Genetics Diagnostic Laboratory. Global MAPS surveys greater than 1000 small molecules in human plasma, pinpointing defects in pathways unmeasurable by standard clinical testing methods, offering a comprehensive and in-depth analysis of patient samples and metabolic status. Our overall goal is to employ Global MAPS analysis in patients with PTHS to identify novel pathway alterations and to understand the basis of TCF4 function in cells. In addition, our proposal will serve as a functional confirmation of current molecular findings in PTHS research, including RNA-sequencing and gene expression profiling, thereby refining the molecular and biochemical targets that would benefit most from therapeutic intervention. Our novel but complementary approach will promote additional analysis to identify points of data convergence among other research groups thereby expediting the process toward targeted therapeutic intervention and clinical trials.
Awarded $50,0000, UPenn, MDBR, 2015-2016: Tõnis Timmusk, Ph.D., Principal Investigator; Mari Sepp, Ph.D., Co-Investigator, Tallinn University of Technology, Estonia
Regulation of TCF4 Transcriptional Activity in Neurons
Transcription factor TCF4 (alias ITF2, SEF2 or E2-2) is a broadly expressed protein involved in the development and functioning of many different cell types. Recent studies point to important roles for TCF4 in the nervous system. Specifically, human TCF4 gene is implicated in susceptibility to schizophrenia and mutations in TCF4 cause Pitt-Hopkins syndrome (PTHS), a rare developmental disorder characterized by severe motor and mental retardation, typical facial features and breathing anomalies. The mutation may be in different parts of the gene, but it appears in only one allele. Whereas in many other genes the other, unaffected allele would be able to compensate for the defect, this is not the case in TCF4. This indicates that the protein encoded by the TCF4 gene is essential for the development of the nervous system, and that human development depends significantly on the amount of this protein in the brain and body. Our previous data have suggested that synaptic activation of nerve cells, that is the basis of brain function, leads to activation and phosphorylation of TCF4 protein. Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation turns many proteins on and off, thereby altering their function and activity. The current project is aimed to find out how the activity and phosphorylation of TCF4 protein is regulated inside nerve cells of the brain, and to characterize the phosphorylation pattern of activated TCF4. Additionally, we want to determine which genes are targeted by TCF4 in nerve cells after synaptic activation. Since Pitt-Hopkins syndrome manifests itself at an early stage, there are better chances for its treatment due to the greater plasticity of children’s brains. Increasing the amount and/or activity of the functional TCF4 protein produced from the healthy allele is among possible approaches to develop drugs for Pitt-Hopkins syndrome treatment. We believe that our project could lead to the discovery of novel possibilities for increasing the activity of TCF4 in nerve cells that could be useful to develop treatments for therapeutic intervention of Pitt-Hopkins syndrome.
2014 – 2015
The PHRF awarded four research grants and two additional research grants through UPenn Million Dollar Bike Ride.
A total of $430,000 awarded for Pitt Hopkins research this year.
$80,000 One Year Grant: Dr. Andrew Kennedy, University of Alabama Birmingham
Investigating Therapies for Pitt Hopkins syndrome
Investigating Therapies for Pitt-Hopkins Syndrome: With the financial support of the Pitt-Hopkins Research Foundation, researchers in the Department of Neurobiology at the University of Alabama at Birmingham have started a research program to investigate the molecular and neural basis of Pitt-Hopkins Syndrome (PTHS). Their research bridges clinical and basic neuroscience by investigating the underlying neurobiology of PTHS and testing potential therapies for cognitive dysfunction using a PTHS mouse model. Specifically, Dr. Kennedy is working toward understanding how histone deacetylase (HDAC) inhibitors may serve to improve cognition in PTHS, and whether the endogenous HDAC inhibitor sphingosine 1-phosphate can serve as a blue print for a more comprehensive therapy. Using next-generation sequencing technology, the genetic and epigenetic consequences of PTHS that result in diminished cognition will be elucidated, which will significantly enhance our understand of the role Tcf4 plays in the learning and memory and help us identify critical downstream transcriptional targets. This grant also serves to broaden the scope of investigation to include potential therapies to improve myelination as well as alleviate the hyperventilation, apnea, and constipation associated with PTHS.
