Abstract Details

Title Generation of Non-Integrating iPS Cells and Motor Neurons from C9orf72 Repeat Expansion Amyotrophic Lateral Sclerosis Patients

Topic Anterior Horn

Presentation(s) S06 - (-)

Poster/Presentation
Number 003

Objective

Background Abnormal expansions of the noncoding hexanucleotide GGGGCC repeat located in the C9orf72 gene represents the most common genetic cause of familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The pathological role of C9orf72 hexanucleotide expansions remains unknown, specifically whether loss or gain of function is a primary mechanism.

Design/Methods We established integration-free iPSC lines from four healthy subjects and four C9orf72 repeat expansion ALS patients using episomal reprogramming factors, OCT3/4, SOX2, KLF4, L-MYC, and LIN28, and shRNA to p53. Colonies were picked and transferred to feeder free growth conditions. They acquired markers of pluripotency, determined by immunocytochemical staining and gene expression array. Lack of episomal plasmid integration and residual transgene expression over multiple passages was confirmed. RNA-seq and rapid amplification of cDNA ends was used to characterize the transcriptome.

Results All iPSC lines differentiated into the three embryonic germ layers determined using embryoid body formation assays. C9-iPSCs maintained genotype by PCR and Southern Blotting. Spin neural aggregate formation protocol of healthy and C9-iPSCs and directed neural differentiation by caudo-ventralizing factors RA and purmorphamine robustly yielded motor neurons, determined by emergence of markers for progenitors (Olig2, Hb9, Islet1, Nkx6.1, and Lhx1/3) and mature MNs (ChAT, and SMI32). Transcriptome analysis using RNA seq and rapid amplification of cDNA ends supported that a gain of function mechanism with toxic RNA containing the repeat is the primary mechanism in C9ORF72 related ALS/FTD.

Conclusions C9orf72 patient-iPSCs provide an opportunity to study C9orf72 function in the context of ALS patient neurons and can provide mechanistic links between the noncoding hexanucleotide expansion and neurodegeneration in ALS. This may one day lead to a cogent drug development strategy that can slow or reverse the devastating loss of motor neurons for C9orf72 ALS patients.

Authors/Disclosures
Robert H. Baloh, MD, PhD, FAAN (Novartis Institutes for BioMedical Research) PRESENTER	Dr. Baloh has received personal compensation for serving as an employee of Roche. Dr. Baloh has received personal compensation in the range of $0-$499 for serving on a Scientific Advisory or Data Safety Monitoring board for Acurastem. Dr. Baloh has received personal compensation in the range of $10,000-$49,999 for serving as an Expert Witness for Irell. The institution of Dr. Baloh has received research support from NIH and CIRM.
	No disclosure on file
	No disclosure on file
	No disclosure on file
	No disclosure on file
Sharday Grant, MD (Home)	No disclosure on file
Steven Levine, MD, FAHA (SUNY Downstate Medical Center)	Dr. Levine has received personal compensation in the range of $500-$4,999 for serving as an Editor, Associate Editor, or Editorial Advisory Board Member for MEDLINK. Dr. Levine has received personal compensation in the range of $50,000-$99,999 for serving as an Expert Witness for Law Firms. The institution of Dr. Levine has received research support from NIH.
	No disclosure on file
	No disclosure on file
	No disclosure on file
Matthew Harms, MD (Columbia)	Dr. Harms has received personal compensation in the range of $500-$4,999 for serving as a Consultant for Biogen. Dr. Harms has received personal compensation in the range of $50,000-$99,999 for serving as a Consultant for Muscular Dystrophy Association. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Invitae. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Variant Bio. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Sarepta. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for Amylyx. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving on a Scientific Advisory or Data Safety Monitoring board for uniQure. Dr. Harms has received personal compensation in the range of $5,000-$9,999 for serving as an Expert Witness for Littlepage Booth. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for O'Connor First. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Searcy Denney. Dr. Harms has received personal compensation in the range of $500-$4,999 for serving as an Expert Witness for Ford, Parshall & Baker LLC . The institution of Dr. Harms has received research support from ALS Association. The institution of Dr. Harms has received research support from Ionis. The institution of Dr. Harms has received research support from ALS Finding a Cure. The institution of Dr. Harms has received research support from Target ALS.
Clive Svendsen, PhD (Cedars-Sinai Medical Center)	No disclosure on file

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