Phase One Research Projects
Necessary Funding: est. $2M in 2019 | Successfully Funded: $1.5M to-date
See Path to a Cure for a detailed explanation on research strategy.
Animal Model Projects:
Project: Establishing and characterizing eight mouse model lines enabling researchers and biotechnology companies to trial therapies for FOXG1 syndrome
DR. JAE LEE, OREGON HEALTH AND SCIENCES UNIVERSITY (OHSU)
Funded: year one | Funding Needed: $200,000 for year two
Dr. Lee is developing and studying eight Foxg1 mouse models representing the entire gamut of FOXG1 human mutations, including a humanized mouse model. These mouse lines will be available to any scientist and biotechnology company interested in the basic biology of Foxg1 and the translational efforts to develop the cure for Foxg1 syndrome. For phenotypic analyses, Dr. Lee will characterize each mouse model using molecular, cellular, brain anatomy, and behavioral analyses.
Explanation of the 8 mouse lines:
Humanized Mouse – A human FOXG1 gene is placed within a mouse model. Primate-specific (like human) sequences of FOXG1 protein are substantially different from murine Foxg1 protein (like mice). Thus, developing a humanized mouse model will contribute to FS pathophysiology and serve as a strong animal model in developing FS-treating strategy.
Early Truncation Mutation – Truncation in 1~180 aa, before DNA binding domain
Middle Truncation Mutation – Truncation in 181~308 aa, within or right after DNA binding domain
Late Truncation Mutation – Truncation after 309 aa (potential) dominant negative
Missense Mutation - Missense with complete loss of function
Missense Mutation – Missense with partial loss of function
FOXG1 deletion – Deletion of the entire FOXG1 gene or FOXG1 associated area
FOXG1 duplication – Duplication of the entire FOXG1 gene
Project: Correcting FOXG1 loss-of-function in post-natal animals. Identification of the etiology of FOXG1 syndrome and the targets for drug discovery
DR. SOO-KYUNG LEE, OREGON HEALTH AND SCIENCES UNIVERSITY
Funded: year one | Funding Needed: $150,000 for year two
Dr Lee is an active contributor to the FOXG1 community. She proposes to use various FOXG1 mouse models understand the impact of loss of FOXG1 in different types of neurons (excitatory and inhibitory) as well as in oligodendrocyte precursors (another important cell type in the brain). She will then perform detailed characterization to examine the overall structure of the cerebral cortex, neuronal connections, electrophysiological activity, and behavioral outcomes in affected animals.
The investigator will also reintroduce FOXG1 at various time points, to determine whether (and when) symptoms can be reversed. Global gene expression analysis will be undertaken to identify any differences as a result of FOXG1 loss. This investigator has previously identified some functional overlap between FOXG1 and the gene FMR1, which causes Fragile X syndrome when defective. Fragile X syndrome is the most common single gene cause of autism and intellectual/developmental disabilities. Fragile X has a very active community that has pushed some drugs through to clinical trials.
Project: Developing, Characterizing and performing high throughput screening on Zebrafish Models
DR. CORRINE HOUART, PHD., KING’S COLLEGE LONDON
Funding Needed $100,000
In order to make good decisions on which drugs and gene therapies to take forth into human clinical trials, we would like to get data from multiple animal models and stem cell models. In addition to our mouse models, we are interested in seeing the effect of FOXG1 on zebrafish models. The characterization and screening work on zebrafish is most helpful to understand the effect of FOXG1 and drugs at a cellular level, for instance the ability to differentiate inhibitory and excitatory neurons. Zebrafish additionally allow us to screen thousands of small molecules quickly. Once we find successful molecules, we can screen those on mouse models and organoids, where the process is more time consuming. We are looking to build the same zebrafish mutants that we have created for our mouse models and organoids for consistency.
Project: Targeting astrocytes in the brain to investigate treatments to sustain neuronal function and survival for FOXG1 syndrome.
DR. SYLVAIN LENGACHER, GLIAPHARM GENEVA, SWITZERLAND
Funded: Aim 1 & Aim 2. | Funding Needed tbd per results
Dr. Lengacher’s team will investigate the role of FOXG1 in the regulation of brain energy metabolism and mitochondrial activity in different brain cell types using GliaPharm’s proprietary platform GliaX™ and expertise centered on the role of brain energy metabolism. The project will investigate the effect of FOXG1 downregulation by siRNA in primary astrocytes metabolism, mitochondrial activity, neuroprotection and synaptic plasticity. If a clear link were to be established between FOXG1 activity and brain energy metabolism, next step would include the test of GliaPharm’s proprietary molecules that stimulate brain energy metabolism in FOXG1 transgenic mice.