$80,000 One Year Grant: Dr. Stephen J. Haggarty, Harvard Medical School, Massachusetts General Hospital
Characterization of Pitt Hopkins Syndrome Stem Cell Models & Therapeutic Screening
(stem cells created from skin-derived fibroblasts of patients with Pitt Hopkins syndrome)
Developing therapeutics for neurodevelopmental disorders is one of the greatest medical needs of the 21st century. Of particular interest for defining underlying molecular and cellular mechanisms of these disorders is Pitt-Hopkins syndrome (PTHS), a neurodevelopmental disorder characterized by severe intellectual disability, seizures, as well as a other peripheral symptoms including failure to thrive, chronic constipation and recurrent facial dysmorphology. PTHS is tightly associated with mutations in the TCF4 gene encoding isoforms of the basic helix-loop-helix (bHLH) transcription factor 4 (TCF4), and recent genetic studies are beginning to connect other genes within the PTHS pathway, including patients with mutations in NRXN1 (neurexin 1) and CNTNAP2 (contactin-associated protein-like 2) that exhibit PTHS-like symptoms. The overall goal of our project is to advance a personalized medicine program for PTHS that seeks to identify novel targets and experimental therapeutics by testing approved drugs and novel synthetic compounds in patient-derived stem cell models in order to select compounds for testing in the animal model of PTHS, and eventually in PTHS patients given appropriate efficacy and safety properties. To this end, we have established a PTHS biorepository consisting of patient skin biopsy-derived fibroblasts and begun to use reprogramming technologies to create a panel of induced pluripotent stem cells (iPSC). The development of these powerful new in vitro culture systems allows: i) investigation of the step-by-step development of neuronal phenotypes due to TCF4 mutations; ii) determination of the role of TCF4 in neuronal gene expression; and iii) the opportunity to screen for experimental therapeutics that can enhance or restore TCF4 expression. Deep phenotyping of neurons derived from PTHS patient iPSCs is on-going using a variety of strategies ranging from imaging of their morphology and neurodevelopmental capacities using automated microscopy to genome-wide analysis of their transcriptome. With our preliminary findings, we have identified a set of novel pathways and targets for modulating TCF4 expression and are currently working to understand their precise mechanism of action as well as to identify compounds with improved efficacy and pharmacological properties. On-going efforts are also to expand the collection of PTHS iPSC models with a range of different genetic mutations and clinical symptoms as well as to generate isogenic controls of patient-derived iPSC models with the use of genome-editing methods to ‘correct’ mutations
$80,000 One Year Grant: Dr. Hazel L. Sive, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology
Characterization and Therapeutic Screening of Pitt-Hopkins Syndrome Using the Zebrafish
Our research has two goals: first, to understand how each variant form of TCF4 leads to specific symptoms of a PTHS patient, and second, to identify chemicals that change increase levels of TCF4, and that in the long term, may lead to therapies. We use the powerful zebrafish system where the transparent larva allows detailed analyses in single cells of the living animal. Fish can show many aspects of PTHS and can connect particular TCF4 mutations and particular symptoms. Zebrafish tcf4 and human TCF4 genes are similar and ‘humanized’ fish can be made where the human gene has been substituted for the fish gene, and so readily studied. Since zebrafish are small, they can be used in whole animal screens to test chemicals that may be potential medications. We have developed fish with reduced or no activity of the fish tcf4 gene, and will test these for analysis of brain development and function, face formation, digestive system formation and function, and muscle function, all of which can be affected in PTHS patients. We will work collaboratively with Dr. Stephen Haggarty (MGH) who uses patient-derived stem cells, making a powerful coordinated effort to understand and treat PTHS.
