The objective of this work is to evaluate an adeno-associated virus (AAV)-based therapeutic strategy targeted to Purkinje cells to reduce the molecular, cellular, and motor phenotypes of the Wistar Furth Shaker rat.

Degenerative cerebellar ataxias affect 1 in 5000 individuals and present with severe motor symptoms including incoordination and tremor. Despite decades of research, there are no widely effective therapeutic strategies. The Wistar Furth Shaker rat is a naturally occurring, x-linked, recessive model of Purkinje cell degeneration and consequent ataxia and tremor. KPF discovered a loss-of-function frameshift mutation in the SLC9A6 gene through positional cloning and transcriptomic analyses. We sought to confirm the identity of the gene by functional complementation supplying the NHE6 protein via AAV transduction.

We generated an AAV containing the unmutated SLC9A6 gene targeted to Purkinje cells with an L7 promoter using the Php.eB capsid. We performed motor analyses using a force plate-based methodology of our design and tested whether we could reduce motor phenotype through intracerebroventricular AAV administration prior to Purkinje degeneration. Ongoing work examines molecular and cellular phenotypes through qRT-PCR, Western blots, and slice electrophysiology.

We identified a frameshift mutation in the SLC9A6 gene, which encodes NHE6, an important sodium/proton exchanger in regulating cell acidity, with high levels of expression in Purkinje cells. Comparing L7-SLC9A6-GFP AAV-treated shaker rats and shaker rats administered an injection control (L7-GFP AAV), we found that L7-SLC9A6-GFP AAV substantially reduced motor dysfunction. In fact, several treated rats maintained motor performance indistinguishable from wild type rats.

A frameshift mutation in SLC9A6, which causes Christianson Syndrome in humans, is responsible for the Shaker phenotype, and we have successfully treated the motor phenotype using an AAV. Ongoing work pertains to cellular and molecular analyses of therapy, as well as defining the therapeutic window of treatment.