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C6 - Gene Targeting and Gene Correction New Technologies

16: Selective Repression of C9ORF72 Repeat Expansion-Containing Sense and Antisense Transcripts in an ALS Mouse Model

Type: Oral Abstract Session

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Session Title: New Technologies for Gene Targeting and Gene Correction

Amyotrophic lateral sclerosis (ALS) is a progressive disease characterized by degeneration of motor neurons in the brain and spinal cord, leading to paralysis and early death. The most frequent genetic cause of ALS is the expansion of the hexanucleotide GGGGCC (G4C2) repeats in the first intron of C9ORF72 gene. Healthy individuals carry up to 30 hexanucleotides repeats, but expansions of this region to hundreds or even thousands cause familial ALS and frontotemporal dementia. There are several mechanisms that may contribute to the disease progression as a result of this expansion. G4C2 repeats undergo bidirectional transcription generating sense and antisense expansion containing RNA and RNA foci as well as repeat-derived peptide translation products, suggesting a pathological, gain-of-function mechanism. To decrease the levels of expansion-containing transcripts in cells, while maintaining expression of healthy C9ORF72 mRNA levels, we previously identified an engineered transcription factor comprised of a zinc finger protein (ZFP) specifically targeting the G4C2 repeat region fused to a DNA-binding repressor protein (KRAB), named ZF-R. We showed >90% reduction of both sense and antisense G4C2 containing transcripts in patient-derived fibroblasts and motor neurons from patient-derived iPSCs carrying 1200 G4C2 repeats with minimal detectable off-target activity as measured by global transcriptomics analyses. To evaluate the potential use of this approach therapeutically, we studied the effect of ZF-R in neonate and adult C9500-BAC transgenic mice. ZF-R was delivered via ICV in neonates, and 5 weeks post injection animals were euthanized for molecular analysis. Using this approach, we reached ~70-80 % reduction of expanded repeat RNA (sense and antisense) in the cortex, associated with 40% of C9ORF72 protein reduction. We also observed ~50% decrease in sense and antisense RNA foci in the same brain region. The repression of sense and antisense containing RNA in the cortex was not associated with any changes in the molecular markers of neuroinflammation or neuronal loss. For the adult study, five month old C9500-BAC mice were injected via ICV with the same construct. Five months post injection animals were euthanized, and molecular analysis was performed. Similar to the neonate study, we found a strong reduction of expanded repeats and also ~60% decrease in Dipeptide repeats (DPR) proteins in the Cornu Ammonis (CA) of hippocampus. Overall, these findings strongly support the use of ZF-Rs for the treatment of familial ALS.

Plain Language Summary
Amyotrophic lateral sclerosis (ALS) is a progressive motor neuron degeneration disease leading to paralysis and early death. Its most frequent genetic cause is hexanucleotide GGGGCC (G4C2) repeat expansions (HRE) in the C9ORF72 gene. Through bidirectional transcription, HREs generate sense, antisense, RNA foci and dipeptide repeat products (DPR), suggesting gain-of-function disease mechanisms. To decrease levels of expanded disease transcripts and retain normal C9ORF72, we validated an engineered zinc finger protein targeting HREs fused to a DNA-binding repressor protein (ZF-Rs) in relevant cell systems and in vivo. Over 90% of sense/antisense G4C2 containing transcripts were reduced in fibroblasts and patient motor neurons. Further mouse studies demonstrated a 80% reduction of expanded repeat RNA, 40% reduction in C9ORF72 protein and ~50% decrease in RNA foci in the cortex with neonatal dosing, and a 60% reduction of expanded repeats and hippocampal DPRs with adult dosing. Our findings support ZF-Rs for treatment of familial ALS.

Amrutha Pattamatta1, Mohammad Samie2, Awdhoot Godbole3, Chen Ting-Wen1, Anagha Sawant1, Susan Ping4, Alexandra Srnka1, Siobhan Ford4, Lujain Al-Sowaimel1, Ricardos Tabet4, Robert Bell1, Lisa R. Keyes5, Sheila Rao-Dayton5, Rob Moccia3, Claudia H. Huichalaf4, David Shivak6, Bryan Zeitler2, Amy Pooler2, Christine Bulawa1

1Previous affiliation, Pfizer Inc, Cambridge, MA,2Sangamo Therapeutics, Inc., Richmond, CA,3Pfizer Inc, Cambridge, MA,4Genomic Medicine, Alexion AstraZeneca Rare Disease, Cambridge, MA,5Genomic Medicine, Alexion AstraZeneca Rare Disease, Durham, NC,6Previous affiliation, Sangamo Therapeutics Inc, Richmond, CA"

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