$80,000 One Year Grant: Dr. Brady Maher, Lieber Institute for Brain Development, Johns Hopkins School of Medicine
Exploring Changes in Neuronal Translatomes in both Cell and Non-Cell Autonomous Animal Models of Pitt Hopkins Syndrome
PTHS syndrome results from a mutation in one copy of the TCF4 gene that results in haploinsufficiency. TCF4 is a transcription factor that depending on the target gene can either produce repression or activation of target gene transcription. Therefore, PTHS results from dysregulation of target gene expression during fetal development. To begin to identify therapeutic targets for the treatment of this disorder we propose to identify target genes that are dysregulated downstream of TCF4 haploinsufficiency. We show that embryonic knockdown of TCF4 expression results in intrinsic excitability deficits in excitatory neurons of the rat frontal cortex. Using a novel combination of molecular approaches (iTRAP), we identify two ion channel genes (Kcnq1, Scn10a) whose expression is increased when TCF4 is suppressed and appear to be responsible for producing excitability defects observed with TCF4 knockdown. These proof-of-principle experiments suggest these methods are effective at identifying TCF4 target genes and therefore we propose to expand this technology to whole-genome RNA sequencing to identify more genes that are regulated by TCF4. To increase the validity of our results we propose to compare to independent animal models of PTHS. A cell autonomous model that uses shRNA constructs to knockdown the expression of TCF4 in a subset of neurons in the frontal cortex and a non-cell autonomous model (TCF4+/- mouse) in which the whole animal expresses only one copy of the TCF4 gene. In both animal models, we will use IUE to transfect a pure population of layer 2/3 neurons with the ribosomal protein L10a-EGFP and use antibodies against EGFP to affinity purify RNA at two distinct developmental time points. The first time point will be at the developmental peak of TCF4 expression and the second time point will be during rodent adolescents. We predict the early time point will identify target genes that are causal for PTHS and the adolescent time point will identify genes that maybe more suitable for treatment in the patient population.
UPenn Million Dollar Bike Ride Awards
$55,000 One Year Grant: Dr. Courtney Thaxton and Dr. Benjamin D. Philpot, University of North Carolina at Chapel Hill
Identification of Genetic and Molecular Targets for Pitt-Hopkins Therapeutics
A major hurdle hindering the discovery of therapeutics to treat Pitt Hopkins Syndrome (PTHS) is the lack of knowledge of the genetic and molecular targets of TCF4 in the nervous system. Current research is utilizing a mouse model that lacks one complete copy of Tcf4, but to date there are no models phenocopying missense or nonsense mutations associated with PTHS. Both missense and nonsense mutations in TCF4 comprise the largest number of causative mutations for PTHS. Therefore, we generated a new mouse model mimicking the most prevalent mutation found in PTHS, as well as a unique mouse model lacking several common mutation sites linked to PTHS. We will use these two new mouse models, as well as a previously developed Tcf4-deletion mouse model, to uncover the genes and molecular pathways regulated by Tcf4. These mice will also be assessed for behavioral changes in order to identify potential genotype-phenotype characteristics. By discovering the genes or common pathways regulated by TCF4, we may be able to identify approved therapeutics that target these affected pathways or design new drugs to reverse the disregulation caused by the absence of TCF4.
$55,000 One Year Grant: Dr. Daniel Marenda, Drexel University, and Dr. Wenhui Hu, Temple University School of Medicine
Understanding TCF4 Function in Post-Mitotic Neuron Synaptic Plasticity
In the study of human disease, animal models often act as surrogates for people when (as is often the case) testing on humans is unethical or not feasible. The common thread of evolution among species allows for discoveries that are made in nonhuman species to be applied across the diversity of life forms, from single-celled bacteria and yeast to large, complex, many-celled human beings. These nonhuman surrogates are collectively known as “model organisms”. They bring with them an enormous battery of sophisticated experimental tools that allow for the study of human diseases and offer clues about the underlying factors that contribute to these diseases.
In collaboration with Dr. Wenhui Hu’s laboratory at Temple University, Dr. Daniel Marenda at Drexel University is using two of these model organisms (the fruit fly and the common mouse) to further understand the function of the TCF4 gene. TCF4 is well known to function during development, to help form proper muscle cells, nerve cells, and other tissues in the body. After these cells develop, they form mature cells (that is, cells that do a job). Dr. Marenda and Dr. Hu have discovered that while TCF4 does function to help cells develop, TCF4 also functions in mature cells as well (particularly mature nerve cells). This is exciting, as there previously has been no described function of TCF4 in mature nerve cells. Further, this opens the possibility of therapeutic intervention for Pitt-Hopkins outside of embryonic development. The hope is that once we better understand what TCF4 is doing in mature nerves, we will be better able to intervene with drugs to fix TCF4 function in those mature cells when TCF4 function is lost due to mutation.
