349. The Cure of Canavan Disease: Is It a Scientific Fiction or Clinical Reality?

Canavan Disease (CD) is a rare and lethal inherited pediatric CNS disorder with recessive mutations in the aspartoacylase (ASPA) gene. Traditionally, its variable disease phenotypes were descripted with the congenital sub-form showing neonatal onset and the severest phenotype with early death. The two other sub-forms, infantile, and juvenile, manifest with delayed onset and milder symptoms. Some CD patients are now 20 years of age and older. To date, there is no effective treatment available. Thus, gene replacement therapy is an attractive approach for treating this devastating disease. Previously, we have shown that a single intravenous (i.v.) injection of recombinant adeno-associated virus (rAAV) expressing human ASPA (hASPA) rescues early lethality and partially restores motor function (1st generation gene therapy) in the CD knock-out (CD KO) mouse, which resembles the congenital sub-form of CD and displays the severest phenotype of all available CD mouse models, with early death at around post-natal day (p) 28. After this remarkable improvement of symptoms with our 1st generation treatment, we further optimized our gene therapy. Now, in its 2nd and 3rd generation, our gene therapy cures the disease in the CD KO mouse. Interestingly, our 3rd generation gene therapy turns CD KO mice into “supermice”, outperforming WT mice on rotarod motor function test. This rescue is persistent and currently mice at 1.5 years of age still show no signs of disease reoccurrence. CNS pathology and magnet resonance imaging (MRI) at p25 and p365 show complete normalization. To further support the efficacy of our 3rd generation gene therapy, we performed neurometabolome profiling with over 400 characterized metabolites that showed reversal of the Canavan disease related metabolic changes including myelin associated lipids. To further evaluate the potency of our 3rd generation gene therapy, we tested different doses and routes of administration. Of note, 200-fold lower doses intraventricularly (ICV) administered still rescues lethality, while mice treated ICV with 20-fold reduced dose draw even with WT mice on motor function testing. Next, we moved to the Nur7 mouse model (resembles infantile and juvenile sub-form) that displays a similar disease pattern as the CD KO mouse with respect to growth curve and neurologic symptoms but eventually re-gains weight and shows survival similar to wild-type mice. Again, we treated mice i.v. with a single dose of rAAVhASPA at p1 as our gold standard and subsequent groups at 6 and 12 weeks of age to determine the therapeutic window. Of note, mice treated at 6 weeks of age recovered within 4 weeks post-treatment. Mice treated later than 6 weeks require more time to recover but still showed significant improvement over Nur7 mutants. This recovery was also correlated by CNS pathology and MRI. Currently, we are evaluating mice that were treated at 24 weeks of age to determine if there is a time point of no return. Overall, our data show clear evidence for the cure of the disease at early and late stages of the disease in two different mouse models. In addition, this is confirmed on different levels of cellular complexity by MRI, fMRI, CNS pathology, and neurometabolic profiling.