Résumé :
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Intravenous (IV) delivery of self-complementary AAV9 (scAAV9) has been reported to be highly efficient for CNS gene transfer due to effective crossing of the bloodbrain- barrier (BBB). We and others recently showed that a single IV injection of an SMN-encoding scAAV9 dramatically rescued an SMA mouse model.Intramuscular (IM) injection is an alternative way to overcome the problem of the BBB via axonal retrograde transport of the AAV particles from the neuromuscular junctions to the motor neuron (MN) soma. However, IM injection of AAV1 to AAV8 only mediates a moderate transduction of the spinal cord MN. Given the superiority of scAAV9 for IV MN gene transfer, we investigated its efficiency for targeting MN after IM injection. As expected, we showed that a single injection of scAAV9-GFP into the gastrocnemius of adult mice mediated MN transduction in the lumbar spinal cord. Surprisingly, the thoracic and cervical spinal cord cells (which are not connected to the gastrocnemius) were also highly transduced, with the highest expression being observed in the cervical segment. MN transduction therefore results not only from axonal retrograde transport but also from diffusion of the vector from the injection site into the bloodstream. IM injection of scAAV9 thus represents an alternative way to IV injection for achieving widespread MN gene transfer in the spinal cord.Although systemic delivery is highly promising for treating CNS disorders, it also mediates transduction of non-nervous tissues such as liver, heart or muscles. The restriction of transgene expression to CNS tissues could offer a safer gene therapy approach by avoiding potential side effects due to ectopic transgene expression. In this aim, we designed (1) scAAV9 vectors expressing GFP under control of a small fragment of the human Synapsin 1 promoter (scAAV9-SYN-GFP) and (2) scAAV9-GFP constructs in which 12 "Neuron-Restrictive Silencer Elements" (NRSE) sequences were placed upstream of the ubiquitous phosphoglycerate kinase (PGK) promoter (scAAV9-NRSE-GFP). Transduction efficacy and neurospecificity of both scAAV9-NRSE-GFP and scAAV9-SYNGFP vectors were analyzed in astroglial and neuronal cultures, and in vivo, following stereotaxic injection into the brain and the spinal cord, or after intravenous injection. Our preliminary results showed that, although the NRSE-PGK promoter mediated efficient GFP expression into neurons, it induced a residual expression in non-neuronal cells. In contrast, transgene expression mediated by the Synapsin promoter appeared highly restricted to neuronal cells both in vitro and in vivo. The next step of this project will consist in the analysis of the neurospecificity and therapeutic efficacy of a systemically injected scAAV9 vector expressing SMN under control of the SYN promoter.
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