2013 – 2014
$80,000 One Year Grant: Dr. David Sweat, University of Alabama Birmingham
New Research Into the Neurobiological Basis of PTHS
The underlying genetic basis of Pitt-Hopkins Syndrome (PTHS) has recently been discovered – PTHS is caused by heterozygous null mutation or deletion of the TCF4 gene on human chromosome 18. The identification of the dysfunctional TCF4 transcription factor gene as the genetic basis of the disorder is a critical step forward in beginning to understand the diagnosis, etiology and molecular biology of PTHS. This project encompasses a set of studies to investigate the cognitive dysfunction associated with PTHS, focusing on mechanistic studies to understand the role of the TCF4 transcription factor in central nervous system function, using genetically engineered mouse models for PTHS. The project is focused on investigating: 1. Cognitive dysfunction in the domain of learning and memory; 2. Alterations in CNS neuronal function and the regulation of long-term gene readout in the CNS, and; 3. Testing one specific hypothesis concerning a potential new drug treatment for PTHS. The longer-term priorities for the proposed project will be two-fold: rapid drug development of a new PTHS treatment and understanding the molecular biology underlying PTHS intellectual disability.
$80,000 One Year Grant: Dr. Stephen J. Haggarty, Harvard Medical School, Massachusetts General Hospital
Generation and Characterization of Pitt-Hopkins Syndrome Stem Cell Models
Developing therapeutics for neurodevelopmental and intellectual disability disorders presents significant challenges and is one of the greatest areas of unmet medical need in the 21st century. Of particular interest for gaining insight into the underlying molecular and cellular mechanisms of neuroplasticity and cognition is Pitt- Hopkins syndrome, an autism-spectrum disorder, known to be caused by mutations in the TCF4 gene on Chromosome 18q21. The TCF4 gene is known to encode multiple isoforms of the basic helix-loop-helix (bHLH) transcription factor 4 (TCF4). Gaining a more complete understanding of the regulation and function of TCF4 in the human nervous system may provide critically needed insight into the pathophysiology of multiple human disorders sharing the features of intellectual disability and developmental delay.
With the overarching goal of advancing a Personalized Medicine Program for Pitt-Hopkins syndrome that seeks to identify novel targets and experimental therapeutics for testing in pre-clinical animal models and eventually in patients, we have established a Pitt-Hopkins syndrome biorepository and are actively collecting skin-derived fibroblasts from patients from which we have created multiple patient-specific, induced pluripotent stem cell (iPSC) models. The development of these powerful new in vitro culture systems now allow: i) investigation of the step-by-step development of neuronal phenotypes due to TCF4 mutations; and ii) determination of the role of TCF4 in neuronal gene expression. In addition to generating cellular tools to study TCF4 function, we are also developing and validated molecular probes to quantify the expression of multiple TCF4 mRNA transcripts in response to experimental therapeutics along with molecular tools for silencing and nucleotide-specific genome editing at the TCF4 locus to create isogenic TCF4 mutant and corrected iPSCs.
Using these novel cellular and molecular tools, we aim to address whether the loss of function of TCF4 leads to deficits in the regulation of genes important for synaptic functions that are known to play an important role in cognition. We will also be able to compare the phenotypes of our Pitt-Hopkins syndrome patient-derived neurons to those of other neurodevelopmental disorders, such as Fragile X syndrome and Rett syndrome, that have been developed in our lab in order to identify potentially distinct, as well as shared, aspects of each disorder.
Overall, we anticipate that our research, in combination with studies using animal models, will help lay the foundation for the discovery of therapeutic agents targeting early steps of the pathophysiological mechanisms associated with Pitt-Hopkins syndrome. In doing so, these efforts are likely to advance our understanding of the molecular and cellular neurobiology of neurodevelopmental disorders that share common mechanisms of aberrant chromatin-mediated neuroplasticity.
