Oligonucleotide-mediated Modulation of C9ORF72 Repeat Expansion in Amyotrophic Lateral Sclerosis

Leon M Larcher

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease affecting motor neurons. A hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic cause of ALS. The C9ORF72 gene encodes three protein-coding transcripts, the expression of which is altered in C9ORF72 ALS. The proposed molecular mechanisms by which the repeat expansion induces neurodegeneration are; (1) C9ORF72 loss-of-function; (2) RNA toxic gain-of-function; and (3) accumulation of toxic dipeptide repeat polypeptides produced through repeat-associated non-ATG (RAN) translation. Removal of the expansion from C9ORF72 transcript or reduction of expansion containing transcripts can partially ameliorate the gain-of-function mechanisms. In this project, we explored catalytic oligonucleotides (DNAzymes) and steric-blocking antisense oligonucleotides to mediate the reduction of expansion containing C9ORF72 transcripts and to indirectly modulate dipeptide repeat production in various cell models. Treatment of neuroblastoma-derived SH-SY5Y cells with DNAzymes designed to cleave the disease-causing transcripts selectively reduced levels of the hexanucleotide repeatcontaining transcript variant 3, compared to the sham control cells, and minimally affected levels of the functional transcript, variant 2. Antisense oligomers designed to alter C9ORF72 splice site selection to modulate expression of the repeat expansion were transfected into patient fibroblasts and iPSCderived motor neurons. Analysis of C9ORF72 transcript variant 3, variant 2 (‘nonpathogenic’ variant), and total levels of C9ORF72 transcript variants following treatment with our antisense sequences revealed that morpholino oligomers could selectively reduce levels of variant 3 and formation of RNA foci. Reduction of variant 3 levels was comparable to that resulting from treatment with an RNase H-dependent positive control antisense oligonucleotide. Our data demonstrate that steric blocking morpholinos can effectively and specifically reduce levels of expansion-containing transcripts and transcript variant 3, without reducing overall expression of C9ORF72 variant 2 or total C9ORF72 transcripts and thus may partly address C9ORF72 haploinsufficiency. We also demonstrated that morpholino antisense oligomers designed to reduce PAF1 RNA and protein levels to indirectly modulate RAN translation could effectively reduce the available C9ORF72 repeat expansion transcripts available to undergo RAN translation; consequently reducing dipeptide repeat levels in cell models. This work presents potential therapeutic strategies to ameliorate C9ORF72-associated amyotrophic lateral sclerosis and supports ongoing efforts to further develop these strategies.

Oligonucleotide-mediated Modulation of C9ORF72 Repeat Expansion in Amyotrophic Lateral Sclerosis

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