Aim 1: FOXG1 siRNA knock-down in astrocyte mouse primary culture.
Aim 2: metabolic characterization
This project is in collaboration with The BLACKSWAN Foundation
Our data indicate that siRNA-mediated down-regulation of FOXG1 in astrocytes dramatically affects brain energy metabolism. Of particular interest, treatment of astrocytes with glutamate, which leads to the secretion of the neuroprotective molecules lactate under normal conditions as postulated by the ANLS (astrocyte-neuron lactate shuttle) actually has the opposite effect. Hence, glutamate leads to the reduction of lactate secretion in FOXG1 impaired astrocytes. The ANLS model explains how energy is transferred from astrocytes to neurons when it is required, i.e. when neurons are active. ANLS was found to be essential for physiological processes including neuroprotection, synaptic plasticity, memory consolidation and cognition. These interesting results open unexplored avenues of research for the role of ANLS in the development of FOXG1 syndrome, and potential new therapeutic strategies for this debilitating pathology.
human stem cell model projects:
Project: Disease Modeling and Characterization of Induced Pluripotent Stem Cell Lines (iPSC) derived from patients with FOXG1 Syndrome - 1 Year project
DR. ALYSSON R. MUOTRI, PHD, UNIVERSITY CALIFORNIA SAN DIEGO (UCSD)
Funding Needed $230,000
This project is a needed step before we can conduct high throughput screening of drugs and gene therapies on human stem cells. Project aimed at creation and differentiation of the iPSCs into relevant forebrain neurons and/or 3D modeling of human forebrain development. IPS lines are created from fibroblasts. Fibroblasts for this project will be established by NGIMS and will be the same mutations as are studied in the mouse models. Thus, we will have rich data from mutations of various categories. These studies aim to provide detailed analysis of the molecular and electrophysiological properties of patient derived neurons. We will gain further mechanistic insight into cellular and molecular events that could potentially be targeted for therapy. Thorough characterization of the lines is expected to establish robust models for future drug screening, assessment of candidate therapies for efficacy and safety assessments, and other pre-clinical studies that will inform clinical trial design.
Project: FOXG1 as target for Autism. Gene targets of FOXG1 in human brain progenitors
DR. FLORA VACCARINO, YALE SCHOOL OF MEDICINE
Funded year one $135,000 | Funding Needed $135,000 for second year
Dr Vaccarino has previously demonstrated that patient-derived iPS neurons from individuals with autism spectrum disorder and macrocephaly (large head size) have higher levels of FOXG1 gene expression. She also showed that there was an imbalance in the proportion of inhibitory and excitatory neurons. Restoring FOXG1 therapy to normal using RNAi reversed these findings, suggesting that FOXG1 may be important for how autism spectrum disorders develop and progress.
The current proposal would use iPS neurons to model the effect of reduced and elevated expression of FOXG1 on global gene expression and the balance of inhibitory/excitatory neurons. Characterizing these pathways would help us understand the role of FOXG1 on important biological pathways. These target pathways could be candidates for drug therapies.
Project: RNA gene therapy to correct FOXG1 symptoms in iPS cells. Assessing the therapeutic potential of small activating RNAs in a patient-derived cellular model of FOXG1 syndrome
DR. ANGUS CLARKE, UNIVERSITY OF CARDIFF
Funded year one
Dr Clarke has already completed much of the groundwork for this project thanks to funding supplied the UK FOXG1 Foundation. iPS cells have been derived from FOXG1 individuals with a variety of mutation types (deletion, missense, frameshift) and differentiated into neurons. The impact on target gene expression (candidates previously identified) will be investigated, as will the electrical activity of these neurons, compared to ‘normal’.
RNAa will be used to boost expression of FOXG1 to determine whether this can correct any symptoms identified in the patient-derived neurons. The electrical analysis methodology would represent a unique feature of this proposal.
We were able to generate stem cells from patient skin cells which we confirmed by measuring levels of genes that reflect the new cell identity, and looking at the appearance of the cells under a microscope. We also examined whether the cells showed the potential to form many different cell types, a characteristic that defines stem cells. Figure 1 shows microscope images of a colony of iPSCs derived from a skin cell biopsy. The identification of these cells as iPSCs was confirmed by two markers: OCT4 (staining the cells green) and SSEA4 (staining the cells red). It is important to do this before continuing with the second part of the process, the generation of brain cells.
Increasing the amount of FOXG1 in our cell model
The nature of the saRNAs (the small messengers used to increase FOXG1 expression) means that they require careful design. Their mechanism of action is not yet well understood, so we are currently uncertain as to what makes some of them work and others not so. We tried 26 different saRNAs in this study and of these, three led to increased FOXG1 expression – it approximately doubled.
