Abstract
Huntington's Disease HD is an inherited neurodegenerative disease caused by trinucleotide repeat (CAG/glutamate) expansion in the huntingtin HTT gene. Clinical symptoms present as motor, cognitive, and neuropsychiatric impairments. Subtle cognitive changes and neuropsychiatric disturbances may be observed preceding a clinical diagnosis. The neuropathology of HD is striatal loss followed by atrophy in other parts of the brain. There is no cure for HD, and current treatments only offer palliative care focusing on symptom management. Since the disease originates from a dysfunctional HTT gene, using a disease-modifying therapy that directly targets the mutant gene would make sense. Although the HTT gene is highly conserved, the exact function of the gene is still ambiguous but known to be associated with a variety of cell functions. Therefore, any HD gene modification should target the mutant huntingtin gene while avoiding the complete suppression of healthy huntingtin. The Fink lab developed an AAV gene therapy using CRISPR/Cas9. This platform uses dxCas9, a variant of the Cas9 protein that recognizes a broad photospacer adjacent motif (PAM) target paired with KRAB, a powerful transcription repressor. It is our goal to deliver KRAB to the mutant HTT gene site so as to reduce its expression by epigenetic editing. Evaluation of the AAV-dCas9 in neuronal stem cells derived from HD patients and dispersion of the AAV-dCas9 among HD animal models has shown positive results. Designing an efficacious platform for dCas9 delivery in vivo will be critical to the specific targeting of disease alleles without unwanted changes to the genome. AAV has become the most chosen vector for gene therapy. AAV has a history of being safe and primarily non-integrating into the target genome. However, traditional AAV vectors have limited packaging capabilities, preventing them from being vehicles for the expression of large proteins such as Cas9. Thus, dCas9 was engineered to be split among two AAV vectors for co-transfection to bypass this limitation. Preclinical in vivo studies with animal models have also been important in determining the overall safety of gene therapies. Therefore, we evaluated the AAV-dCas9 therapy in the YAC128 mouse model. This transgenic mouse has an artificial chromosome with the full-length human huntingtin's gene with 128 GAC repeats. This HD mouse model autogenously follows the disease progression seen in humans, making it ideal for translational studies. For this study, the YAC128 mouse striatum was implanted with either the AAV vector system containing a guide RNA which should target the human mutant HTT gene, or the AAV vector without the guide RNA serving as a negative control. Molecular and behavior assays were performed in parallel to evaluate the effect of the AAV-Cas9 treatments on huntingtin expression and functional deficits of the transgenic mouse. None of the molecular tests had any statistically significant differences in expression levels. On the other hand, the rotarod behavioral assay, which determines the latency to fall off a rotating rod, did show a statistically significant difference in the time the transgenic mice stayed on the accelerating rod regardless of treatment compared to their wild-type counterparts. This result was unexpected because this showed that there was not an improvement in behavior deficits with the treatment. Even so, this treatment platform should still be considered since epigenetic modifications have been shown to downregulate HTT expression in previous studies using both dCas9 and KRAB.