Reduction of the polyQ repeats in the P/Q type Calcium Channel Splice Specific Variant causing Spinocerebellar Ataxia Type 6 using CRISPR-Cas9 System.

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Spinocerebellar ataxia type 6 (SCA6) is an autosomal dominant, debilitating neurological disorder caused by CAG expansions of 20-33 repeats in the C terminus (CT) of the P/Q type calcium channel gene. CAG encodes the amino acid glutamine, leading to poly-glutamine repeats in the calcium channel protein. In humans, an alternative splicing event occurs at C-terminal end of P/Q type calcium channel gene resulting in two isoforms, one devoid or one containing the CT CAG expansion (1, 2). Both isoform transcripts are equally abundant in adult cerebellar Purkinje cells (PC). However in individuals suffering from SCA6, the diseased isoform transcript containing the CAG expansion is increased (2) compared to control individuals. Moreover, the CT of the alpha subunit of the P/Q type calcium channel (Cava12.1) undergoes proteolytic degradation leading to a more stable diseased CT peptide fragment which specifically accumulate in nuclear and cytosolic PC protein aggregates in adult SCA6 patients (2-4). These diseased CT fragments may potentially be acting as a dominant negative mutant to interfere with the normal interactions between P/Q type calcium channels and its substrates (binding partners) which eventually leads to the SCA6 disease state. I have recently demonstrated in mice that PC specific expression of only the CT polyQ27 fragment (containing a stretch of 27 glutamines as found in SCA6 patients) is sufficient to cause SCA6 like symptoms such as PC degeneration, ataxia and motor learning deficits where the CT fragment lacking the polyQ domain did not (5). Since there are no therapeutic treatments available for these individuals, I propose to develop a potentially, new therapeutic method where I specifically reduce the CAG repeats in the CACNA1A gene using the CRISPR-Cas9 (Cas9 nucleases from microbial clustered regularly interspaced short palindromic repeat) system. I will first test the targeting efficiency of the method in a neuroblastoma cell line. Then I will introduce a viral (adeno-associated virus) form of these CRISPR targeting vectors into the cerebellum of our transgenic SCA6 mouse model expressing the diseased polyQ27 expansion and examine the extent and efficiency of eliminating the production of the diseased CT fragment and the SCA6-like symptoms.