Grant reports

2014

Fragile X gene therapy using Adeno-Associated Viruses

Fragile X syndrome (FXS) is a genetic disorder that is the most common single leading cause of autism and intellectual disability in males. Caused by a severe down-regulation of the Fragile X mental retardation protein (FMRP), FXS does not have any long term pharmaceutical cures. However, recent advances in gene therapies using Adeno-Associated Viruses (AAV) show promise for future treatments of FXS and a number of other neurogenetic diseases. We thus aim to construct and test a number of biopharmaceuticals that fuse AAV with FMRP so that it can be delivered into the brain cells for the long term amelioration of FXS syndrome. The Fragile X mouse model (FMRP KO C57/BL6 mice) displays a wide range of behavioral characteristics (phenotypes) and are excellent models to test this AAV-FMRP strategy. Using molecular biology techniques and protein engineering, we aim to construct a number of AAV-FMRP virus particles, which will be initially tested on stable cell lines and ex-vivo cortical cells (brain cells in a test tube).  We will look for “selective tropism”, which refers to the virus’ ability to target the right kinds of cells, and then measure FMRP production rates in these cells. We will then identify the most promising selection of AAV-FMRP combinations, and inject them into the brains of Fragile X newborn mice.  This will allow us to observe them, to document any “behavioural rescue” during adulthood, and measure rates of functioning of this transgene (the replaced gene) using immunohistochemistry and western immunoblotting imaging techniques. We will then look for correlations between behavioral tests and local neuroanatomical production rates of FMRP in order to gain insight into the precise brain regions and cell types associated with this disorder.  These results will improve our understanding of the neuropathological defects in FXS, and help us devise better gene therapy approaches and approaches that may eventually be used to treat people

A definitive method for FMRP Measurement in Fragile X syndrome individuals

All of the core symptoms of Fragile X syndrome are caused by the absence of FMRP(Fragile X Mental Retardation Protein) in the brain cells of affected individuals.  This in turn is a direct result of a mutation in the FMR1 gene that renders it incapable of directing the production of FMRP. Researchers now know that there is no direct correlation between the size or length of the FMR1 mutation and the severity symptoms in individuals with Fragile X. However there is a direct correlation between the levels of FMRP in the peripheral blood, and the degree of intellectual disability.  Therefore, this lab is developing a simple, accurate technique to measure FMRP levels in the platelets in the peripheral blood.  This will make it easier to diagnose affected individuals, predict cognitive function and may be used as a tool to measure the effectiveness of future treatments.

The rescue of FMRP-related presynaptic long-term potentiation, social interaction, learning and memory deficits in Fmr1 knockout mice

This lab has used a previous FXRFC grant to characterize the phenomenon of long term potentiation (LTP) of synaptic strength, and its correlation with learning and memory function in mice. Dr. Zhuo’s lab reported that the Fragile X mouse model exhibits altered postsynaptic LTP in a part of the brain that is critical for learning and memory, known as the anterior cingulate cortex (ACC) . As well, they found that Fragile X mice have a deficit in fear learning behaviour.  Although postsynaptic mechanisms (ie. mechanisms in the receiving neuron) have been studied in Fragile X mice, the presynaptic mechanisms of LTP were unclear (ie. mechanisms in the sending neuron). To address this gap in knowledge, a new procedure was developed for inducing presynaptic LTP (pre-LTP) in the ACC, and some molecular mechanisms involved in this process were identified. When pre-LTP stimulation was applied to Fragile X mice, pre-LTP was observed to be defective. Preliminary data are suggestive that after induction of pre-LTP, the distribution of parts of an enzyme known as of PKA (Protein Kinase A) in the pre-synapse is abnormal. This can in turn adversely affect neuronal plasticity and communication between neurons.
 
The FXRFC has renewed this grant so that Dr. Zhuo’s lab can continue to characterize and compare the pre-LTP in normal mice to that of Fragile X mice. They will also study the functional  roles of arachadonic acid (ARA) in synaptic plasticity, the molecular mechanisms involved, and effects on social behaviour in Fragile X mice. The idea for investigating the effects of ARA was prompted by results from a pilot publication reporting that administration of commercially available Aravita (high dose ARA in combination with docosahexaenoic acid (DHA) and a low dose of astaxanthin (an antioxidant) because it showed beneficial effects on social behaviour in people with autism. ARA, a poly-unsaturated omega-6 fatty acid, is a key pro-inflammatory intermediate. DHA is an omega-3 fatty acid. Both ARA and DHA have been found to be important in neurochemical signal transduction related to brain cell maturation in animals and humans.