Evaluation of a Targeted Gene Therapy Strategy in a Transgenic Mouse Model of Huntington’s Disease

Abstract

Huntington’s disease (HD) is a progressive, dominantly inherited neurodegenerative disorder caused by an expansion of greater than 36 cytosine-adenine-guanine (CAG) repeats in the gene encoding the huntingtin protein (HTT). This expanded trinucleotide repeat encodes an expanded polyglutamine tract within the HTT protein which confers a toxic gain of function. HD is characterised by a triad of symptoms: a decline in motor control, decaying cognitive function, and neuropsychiatric disturbances. Almost three decades have passed since the mutation for HD was discovered, yet no effective treatments exist for this disorder and current therapies only provide temporary relief from some symptoms. This is problematic as HD is symptomatically multi-faceted and progressively neurodegenerative. Because mutant HTT is wholly responsible for pathogenesis, it would seem ideal to silence HTT gene expression to prevent further neurodegeneration and thus halt disease development. Gene therapy is therefore an appealing treatment approach. This project initiates the process of examining a viral vector-based gene therapy, under the control of a physiologically regulated gene expression mechanism, containing micro RNA (miRNA) against HTT mRNA, in the transgenic YAC128 (yeast artificial chromosome, 128 CAG repeats) mouse model of HD. Aim one of this project sought to optimise infusion parameters, such as injection coordinates, for delivering vectors to the striatum and not the surrounding brain regions and also to examine potential neurotoxicity associated with treatment. Following stereotaxic injection into the striatum, immunolabeling assays revealed strong transduction of neurons within the striatum which was not met with a neuroinflammatory response. Results also revealed one vector injection coordinate which limited most transduction to the striatum and not surrounding brain regions. Aim two of the project investigated whether an unregulated form of the gene therapy produced enough miRNA against HTT to produce HTT knockdown. Following stereotaxic injection with vector, droplet digital PCR analyses revealed no apparent knockdown of HTT. Modifications were made to the miRNA sequence and this new sequence was co-transfected with a mutant huntingtin-expressing plasmid into cells. Immunocytochemistry revealed potent HTT silencing under regulated and unregulated plasmid constructs. This new miRNA sequence is a promising candidate for future gene therapy research into mutant HTT silencing.