$50,000 One Year Grant: Dr. Hazel L. Sive, Whitehead Institute for Biomedical Research, Massachusetts Institute of Technology
Characterization and Therapeutic Screening of Pitt-Hopkins Syndrome using the Zebrafish
We propose to develop the zebrafish as a tool to understand the biology of and to assess potential therapeutic treatments for Pitt-Hopkins Syndrome (PTHS), a rare but devastating disorder associated with the TCF4 gene. While there are common symptoms associated with PTHS including intellectual disability, locomotion deficits, epilepsy and chronic constipation, the severity of these can vary between patients. The connection between the multiple mutations observed in the TCF4 gene in individual patients and the symptoms of each patient is not understood. This study will contribute to a “personalized” approach in categorizing PTHS TCF4 genes and patient symptoms. The study will also use a whole animal screening approach to identify chemicals that may lead to treatments for PTHS.
$50,000 One Year Grant: Dr. Courtney Thaxton and Dr. Benjamin D. Philpot and Dr. Mark Zylka, University of North Carolina at Chapel Hill
Identification of Genetic and Molecular Targets for Pitt-Hopkins Therapeutics
Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder on the autism spectrum. Since the discovery of TCF4 as the causative gene for Pitt-Hopkins syndrome (PTHS), little progress has been made towards uncovering the genetic and molecular pathways regulated by TCF4 and their involvement in brain functions. Progress has been further limited due to the embryonic lethality observed in mouse models that entirely lack expression of the TCF4 gene. Here, we will uncover the genes and molecular pathways regulated by TCF4 in primary brain neurons, a principal cell type affected in PTHS, with the expectation that this information will provide the most rational path towards designing therapeutic strategies. We will use whole- genome analyses to identify every gene affected by different genetic models of PTHS. To mimic large deletions of TCF4, we will determine the genetic and molecular consequences of TCF4 haploinsufficiency. To mimic PTHS-causing mutations, we will establish the consequence of common and recurring TCF4 point mutations on gene expression. By comparing the genes affected by these diverse manipulations associated with PTHS, we will better pinpoint the relevant genetic and molecular pathways to be targeted for future therapeutics.
$20,000 One Year Grant to develop new antibodies and testing those antibodies with the specific goal of identifying a ChIP-grade antibody. Once ChIP grade antibodies are identified, funding to complete aims 1 and 2 could be negotiated, at 30-50K to continue project: Dr. Joseph D. Buxbaum, Icahn School of Medicine at Mount Sinai
Functional Analysis of TCF4 in Neurons
Pitt Hopkins syndrome is a debilitating childhood disorder caused by deficiency of a protein called TCF4. To date, how TCF4 participates in the development of the brain and the connection between the brain cells (neurons) is not known. Here, we propose to identify the genes regulated by TCF4 in human and mouse, and to define if these target genes are turned on or turned off. In addition, we propose to study the functions of TCF4 at synapses, which provide for communication between adjacent neurons. We will study if depleting TCF4 in a mouse model alters the biochemical composition, the architecture, and the function of the synapses. Our study will help in the understanding of what are the specific defects caused by deficiency of TCF4, increasing the current knowledge on the pathological processes occurring in patients and laying the groundwork for the development of novel therapeutic strategies.
Aim 1. Identify the target genes of TCF4 in purified neuronal nuclei from human brain.
Aim 2. Identify the target genes of TCF4 in purified neuronal nuclei from mouse brain and correlate with RNA expression in Tcf4+/- mice and littermate controls.
Aim 3. Dissection of Tcf4 function in mouse.
$50,000 One Year Grant: Dr. Tonis Timmusk, Tallinn University of Technology, Estonia
Signaling Pathways and Compounds Regulating Transcriptional Activity and Phosphorylation of TCF4 Protein in Neurons
Pitt-Hopkins syndrome is a cognitive functional disorder (a form of mental retardation) diagnosed in less than 200 people in the world. Polymorphism or variation in the DNA sequence of the same gene has been linked to schizophrenia. Pitt-Hopkins syndrome is caused by a so-called de novo genetic mutation – one that is non-hereditary. The mutation may be in any part of the gene, but it appears in only one allele. Whereas in many other genes the other, unaffected allele would be able to compensate for the defect, this is not the case in TCF4. This indicates that the protein encoded by the TCF4 gene is essential for the development of the nervous system, and that human development depends significantly on the amount of this protein in the brain and body.
