Virus-mediated Delivery of the Fmr1 Gene as a Tool for the Treatment of Fragile X Syndrome

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by a trinucleotide repeat expansion in the FMR1 gene that codes for fragile X mental retardation protein (FMRP). The goal of the present study was to determine whether restoring FMRP expression in the brains of the Fmr1 knockout (KO) mouse model of FXS could correct the disorder. Initially, we investigated major factors affecting tropism, expression level, and cell-type specificity of adeno- associated viral vectors (AAV), including encapsidation of different AAV serotypes, promoter selection, and the timing of vector administration, using intra-cerebroventricular injections of AAV vectors expressing enhanced green fluorescent protein. Our results favored the use of AAV serotype 9 (AAV9) vectors containing the neuron-selective synapsin-1 promoter, injected into the lateral ventricles of neonatal mice. To determine if FMRP expression in the central nervous system could reverse phenotypic deficits in the Fmr1 KO mouse model of FXS, we used a single-stranded AAV9 that contained a major isoform of FMRP. The vector was delivered to the brain via a single bilateral intra-cerebroventricular injection into neonatal Fmr1 KO mice on postnatal day 5 (in the first phase of the study) or 0-1 (in the second phase). Transgene expression and behavioral assessments were conducted either 22-26 or 50-56 days post-injection. Western blotting and immunocytochemical analyses of AAV-FMRP-injected mice revealed neuron-specific FMRP expression in the striatum, hippocampus, retrosplenial cortex, and cingulate cortex. AAV-FMRP injections reversed the pathologically elevated repetitive behavior and the deficit in social dominance behavior seen in Fmr1 KO mice in phase 1, as well as the elevated startle response and lower anxiety in phase 2. These results provide the first proof-of-principle that gene therapy can correct specific behavioral abnormalities in the mouse model of FXS.Ph.D