Adeno-Associated Virus Type 9-Mediated Gene Therapy of Choline Acetyltransferase-Deficient Mice.

The enzyme choline acetyltransferase (ChAT) synthesizes acetylcholine from acetyl-CoA and choline at the neuromuscular junction and at the nerve terminals of cholinergic neurons. Mutations in the ChAT gene () result in a presynaptic congenital myasthenic syndrome (CMS) that often associates with life-threatening episodes of apnea. Knockout mice for die at birth. To circumvent the lethality of this model, we crossed mutant mice possessing sites flanking exons 4 and 5 with mice that expressed . Injection of tamoxifen (Tx) at postnatal (P) day 11 in these mice induced downregulation of , autonomic failure, weakness, and death. However, a proportion of mice receiving at birth an intracerebroventricular injection of 2 × 10 vg/kg adeno-associated virus type 9 (AAV9) carrying human (AAV9-) survived a subsequent Tx injection and lived to adulthood without showing signs of weakness. Likewise, injection of AA9- by intracisternal injection at P28 after the onset of weakness also resulted in survival to adulthood. The expression of Chat in spinal motor neurons of mice injected with Tx was markedly reduced, but AAV-injected mice showed a robust recovery of ChAT expression, which was mainly translated by the human RNA. The biodistribution of the viral genome was widespread but maximal in the spinal cord and brain of AAV-injected mice. No significant histopathological changes were observed in the brain, liver, and heart of AAV-injected mice after 1 year follow-up. Thus, AAV9-mediated gene therapy may provide an effective and safe treatment for patients severely affected with CMS.