FPWR is working to eliminate the challenges of PWS by speeding up development of two types of therapies: Genetic therapies seek to compensate for the lack of expression of the paternally expressed PWS genes on chromosome 15. Pharmaceutical therapies act on pathways downstream of the missing PWS genes.
Pharmaceutical therapies are challenged by the need to identify the targets of PWS genes and develop robust disease-relevant cell models. FPWR has recently developed a new translational research program, the PWS Cellular Network, to address these challenges. This summer, the newsly established Cellular Network called for the formation of a central repository of high-quality, induced-pluripotent stem cells derived from people with PWS. This repository is currently underway.
The Need for Relevant PWS Cell Models
While the PWS genes were identified more than two decades ago, the targets and pathways downstream of the PWS genes are still unknown. In two groundbreaking studies performed in 2013 and 2015, Dr. Potts and collaborators identified two proteins called TRIM27 and USP7 that bind with MAGEL2 to regulate protein recycling and protein trafficking. Recently, Dr Wevrick and collaborators identified a new pathway by which Necdin and MAGEL2 regulate the abundance of the leptin receptor at the cell surface shedding light on a possible pathophysiological mechanism underlying PWS. Indirect evidence suggests that the pro-hormone convertase PCSK1 and NHLH2 could be the targets of SNORD116 leading to the hallmarks of PWS. More work needs to be done to establish robust links among gene expression, pathways and phenotypic abnormalities of PWS. This relies on developing relevant cell models for PWS.
An alternative approach to identify disease relevant targets is to use phenotypic drug screening, defined by screening and selection of compounds based on quantifiable phenotypic endpoints without prior knowledge of the drug target. Phenotypic screening can be done in a small animal model or via readouts in cell behavior, morphology, or change in cellular products. Whereas phenotypic screening allows, in principle, the identification of multiple targets and pathways underlying a complex phenotypic trait abnormality, it requires modeling phenotypic abnormalities of the disease in an appropriate tissue or cellular model.
Use of Stem Cells for Studying PWS
The lack of robust cell models for PWS is the main challenge for developing and screening small molecules for PWS in a high-throughput manner. Most cell models are derived from mouse models of PWS. Other models comprise immortalized cell lines that have been genetically modified to mimic PWS. Although these cells do not originate from human, they have allowed a better understanding of the defects that are characteristics of PWS at the cellular and tissue level. The recent development of stem cells derived from people with PWS offers a potential new tool to model and study human PWS in a dish.
Induced-pluripotent stem cells (iPSCs) are derived from adult somatic cells (e.g. fibroblasts that can be extracted from a skin biopsy) before they are reprogrammed into pluripotent stem cells capable of theoretically indefinite self-renewal and become any cell type of the body. It is important, though, to demonstrate that these reprogrammed cells maintain their characteristics in particular the genomic imprinting properties. iPSCs derived from patients with PWS have have been shown to maintain the appropriate methylation imprint, according to research by Chamberlain, et al., and Okuno et al.
The potential unlimited source of cell material carrying the patient`s genetic defects and the capabilities of iPSCs to differentiate into neurons and other cell type of interest make human iPSCs derived from PWS an attractive disease model. Several iPSCs lines derived from people with PWS have been created, some of which have been differentiated into hypothalamic neurons. Stem cells originating from dental pulp of people with PWS are also a model of interest for PWS. Dental pulp stem cells are multipotent neural crest cells that do not need to be reprogrammed to differentiate into neurons. Their limited self-renewal capabilities need, however, to be addressed before they could be considered as cellular assays for drug screening.
Cellular Network Calls for Stem Cell Repository
Stem cells derived from people with PWS are of great interest for better understanding the cellular defects that are at play in PWS and serving as assays for drug screening. These models need to be thoroughly characterized to ensure they model the phenotypic characteristics of PWS. A Cellular Network Program was recently formed, bringing together researchers with different areas of expertise (see list below). The network seeks to identify targets and foster drug discovery for PWS to support future cell-based high throughput screening. Members are collaborating to characterize PWS cellular phenotypes and foster the development of cellular assays using iPSCs derived from people with PWS.
The Cellular Network was launched at a workshop in August 2017 in Indianapolis, in conjunction with the FPWR scientific day and conference. The workshop began by summarizing current cellular models and targets of interest for PWS: Most cellular work has been performed until now in tissue, immortalized cell lines, primary cultures, slices or explants derived from animal models or embryos from brain or muscle tissues with a few of them using human fibroblasts. Impairment in cell development, migration and differentiation have been described suggesting neurodevelopmental defects in PWS. Several targets of interest (described above) have been discovered which could potentially serve as read-outs for high throughput drug screening. Although several iPSC lines from PWS patients have been developed, few cellular phenotypes have been described. Their immaturity in cultures is a limitation factor for assessing neuronal function in a dish. In addition, the reprogramming methods, the genetic variability and the variability due to culture methods need to be addressed.
In the context of the current cellular models and targets of interest for PWS, at the workshop, the Cellular Network recommended the development of a central repository of high-quality, induced-pluripotent stem cells derived from people with PWS. This repository would support the development of assays that are biologically relevant, reproducible and scalable for high throughput drug screening. Efforts are currently underway to set up such a repository and allow for further in-depth characterization of iPSCs originating from people with PWS.
Cellular Network Program Participants
- Dr. Anne Bang, Director, Cell Biology, Sanford-Burnham Medical Research Institute, La Jolla
- Dr. Ryan Potts, Associate Professor, Department of Cell and Molecular Biology St. Jude Children's Research Hospital, Memphis
- Dr. Christian Schaaf, Assistant Professor, Molecular and Human Genetics, Baylor College of Medicine, Houston
- Dr. Valter Tucci, Senior Group Leader, Neurobehavioural Genetics Group, Istituto Italiano di Tecnologia, Genova, Italy
- Dr. Janine LaSalle, Associate Director of Genomics, Genome Center Professor, MIND Institute, University of California, Davis.
- Dr. Robert D. Nicholls, Director, Birth Defects Laboratories, Professor, Division of Medical Genetics, Department of Pediatrics, Children's Hospital of Pittsburgh of UPMC, Pittsburgh,
- Dr. James Moresco, Staff Scientist, Department of Molecular Medicine, The Scripps Research Institute, La Jolla (TBC).
- Dr. Rachel Wevrick, Professor, Department of Medical Genetics, University of Alberta, Edmonton, Canada
- Dr. Sebastien G. Bouret, Associate Professor of Pediatrics, Scientific Director of Animal Care Facility and Rodent Metabolic Core, Children’s Hospital of Los Angeles, University of Southern California, Los Angeles
- Dr. Guo-li Ming, Professor, Department of Neuroscience, University of Pennsylvania, Adjunct Professor, Departments of Neurology and Neuroscience, Institute for Cell Engineering, Johns Hopkins University
- Dr. Rudolph L. Leibel, Professor of Pediatrics and Medicine, Director, Division of Molecular Genetics and the Naomi Berrie Diabetes Center, Columbia University
- Dr. Stormy J. Chamberlain, Assistant Professor, Dept. of Genetics and Genome Sciences, University of Connecticut Health Center, Farmington
- Dr. Larry Reiter, Associate Professor, Dept. of Neurology, Dept. of Anatomy and Neurobiology, The University of Tennessee Health Science Center, Memphis
- Dr. Theresa Strong, Director of Research Programs, FPWR, USA
- Dr. Nathalie Kayadjanian, Director Translational Research, FPWR, USA