Abstract

1. Generation of Isogenic Huntington’s Disease Cell Models Through Targeted Gene Modification of Patient-Derived iPSCs Mahru C. An, Ningzhe Zhang, Gary Scott, Daniel Montoro, Sonnet Davis, Simon Melov, Arvind Ramanathan, and Lisa M. EllerbyBuck Institute for Research on Aging, 8001 Redwood Blvd., Novato, CA 94945 Abstract Results Results 2. Global gene expression analysis of normal vs expanded lines of iPS cells 5. Transplantation of corrected NSCs into stritum of HD model mice Huntington’s Disease (HD) is a rare genetic disorder caused by a polyglutamine (CAG) expansion in the first exon of the huntingtin gene. The disease is inherited in an autosomal dominant fashion, and the majority of HD patients carry only one expanded allele resulting in severe motor dysfunction, cognitive and behavioral impairment, and death. Recent advances in the development of patient derived induced pluripotent stem cell (iPSC) lines to model genetic disease, coupled with the ability to introduce targeted genetic corrections to iPSCs has led us to establish a method to introduce corrections or modifications to the disease-causing expanded CAG region via homologous recombination. Using this approach, we previously genetically corrected an iPSC line derived from a HD patient by replacing the disease-causing 72-CAG length allele with a normal 21-CAG length allele. The resulting isogenic pair of patient-derived stem cells are capable of differentiation into neural precursors or DARPP32 positive striatal neurons and initial characterization of these cell lines confirms rescue of phenotypes previously associated with HD models. We have further expanded upon this approach to generate additional isogenic lines representing an allelic series (Q21, Q42, Q72, Q100), allowing us to take a highly detailed systems-level approach to understanding the network of molecular perturbations associated with mutant huntingtin expression. HD patient iPSC vs normal iPSC (unique genetic backgrounds) HD patient iPSC vs genetically corrected iPSC (isogenic backgrounds) Global gene array analysis plotting relative expression level of normal vs expanded pairs of iPSC lines. Comparison of non-isogenic backgrounds results in 10-fold higher number of differentially expressed genes over threshold compared to an isogenic comparison. NSCs derived from corrected iPSCs are injected into the striatum of an R6/2 HD model mouse. Fixed, immunostained brain sections show that injected (STEM121-green) cells also express GABA, DARPP-32, or GFAP, suggesting that these cells are able to differentiate into specific subtypes of cells important for the pathology and possible repopulation of the HD affected striatum. 3. Directed differentiation of corrected HD-iPSC clones Results 6. Metabolomic analysis of isogenic NSC lines, HD vs normal. 1. Targeted modification of Htt CAG repeat size in HD patient-derived iPSCs NSCs were derived from corrected HD-iPSCs through embryoid body formation and isolation of neural rosettes and stained for NSC markers Sox1 and Nestin. NSCs were further induced to striatal neurons expressing DARPP32. Calbindin and GABA shown by ICC staining. 4. Targeted correction of HD-iPS cells corrects known disease endpoints Nanog Oct4 Sox2 SSEA4 Preliminary metabolomic analysis of NSCs derived from HD-iPSCs compared with disease corrected iPSCs (n=5) showing increased relative abundance of amino acids, glutathione synthesis, and TCA cycle metabolites. Summary Gene targeting strategy for modification of CAG repeat length in the human Huntingtin gene. Southern blotting analysis of HindIII digested HD-iPS clone genomic DNA and control samples. Expanded polyglutamine correction revealed by western blot analysis of lysates from HD-iPS and corrected clones C116 and C127 using antibodies for Huntingtin (Htt2166) and expanded polyglutamine (IC2). HD-iPS cells express classic ES cell markers. HD-iPS cells were cultured in the same conditions as human ES cells and immunostained for classic ES cell markers Oct4, Nanog, Sox2 and SSEA4. Embryoid bodies from corrected iPS cells give rise to progenies of all three germ layers. Immunostaining of clone 127 derived embrioid bodies shows differentiation into ectoderm (Nestin), mesoderm (smooth muscle actin, SMA) and endoderm (Sox17) fates. Karyotyping and G-banding analysis confirms the absence of genetic abnormalities in one line (C127) of corrected HD-iPSC clones. • We have developed methods for genetic modification of HD patient derived iPSCs to generate isogenic human cell lines which maintain pluripotent characteristics and are globally similar in gene expression to the original HD iPSC. • We have shown that correction of the disease causing CAG length in HD-iPSCs normalizes pathogenic HD signaling pathways and reverses HD disease phenotypes such that the neurons are no longer susceptible to cell death or display altered mitochondrial bioenergetics. • Proceeding from this approach, we have generated additional isogenic lines reflecting multiple CAG lengths and are working to analyze multiple genomic and metabolomic data sets from isogenic iPSCs and NSCs. NSCs derived from corrected HD-iPSCs are assayed for a number of endpoints that have been previously established to be associated with the HD state. In all cases, genetic correction of CAG length re-normalizes these disease states relative to expanded lines. We thank Dr. George Q. Daley for the HD-iPS Cells (Park et al. Cell 134, 877-886, 2008)