713. IntraCSF Administration of AAV9/MeCP2 Extends Lifespan of MeCP2-Null Mice While Preventing Toxicity Associated With IV Administration

Rett Syndrome (RTT) is a neurodevelopmental disorder that shortens the lifespan of patients while depriving them of the ability to walk, talk, or interact with others. RTT is caused by inactivating mutations in the X chromosome-linked gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcription regulator that is highly expressed in neurons. Patients with RTT express mutant MeCP2 in ~50% of their cells due to random X-chromosome inactivation in each cell. The remaining “healthy” cells have an activated chromosome encoding functional (WT) MeCP2. Although researchers have already used MeCP2 gene therapy to reverse the RTT phenotype in mosaic mice, concerns about side effects (i.e., motor and learning deficits) resulting from MeCP2 overexpression in “healthy” cells persist. Indeed, humans suffering from MeCP2 duplication syndrome have been shown to suffer from muscular spasticity and intellectual disabilities. In addition to persistent concerns about MeCP2 overexpression, IV administration of the scAAV9/hMeCP2 vector (1×1011 vg/mouse) has been shown to cause significant liver toxicity in MeCP2-null mice (Gadalla et al, Mol Ther, 2013). We tested the hypothesis that by moving from an IV route of administration to an intraCSF route, we could lower the dose of AAV vector enough to avoid the high peripheral organ transduction that generates toxic side effects. Modeling our earlier experiment with an IV route, juvenile (4-5 week old) knock-out (KO) mice and WT littermates received a single intrathecal injection of AAV9/MeCP2 vector. RTT mice dosed intrathecally with 1×1010 vg had a 48% increase in median survival compared to vehicle-injected mice (n=6 treated; n=11 vehicle; P=0.008), nearly identical to the rescue we previously observed following the IV approach but at 1/10th the dose and avoiding overexpression-related toxicity in the liver. In conclusion, by changing the route of administration of the AAV9/MeCP2 vector from IV to intraCSF, we were able to generate a nearly identical therapeutic outcome with a 10-fold lower dose, and without excessive (toxic) gene transfer to peripheral organs. As a next step to utilize the intraCSF route of administration, DNA shuffling and directed evolution were utilized to select for AAV capsid variants that would have higher efficiency and improved biodistribution in the RTT mice after intraCSF administration. Multiple clones were selected that have CNS transduction equivalent to AAV9, but importantly, all had highly reduced peripheral organ biodistribution. Preliminary studies using two clones, RTTF1.04 and RTTF1.11, were able to confer significant survival benefits to male RTT KO mice (34% and 25% prolongation of lifespan, P = 0.03 and 0.05, respectively) after delivery of the MeCP2 transgene at a dose of 1×1010 vg. Overall, our studies support the use of an intraCSF route of administration to treat RTT using AAV vectors, and the potential to develop safer and more effective AAV-based vectors tailored to the intraCSF route of administration.We acknowledge generous support from IRSF and RSRT.