Our previous data have suggested that synaptic activation of nerve cells, that is the basis of brain function, leads to phosphorylation of TCF4 at specific sites of the protein. Phosphorylation is the addition of a phosphate group to a protein or other organic molecule. Phosphorylation turns many proteins on and off, thereby altering their function and activity. The current project is aimed at identification of signalling pathways inside nerve cells of the brain that regulate the phosphorylation and activity of TCF4 protein, characterization of the phosphorylation pattern of activated TCF4 and assessment of the efficiency of chemical compounds targeted at different components of the identified signalling pathways to increase TCF4 activity and correct PTHS deficiencies in neurons.
Since Pitt-Hopkins syndrome manifests itself at an early stage, there are better chances for its treatment due to the greater plasticity of children’s brains. If previously it was believed that mental retardation cannot be treated, then the results of recent tests on mice give hope that it can. In addition, the identification of treatment could be simpler for disorders caused by one gene than for disorders caused by the malfunction of hundreds of genes. Therefore, increasing the amount and/or activity of the functional TCF4 protein produced from the healthy allele is among possible approaches to develop drugs for Pitt-Hopkins syndrome treatment. We believe that current project could lead to the discovery of novel possibilities for increasing the activity of TCF4 in nerve cells that could be useful to develop treatments for therapeutic intervention of Pitt-Hopkins syndrome.
Grant Amount: $63,920: David Sweatt, PhD, Evelyn F. McKnight Chair, Dept of Neurobiology; Director, McKnight Brain Institute; University of Alabama at Birmingham
The identification of the dysfunctional TCF4 transcription factor gene as the genetic basis of Pitt-Hopkins Syndrome is a critical step forward in beginning to understand the diagnosis, etiology and molecular biology of PTHS. This project encompasses a set of studies to investigate the cognitive dysfunction associated with PTHS, focusing on mechanistic studies to understand the role of the TCF4 transcription factor in central nervous system function. For this project we are using genetically engineered mice in which the TCF4 gene has been manipulated in order to mimic human PTHS. This particular project is focused on investigating whether there is aberrant regulation of epigenetic molecular mechanisms, and altered transcriptional regulation of genes and small non-coding gene products in the PTHS model mice. For these studies we are particularly interested in learning and memory function as it relates to these molecular biological mechanisms in the CNS. Toward that end we are using next-generation high-throughput DNA sequencing methodologies coupled with epigenomics and bio-informatics approaches.
Grant Amount: $50,000: Stephen J. Haggarty, PhD, Associate Professor of Neurology | Harvard Medical School and Massachusetts General Hospital
The additional funds will be used to further support a post-doctoral research scientist in the Haggarty laboratory who is developing assays with human patient specific, stem-cell derived neuronal to measure TCF4 expression at the mRNA and protein level. Additionally, methods for mapping TCF4 target genes using state-of-the-art techniques for chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq) are being piloted. These studies are anticipated to provide important new insight into how the loss of TCF4 function may lead to changes in pathways important for neuroplasticity.
2012
- Molecular and Neural Basis of PTHS in TCF4 Mouse Models, with Dr. David Sweatt at the University of Alabama at Birmingham (a mutation or deletion in the TCF4 gene causes PTHS), $85,000
- Determining if TCF4 is necessary for normal cognitive function in a developed mature central nervous system, using mouse models, with Dr. David Sweatt at the University of Alabama at Birmingham, $75,000
- Generation and characterization of isogenic PTHS Stem cell models with Dr. Stephen Haggarty at Harvard Medical School, $85,000
- Creating an accurate description of the physical and behavioral consequences of an abnormality in the TCF4 gene with Dr. Jannine Cody at the Chromosome 18 Clinical Research Center at the University of Texas at San Antonio, partnering with Dr. Stephen Haggarty at the Harvard Stem Cell Institute, using skin cells to create stem cells. For this grant, Dr. Cody budgeted: $5,719 for year one (given in April 2012 by the PTHS Fund).
