Résumé :
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Domestic cats exhibiting a recessive form of lower motor neuron (MN) degeneration, associated with a deletion of the lix1 gene, represent a large animal model of type III spinal muscular atrophy (SMA). In this study, we first analyzed the efficiency of intravenous (IV) and intracisternal (IC) delivery of GFP-expressing self-complementary AAV9 vectors (scAAV9.gfp) in neonatal and adult SMA cats. In the same way, scAAV9-mediated GFP expression was compared to that obtained following IV injection of scAAV5.gfp and scAAV8.gfp in young adult (7 weeks-old) SMA cats. A high number of GFP+ MNs was found in the whole spinal cord after IV injection of scAAV9.gfp in neonatal and adult cats. In contrast, only a negligible number of MNs was transduced in the spinal cord of adult cats after scAAV5.gfp delivery and only GFP+ nerve fibers were observed within the dorsal columns after scAAV8.gfp injection. We subsequently analyzed the efficiency of scAAV9.gfp administration into the cisterna magna to mediate gene transfer into the MNs of both neonatal and adult cats. This approach resulted in MN transduction in the cervical,thoracic and lumbar segments of the spinal cord. These preliminary results suggest that IC scAAV9 injection could potentially be considered as another potential strategy to deliver a therapeutic gene into the feline spinal cord. To analyze the therapeutic potential of LIX1 overexpression into the spinal cord of SMA cats, we constructed an scAAV9 vector expressing LIX1 under control of the CMV or the CMV enhancer/chicken b-actin (CAG) promoter and verified both in vitro and in vivo LIX1 expression. Western blot analyses of HEK293 cells transfected with the plix1 plasmids showed the presence of the LIX1 protein at the expected size. Two newborn SMA-affected kittens have been intravenously injected with the scAAV9.lix1 vector. qPCR analysis revealed the presence of the viral genome in all segments of the spinal cord. The therapeutic impact and the vector expression analysis are ongoing in order to validate the efficiency and the safety of these strategies for SMA gene therapy
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