The research projects selected for funding were selected based on their ability to develop new therapeutics for treating our children with IQSEC2 mutations.
New publication providing logic for molecular engineering of IQSEC2 minigene
In the previous blog in October I briefly described possible approaches for IQSEC2 mediated disease. In this update, I wish to provide an exciting update on research on one of these approaches-gene therapy using adeno-associated virus (AAV).
March 7, 2023
Authors: Reem Jada, Veronika Borisov, Eliezer Laury, Shmuel Halpert, Nina S. Levy, Shlomo Wagner, Shai Netser, Randall Walikonis, Ido Carmi, Shai Berlin and Andrew P. Levy
1 Technion Faculty of Medicine, Technion Israel Institute of Technology, Haifa 3200003, Israel
2 Sagol Department of Neurobiology, Faculty of Natural Sciences, University of Haifa, Haifa 3498838, Israel
3 Department of Physiology, University of Connecticutt, Storrs, CT 06269, USA
* Correspondence: alevy@technion.ac.il
March 5, 2023
Authors: Nina S. Levy, Veronika Borisov, Orit Lache and Andrew P. Levy *
Technion Faculty of Medicine, Technion Israel Institute of Technology, Haifa 3200003, Israel
* Correspondence: alevy@technion.ac.il
February 15, 2023
Authors: Reem Jada, Veronika Borisov, Eliezer Laury, Shmuel Halpert, Nina S. Levy, Shlomo Wagner,
Shai Netser , Randall Walikonis, Ido Carmi, Shai Berlin, and Andrew P. Levy,*
* Correspondence: alevy@technion.ac.il
October 27, 2022
Research Objectives and Programs for Reducing Morbidity in Children with IQSEC2 mutations
This page is written in scientific language and is intended to provide insight into the status of research on reducing morbidity from IQSEC2 mutations. This page will be updated frequently. The challenge is daunting but we are optimistic as progress in this area of medical research is moving at a rapid pace.
To date over 80 different pathological mutations in the IQSEC2 gene have been identified. Most (80%) of these are nonsense mutations and are not believed to produce any protein product. The remainder of IQSEC2 mutations are missense mutations located in the IQ, Sec7, PH or PDZ domains of the IQSEC2 protein. Several recent excellent reviews have been written on the molecular epidemiology of IQSEC2 mutations. At present, there is no clear connection between the severity of IQSEC2 associated disease and any specific mutations. Several groups have created a registry documenting the natural history of IQSEC2 related disease and we will work to unify these registries into a shared database for researchers. We encourage all parents of the IQSEC2 community to participate in these registries. These registries and natural history studies will be essential for selecting proper outcome measures in the design of future clinical trials of treatments for IQSEC2 disease.
The basic research component of this foundation will support translational research whose goal is to find new treatments and ultimately a cure for IQSEC2 disease. The foundation will work with researchers studying IQSEC2 to develop these areas of research in specific Requests For Applications (RFAs). We review below our current knowledge in these areas and future directions for research applications.
Establishment of mutation specific models of disease to serve as a platform for drug development.
In order to understand the pathophysiology underlying IQSEC2 mutations and to test the efficacy of different treatments it is necessary to develop mutation-specific models. We review below the status of these models and current challenges that exist in their development and use.
Mouse models. The IQSEC2 gene is remarkably well conserved across all species with human and mouse IQSEC2 being over 98% identical and even fish and frog IQSEC2 being 80% similar to human IQSEC2. Three independent groups have generated mouse knockout models of IQSEC2 in which a nonsense mutation has been created in the mouse IQSEC2 gene resulting in the loss of production of IQSEC2 (Shoubridge, Frankel). In addition, a murine model of the human A350V missense mutation has been created. Mice from all four of these models have seizures, impaired social interactions and cognitive dysfunction thereby reproducing the most critical aspects of the human phenotype. These models may be used to identify potential targets for therapeutic interventions by examining gene expression and metabolomic profiles. While the murine models are the only means to study behavior and seizures, they are costly to produce and maintain and the ability to conduct high throughput drug screening with the mice is limited.
Human stem cell models. Induced pluripotent stem cells have been produced from skin biopsies of children with different IQSEC2 mutations. These stem cells can then be differentiated into neurons of specific types (i.e. hippocampal dendrite gyrus neurons) and then studied in the laboratory for how the mutation effects neuronal function and behavior. To date one such stem cell model of a child with an A350V mutation, and correction with CRISPR, has been reported with the identification of specific abnormalities in the excitability of the human A350V IQSEC2 neurons and their ability to form connections with other neurons. Multiple groups have collected skin biopsies of IQSEC2 children with the hope of generating neurons carrying specific mutations. A major challenge in integrating these data will be to find common pathways that are affected in children with different mutations and the standardization of the growth and differentiation conditions of the stem cells. Furthermore, due to differences in the genetic background of each child from whom the cell line was derived, it is important that isogenic corrected controls (via CRISPR) be generated for each line.