Project: Worldwide FOXG1 Biobank of Patient Samples
FREE with the US National General Institute of Medical Sciences
Storing FOXG1 patient and relation blood and skin samples. Reprogramming skin samples into Fibroblasts and performing quality controls. Making samples and fibroblasts available to both academic and commercial entities. Linking patient samples to patient registry information.
small molecule testing projects:
Project: Suppressing Nonsense Mutations with Small Molecule Compounds to Cure Nonsense FOXG1 Mutations
DR. DAVID BEDWELL, UNIVERSITY OF ALABAMA AT BIRMINGHAM
Funding Needed $68,000
As part of a collaboration with Southern Research through a study funded by the Cystic Fibrosis Foundation, >771,345 small molecules were recently screened to identify agents that suppress Premature Terminal Codons (PTCs). ~200 primary, validated, non-cytotoxic hits were identified from these screens. 30% of all FOXG1 current mutations consist of nonsense mutations which generate in-frame PTCs. Preliminary data collected at the University of Alabama, Birmingham suggests that a subset of these hits may be effective at suppressing PTCs in multiple contexts. The goal of this study is to identify compounds capable of suppressing FOXG1 nonsense mutations and restoring full-length, functional FOXG1 protein and many symptoms of FOXG1 Syndrome.
Gene therapy testing projects:
Project: RNA Gene Therapies for FOXG1 missense mutations. Developing an integrated platform for scalable, etiopathogenic-clinical profiling of subtle FOXG1 mutations and experimental, RNA-drive rescue of their histopathogenic effects
DR. ANTONELLO MALLAMACI, SISSA AND DR ROBERTO CILIO, UCSF
Funded year one
Dr Mallamaci has a strong history characterizing the role of FOXG1 in brain development. The investigator aims to develop a streamlined protocol that can be used to consistently characterize the impact of various FOXG1 missense mutations. Given the investigators background, the proposed measures are focused around aspects of brain development that FOXG1 has been shown to be important for, like: self-renewal of early neural cells, differentiation into inhibitory and excitatory neurons, etc.
The investigator will also seek to determine whether bad outcomes of FOXG1 missense variants can be corrected using RNA-based gene therapy (either boosting FOXG1 expression, or silencing it).
Project: CRISPR/CAS9 Mediated Gene Editing in FOXG1-mutated patient-derived cells
DR. ALESSANDRA RENIERI, UNIVERSITY OF SIENA, ITALY
Funded by the Petram Foundation.
Prof. Dr. Alessandra Renieri first discovered, in 2008, the association between FOXG1 gene mutations and the severe disease in humans. In the following years she developed expertise in induced Pluripotent Stem Cells (iPSCs) characterization and differentiation in neurons from FOXG1 patients. She follows the majority of FOXG1 patients in Italy and her Medical Genetics Department is a referral center for patients in Europe.
In collaboration with Dr. Silvo Conticello, an expert in DNA/RNA editing, she proposes to use CRiSPR/Cas9 technology to edit FOXG1 mutations. This project will specifically cut the mutated allele and edit it using a donor DNA harbouring the normal sequence. To allow the CRiSPR/Cas9 correction system to enter the cells, a viral system (AAV = Adeno-Associated Virus) will be used as carriers.
Goal: Use a CRISPR/Cas9 gene editing approach to correct FOXG1 mutations in patient-derived tissues.
What Did They Do?
The CRISPR/Cas9 system is used to create small breaks in DNA strands at targeted locations. The body recognizes these breaks as errors that need to be repaired. If a correct piece of DNA is provided at the time of the DNA break, it is possible to replace the existing sequence. This mechanism represents a promising gene editing approach where a ‘mutation’ or genetic variant can be corrected.
The authors tested their gene editing strategy in two different tissue types: patient skin cells, and patient skin cells that have been transformed into neurons. In both tissue types, some degree of correction is observed, meaning that there is more ‘healthy" FOXG1 present following introduction of the CRISPR/Cas9 system. The presented data is largely describing successful editing of a single missense variant. When using a gene editing approach, accuracy is critical. ‘Off-target’ effects are when the gene editing system introduces DNA breaks unexpectedly. The authors use DNA sequencing to show that there are minimal ‘off-target’ effects.
This work is a promising first step in demonstrating the ability to correct FOXG1 mutations in human tissues. Future work is needed to establish: (1) the amount of FOXG1 correction required to treat symptoms; (2) effective delivery to cells of interest; (3) functional impact of FOXG1 correction beyond the DNA level; (4) success in a more complex model (like an animal); (5) optimal window for treatment; (6) appropriate dosing and safety.