Grant Amount: $85,000: Dr. David Sweatt, Department of Neurobiology, the University of Alabama at Birmingham
PTHS Foundation Funding Enables New Research Into the Neurobiological Basis of PTHS
With the help and financial support of the PTHS Foundation Dr. J. David Sweatt, Professor and Chairman of the Department of Neurobiology at the University of Alabama at Birmingham, has started a new project to investigate the molecular and neural basis of Pitt-Hopkins Syndrome (PTHS). Dr. Sweatt is a senior scientist in the field of learning and memory, with an track record of prior discovery related to intellectual disability.
The research in the Sweatt laboratory capitalizes on the recent discovery that PTHS is a neurodevelopmental disorder, the underlying genetic basis of which is mutation/deletion of the TCF4 gene. In PTHS patients the altered gene product is present throughout development but is also present in the fully developed CNS. PTHS is an orphan disease, and at present there is a profound lack of information concerning the molecular neurobiology underlying PTHS. Thus, the Sweatt laboratory will begin to investigate the molecular and cellular basis of nervous system dysfunction in PTHS, to lay a cornerstone of research that will allow the hope of the development of a treatment for these patients in the future.
In their study the Sweatt laboratory will undertake comprehensive behavioral testing of genetically engineered PTHS model mice (TCF4 heterozygous deficiency mice) focusing on learning and memory, and investigating whether there is altered function in the neurons of PTHS model mice. In addition the Sweatt lab will testing whether a specific category of drugs, histone deacetylase (HDAC) inhibitors, might ameliorate any behavioral and neurophysiologic deficits observed in the mice. The proposed studies will yield valuable information concerning the Intellectual Disabilities aspect of PTHS, and whether HDAC inhibitors might be a candidate treatment for this aspect of PTHS.
Partnering with the Simons Foundation Autism Research Initiative
The PTHS research in the Sweatt laboratory is also being supported by a brand-new Simons Foundation Autism Research Initiative (SFARI) Explorer Award. In addition to manifesting intellectual disabilities, PTHS is an Autism Spectrum Disorder (ASD) and the autistic characteristics of PTHS are a crucial aspect of the syndrome. Thus, given their focus on autism the Simons Foundation has generously now begun funding research into PTHS. For their research project, in addition to studying the memory and neuronal characteristics of the PTHS model mice, the Sweatt laboratory will study the PTHS model mice in the domain of autistic behaviors as well. This important aspect of the research project will undertake behavioral studies to assess whether the TCF4 heterozygous deficiency mice manifest autistic-like behaviors, and also address whether HDAC inhibitors might ameliorate autism-like phenotypes in the mice. Studies of the PTHS model mice in terms of ASD-like behaviors are important not only because of their direct relevance to PTHS patients, but also because studies of the TCF4-deficient animals may give fundamental insights into the molecular neurobiology underlying the broader spectrum of autistic disorders. Furthermore, any potential drug treatments that ameliorate autism-like behaviors in PTHS mice might also be useful to consider for possible use with other ASD’s as well.
Additional Grant: $75,000 | Fall 2012
Dr. Sweatt’s group received a second grant to determine if TCF4 is necessary for normal cognitive function in a developed mature central nervous system. This work will lay the ground work for determining if PTHS is a developmental or chemical disorder in learning and memory or both.
Grant Amount: $85,000: Dr. Stephen Haggarty, Harvard Medical School
The Chromosome 18 Clinical Research Center at the University of Texas San Antonio is partnering with Dr. Stephen Haggarty, at the Harvard Stem Cell Institute and Assistant Professor of Neurology at Harvard Medical School, to work on drug development. They will be using skin cells (fibroblasts) from individuals with Pitt Hopkins to create stem cells and then differentiate them into neurons (or other important cell types). They can then use these cell lines to screen a large variety of possible drugs relatively rapidly. This project is very significant, both for its importance for Pitt Hopkins and as a “proof of principle” that we can apply to other conditions involving the central nervous system.
In addition to his collaborative work with Dr. Cody at the University of Texas, San Antonio, Dr. Haggarty’s group is working on identifying common molecular pathways and networks dysregulated due to TCF4 haploinsufficiency, using specially developed isogenic PTHS stem cell models or neurons. This work will set the stage for the discovery of therapeutic targets aimed at improving cognition.
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