IQSEC2 disease models in zebrafish, xenopus and even drosophila are being developed by several groups that are amenable to high throughput drug testing. Several new antiepileptic drugs have been developed using a zebrafish seizure model (including Dravet Syndrome). We will encourage research using these models.
Translational research with the IQSEC2 animal models for precision medicine for IQSEC2 disease
As noted above the purpose of developing the animal models is to serve as a platform for drug discovery. This may be the identification of drugs that were originally used for a different indication or for newly designed drugs or for the use of AAV and CRISPR technology that will be discussed below.
The advantage of repurposing known drugs is that their safety profile is known and the drugs are already FDA approved so that they can be potentially brought to clinical use rapidly. A drawback of this approach is that the drug targets are often involved in multiple cellular processes so that drugs may have considerable off-target effects.
Precision medicine involves the development of treatments based on the pathophysiology of the disease and requires an understanding of IQSEC2 biology and pathophysiology. Two potential examples of the application of this principle for treating IQSEC2 disease are the targeting of Arf6-GTP which appears to be elevated in the majority of mouse models of IQSEC2 mutations and AMPA receptors which are decreased in neonatal hippocampal neurons carrying the A350V mutation. Positive allosteric modulators of AMPA receptors appear to correct in vitro defects in the electrophysiological behavior of neurons derived from with a child with the A350V IQSEC2 mutation. The foundation will solicit focused research programs designed to develop new precision medicines for IQSEC2 using existing and novel models of IQSEC2 disease.
Can we increase the normal IQSEC2 gene activity in females with IQSEC2-related disorders?
The IQSEC2 gene is located on the X-chromosome. Females have two copies of the X-chromosome. Thus, females with IQSEC2-related disorder have both a normal and a mutated copy of the IQSEC2 gene. In contrast to most of the genes on the X-chromosome that are subject to random X-chromosome inactivation to preserve the balance with males, the IQSEC2 gene escapes this X-chromosome inactivation. The normal copy of IQSEC2 may help reduce the severity of symptoms in females with mild IQSEC2 mutations, but this does not seem to be the case in many females who have more severe IQSEC2 mutations. There may be a threshold level of IQSEC2 activity needed for proper brain development. Innovative research has uncovered ways of altering expression of genes on the X-chromosome. Several groups are studying how the IQSEC2 gene is regulated during early neuronal development and whether there may be ways of increasing the levels of normal IQSEC2 gene expression in females. If so, this could potentially provide clinical benefit to many female children with IQSEC2 disorders.
Correction or rescue of IQSEC2 mutations by CRISPR or AAV.
Technology using CRISPR or adeno associated virus (AAV) present possible cures for IQSEC2 disease unlike the precision medicine drug programs described above. CRISPR and related technologies involve the repair of specific mutations in the DNA. Proof of concept for the treatment of human disease with CRISPR has been demonstrated for Duchene’s muscular dystrophy and some rare blood diseases but currently this is limited to cells which are actively dividing (i.e. bone marrow cells) making the use of CRISPR in neurons not possible at the present time. Moreover, every mutation will need its own CRISPR construct and optimization conditions – making this approach potentially very difficult to apply to a small population of patients with many different mutations. However, there exist variations on this theme such as the use of suppressor tRNA that may allow read through of nonsense mutations (80% of all IQSEC2 mutations). AAV offers a promising means to deliver a gene specifically to the brain. Indeed, an AAV expressing IQSEC2 has been used in IQSEC2 knockout mice to rescue the mice from disease. Gene expression from AAV is stable and the AAV doesn’t integrate into the genome so off-target effects are minimal. There are currently over 100 clinical trials in place for treatment of diseases with AAV (primarily neurodegenerative disorders). There are several major barriers limiting implementation of AAV for treatment of IQSEC2 disease. First, the IQSEC2 gene is very large (1488 amino acid coding sequence) making it too big to fit into conventional AAV viruses. Alternative viruses may be possible but these are less well characterized. Second, the cells in the brain which require IQSEC2 will need to be identified in order to allow targeting of the gene therapy to the correct spot and to design the AAV vectors appropriately so that expression will be only in specific neurons. Frankel and colleagues have demonstrated that IQSEC2 is produced in both glutamatergic excitatory and parvalbumin inhibitory neurons and it is unclear if expression in one or the other site is critical for a cure. Understanding this salient point is critical in designing how the AAV virus expressing IQSEC2 will be made with regards to DNA regulatory sequences inserted into the virus (again limited by the size restrictions of the DNA construct used in AAV). Third, the dose of IQSEC2 will need to be considered as either too much or too little IQSEC2 has been associated with neuronal pathology Fourth, it may be necessary to knockdown or eliminate the expression of mutant IQSEC2. At least 20% of pathological mutations involve production of an IQSEC2 protein whose function may be dominant. In other words, supplying a normal copy of the IQSEC2 gene may not be effective as the presence of the mutant protein may still cause disease.
Within the next few months, the IQSEC2 Scientific Advisory Board will be issuing a RFA encouraging collaborative translational research in the areas discussed in this document.
Chief Scientific Officer
Scientific advisory board panel:
References Attached:
