Expansion of a hexanucleotide repeat G4C2 in the non-coding region of chromosome 9 open reading frame 72 (C9orf72) is the most common genetic cause for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). While non-pathogenic C9orf72 alleles possess between 2 to 25 repeats, pathogenic repeat expansions vary from tens to thousands. C9orf72 expansions are bidirectionally transcribed leading to the formation of intracellular sense and antisense RNA repeat expansion (RRE) foci. Moreover, the transcripts are prone to repeat-associated non-ATG (RAN) translation producing dipeptide repeat proteins (DPRs). ALS/FTD patients currently have no treatment options. Our strategy is to target the disease causative factor using the virus carrier adeno-associated virus serotype 9 (AAV9) vector to mediate gene editing of C9orf72 gene. We are pleased to report that all our initial objectives were achieved:
1) We successfully designed vectors to achieve two therapeutic approaches: i) AAV vectors expressing gRNAs editing on either side of the expansion to excise the repeats (selected vector named C901); ii) vectors designed to remove a strong Kosak consensus sequence (~70bp) just upstream of the expansion region, which was demonstrated to be crucial for RAN translation (named C904).
2) C901 treatment of induced-neural progenitor cells (iNPCs) derived cells from ALS/FTD patients with C9ORF72 expansion led to significant reduction in sense RNA Foci, when comparing with controls. Further validation was performed in cortical neurons cultured from C9-500 BAC mouse model. Our studies showed consistent excision of a full-size C9ORF72 pathogenic expansion with C901, in cortical neurons derived from the C9-500 BAC mouse model, leading to significant decrease in RNA Foci. Interestingly, transduction with C904 also decreases the number of RNA Foci when compared with the control groups. Analysis from protein extracts of C9-500 cortical neurons showed that transduction with C901 and C904 therapeutic viruses mediate successful reduction in poly(GP) DPRs.
3) Our data generated in iNPCs and cortical neurons provide strong confidence in the selected therapeutic vectors. To test our concept at the whole organismal level, successful therapeutic (C901, C904) and control (C902) AAV9 vectors were administered in C9-500 BAC mouse model. 5 groups of mice were included in this extensive proof-of-concept study: C901 (n=25), C904 (n=25), C902 (n=25), untreated mice (n=25) and wild type group. The animals were tested for rotarod, catwalk, open field, marble burying, electromyography, and neuroscoring. We employed a double-blind randomization process in which experimental groups were blinded to the person conducting viral delivery and behavioural testing and another blinded person designed to analyse the data. Please note that the mice experiments takes several months because of the slow and delayed phenotype of the C9-500 BAC mouse model. Analysis of the large amount of data generated in our proof-of-concept is ongoing.
We expect to fully analyse the proof-of-concept in vivo data in near future. We also have plan to submit a manuscript for publication and applications to secure follow up funding from the Wellcome Trust Innovator scheme. This would allow us to take our project close to clinical development.
