898. Intracerebral Exon Skipping Restores Dystrophin Expression in CA1 Pyramidal Neurons of mdx Mice
Authors: Elise Peltekian, Karine Ros, Aurelie Goyenvalle, Carole Gruszczynski, Cyrille Vaillend, Serge Laroche, Luis Garcia, Olivier Danos
Journal: Molecular Therapy (2006)

Our goal is to evaluate the consequences of therapeutic gene transfer in the brains of adult mdx mice with memory deficits. We will try to compensate the deficits of mdx mice, by means of intracerebral administrations of vectorized antisense sequences that are potentially able to eliminate the mutated exon 23 of the dystrophin gene in this mutant, thereby allowing a local restoration of brain-dystrophin expression. We have recently developed and validated this technology and obtained a complete, functional and enduring restoration of dystrophin in the skeletal muscle of mdx mice [Goyenvalle et al., 2004]. AAV vector considerably enhanced the proper subcellular localization of antisense sequences and their inclusion into the spliceosome. Transfection of snRNA into target cells is facilitated by the use of gene-tranfer vectors derived from parvovirus (AAV) or lentivirus (HIV). These types of vectors have been shown to be particularly efficient for gene transfer into pyramidal neurons of the hippocampus.We show here that rescue of almost the entire dorsal hippocampus has been achieved by a bilateral intracerebral stereotaxic administration of an AAV-vector expressing antisense sequences linked to a modified U7 small nuclear RNA (snRNA). Restoration of dystrophin expression can be obtained in CA1 pyramidal cells of adult mdx mice after stereotaxic administration of the AAV-vector expressing the appropriate antisense sequences. This result already suggests that the reversal of genetic alterations can be achieved in the mature brain. To confirm and quantify the restoration of dystrophin expression, mRNA analyses and protein detection immunohistochemistry were carried out on serial brain tissue sections. The DNA sequence confirmed that the 688 bp band resulting from RT-PCR corresponds to the exon 23-skipped mRNA.We will now determine whether this treatment may compensate the long-term memory deficits displayed by mdx mice in object recognition tests and massed-training in the water maze. We will also establish the impact of the treatment on the neural mechanisms thought to underly the cognitive deficits associated with dystrophin loss (e.g., LTP, GABAA-receptor clustering, expression of dystrophin-associated proteins). Finally we will determine whether the efficacy of this invasive therapeutic approach may be potentiated by experimental stimulation of functional brain plasticity or by pharmacological modulation of GABAergic transmission. One main interest of this approach is the restoration of the lost protein not only in the target structure of the brain but also in the target cells, within the complex cell population of the brain tissue. Potentially, exon skipping may also be used to selectively inactivate the expression of genes involved in cognitive processes and synaptic plasticity, to undertake a precise analysis of these phenomenons and open new opportunities for therapeutic strategies to rescue cognitive alterations.