496. From Gene Therapy to Gene Enhancement: Optimized Pre-Clinical Gene Therapy Transforms Mice with the Severest Canavan Disease Phenotype Into “Supermice”

Abstract

Canavan Disease (CD) is a rare and lethal inherited CNS disorder caused by autosomal recessive mutations in the aspartoacylase gene. To date, there is no effective treatment available. Gene replacement therapy is an attractive approach for this devastating disease, although the physiologic function of AspA and its metabolite N-acetylaspartate (NAA) is still not fully understood.We previously reported that systemic delivery of the human aspartoacylase (hAspA) gene by recombinant adeno-associated virus (rAAV) to the CNS of CD mice ameliorated symptoms, partially restored motor function and increased life expectancy to the extent of wild-type (WT) mice. We further sought to investigate the mechanism behind this pre-clinical gene therapy for CD and to improve therapeutic potency. We hypothesized that Kozak sequence and codon modification of hAspA cDNA enhance therapeutic expression to transform our 1st generation AspA expression cassette into a more potent 2nd generation pre-clinical remedy and a better tool to study the physiologic role of AspA.We created expression cassettes containing codon-optimized (Opt) hAspA cDNA together with different Kozak sequences (i.e. HKz and FKz). Our in vitro and in vivo data demonstrate stronger hAspA expression and enzyme activity of the FKzhAspA-Opt vector over all other tested constructs as well as decreased NAA in the CNS below WT level by MRS analysis. Interestingly, rAAVFKzhAspA-Opt treated CD mice perform equally on inverted screen and balance beam but significantly (>50%) better than WT at p28, p90 and p180 on rotarod, suggesting that ubiquitously high expressed hAspA exerts some enhancing effect on treated mice. To confirm these findings, we moved to another mouse model of CD (Nur7 mouse). FKzhAspA-Opt treated Nur7 pups show a similar trend as seen in the CD mice on motor function tests. In addition, open-field testing to measure overall activity and anxiety demonstrates that treated Nur7 mice are more mobile with significantly longer travel distance but anxiety levels equal to WT mice. Currently, we are investigating the molecular mechanism underlying this enhanced performance of treated versus WT mice by studying the metabolome, microRNAome and the possibility of achieving therapeutic efficacy with reduced doses of the optimized vector.Overall, our data demonstrate that our 2nd generation gene therapy for CD not only completely restores the disease phenotype but also achieves motor function performance and overall activity superior to WT mice in two different CD mouse models. Using the same AAV serotype with identical tissue and cell tropism as previously published by our group, implies that excessive NAA is highly detrimental to normal physiology and the NAA re duction independent of the hAspA expressing cell type is sufficient for therapeutic success, which might also suggest a positive correlation of higher AspA activity and lower NAA levels with gene therapeutic outcome for this inborn error of NAA metabolism.