Dr. Derek Bowie, McGill University ~ 5 year Grant $725,466
Rett syndrome and Fragile-X mental retardation are debilitating neurodevelopmental disorders that strike young infants after apparently many months of normal growth and development. Although there is currently no effective medication, the time delay suggests a “window of opportunity” for therapeutic intervention may exist. Each disorder presents with a spectrum of symptoms, however, afflicted children commonly possess autistic features. This finding has suggested to some that Rett syndrome and Fragile-X mental retardation may have a common molecular basis. In this grant application, we provide evidence for a novel signaling pathway triggered in early brain development by the neurotransmitter, L-glutamate. Importantly, this pathway is absent in a mouse model of Fragile-X mental retardation. We hypothesize it is also deficient in Rett syndrome. Experiments are planned to study the properties of this signaling pathway with the aim of rescuing its expression. Our work demonstrates for the first time a missing molecular event in the brains of Fragile-X sufferers that may also be lacking in Rett syndrome. The benefit of this work is that it may provide a basis for the development of clinically relevant compounds to treat these disorders early on in childhood.
Dr. James R. Ellis, Hospital for Sick Kids, Toronto ~ 1 year Grant $100,000
Induced Pluripotent Stem (iPS) cells are generated by inducing skin cells to change into stem cells that resemble embryonic stem (ES) cells. This “reprogramming” is performed by infecting adult skin cells with viruses that deliver a combination of “pluripotency” genes that confer stem cell properties. We have made mouse and human iPS cells using these pluripotency genes, and have developed a new EOS virus vector that signals when the skin cells are reprogrammed into stem cells. This technology can be immediately used to generate cell models of human disease by reprogramming patient skin cells into iPS cells, and then differentiating the iPS cells into the affected cell type. Rett Syndrome (RTT) is an Autism Spectrum Disorder (ASD) caused by defects in the MECP2 gene that affects girls with devastatingly early onset. RTT is an outstanding candidate for an iPS cell model of disease because it is not ethically possible to obtain nerve cells from the brains of these individuals. To create cell models for RTT, our first aim is to generate mouse and human iPS cells using skin cells from RTT knockout mice, RTT patients and normal controls. Our second aim is to differentiate these iPS cells into nerve cells for comparative studies to identify abnormal functions of RTT nerve cells in culture. Our third aim is to genetically rescue the RTT nerve cells by viral delivery of a normal MECP2 gene to rescue the nerve cell abnormalities. Overall, these exciting experiments will serve as a cell model to determine the functional defects in RTT nerve cells, and will test the ability of gene therapy to correct them in a patient specific context. The iPS cell lines may prove useful for screening potential therapies and for future cell therapies.
Dr. James Eubanks
University Health Network, Toronto Western Research Institute ~ 5 year Grant $748,827
Rett syndrome is one of the most common genetic forms of developmental delay affecting girls. We know the gene responsible for the condition, and there are mice that develop a Rett-like condition. Even though we have known the gene responsible for Rett syndrome for over 10 years, we still do not which types of cells in the brain play a key role in causing Rett syndrome, nor do we really know if the condition can be corrected. Our previous work suggests that correcting Rett syndrome is possible, as we showed that if one fixes the mutated gene in the brain of affected mice, some behavioural improvements are seen. But our rescue was incomplete – and we do not know why. Perhaps we targeted the wrong types of cells, or not enough cells in the brain to fix the condition, or perhaps not all of the problems with Rett syndrome can be fixed. Our current project will determine how well some of the most problematic symptoms of Rett syndrome can be corrected, and test which types of cells in the brain must be considered for the best treatment strategies.
Dr. N. Farra, Dr. J.R. Ellis
Hospital for Sick Children, Toronto ~ 3 year grant $105,000
Rett Syndrome (RTT) is a severe neurological disorder caused by mutations in the MECP2 gene. Primarily affecting females, this common cause of mental retardation may be treated by “gene therapy” that introduces the normal gene into affected cells. Although RTT is reversible in mice, gene therapy for this autism spectrum disorder will be a challenge because too much MeCP2 causes disease. By virtue of its position on the X chromosome, random X-inactivation in females leads to mosaicism where neurons express only mutant or only wild-type MeCP2. Consequently, vectors designed for RTT therapy must deliver MeCP2 to neurons while avoiding over-expression. For my thesis, I will generate novel lentivirus vectors that preserve overall levels by expressing therapeutic human MeCP2 while simultaneously expressing a microRNA (miRNA) that specifically degrades equivalent amounts of endogenous mouse Mecp2. Lentiviruses will be produced and these “co-transcriptional” vectors will be evaluated following infection of neurospheres and neurons derived from induced pluripotent stem cells (iPSCs). iPSCs, which like embryonic stem cells are capable of differentiating into any adult cell type, have been generated by reprogramming differentiated mouse and human fibroblasts using specific pluripotency-determining transcription factors. To model RTT, I have generated iPSCs from Mecp2308 mice and will differentiate these mutant cells down the neuronal lineage. These mice express a truncated form of MeCP2 and represent a mouse model of RTT. These differentiated neurons will be infected with co-transcriptional viruses and I will evaluate whether mutant neuronal phenotypes are rescued in vitro. To correct RTT symptoms in vivo, these cells will be delivered into the brains of postnatal mice, and memory, learning, and behaviour will be assessed. Our anticipated outcome is a proof of principle that co-transcriptional gene therapy produces neurons that express correct levels of normal MeCP2.