HORIZON DISCOVERY

t + 44 (0)1223 655580f + 44 (0)1223 655581e info@horizondiscovery.comw www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

INTRODUCTION

Familial Hypercholesterolemia (FH) is an autosomal dominant disorder, characterized by plasmaaccumulation of cholesterol. Worldwide, about one in 200 people has FH, and if untreated, aroundone in two men and one in three women will develop premature coronary heart disease. Themajority of FH patients are carriers of mutations in the Low-density Lipoprotein Receptor (LDLR).Phenotypes appear to be correlated with the level of expression of LDLR: heterozygotes have twicethe normal plasma levels of LDL-cholesterol, and homozygous individuals four times the normalamount. A common LDLR mutation is c.301G>A, which results in substitution of a glutamic acidwith a lysine at amino acid 101 (p.E101K). Here we report the generation of isogenic humaninduced pluripotent stem cells (hiPSCs) carrying the E101K mutation, followed by in vitrodifferentiation into hepatocyte-like cells to model FH. Using the CRISPR-Cas9 genome–editingtechnology in healthy donor hiPSC cell lines, the following genotypes were achieved: LDLR(E101K/E101K), LDLR (E101K/+), LDLR (E101K/-), LDLR (-/-) and LDLR (+/-). A defined four-stageprotocol was used to differentiate these isogenic hiPSCs into liver cells. Generated isogenichepatocyte-like cells have been validated at both the genotypic and phenotypic levels using anarray of biochemical methodologies. This renewable and scalable source of patient relevantisogenic hepatocytes provides a biologically relevant platform to expedite in vitro disease modellingand novel drug discovery.

PLURIPOTENCY ASSESMENT AND EMBRYOID BODY DIFFERENTIATION

hiPSC GENE EDITING USING CRISPR

Figure 2: A) Quantitative PCR analysis of Nanog, Oct4 and Sox2 demonstrate that targeted cells exhibit normalexpression of pluripotency markers. B) Embryoid body differentiation of parental and LDLR (E101K/E101K) hiPSCsshows that differentiation potential in all three germ-layers is maintained after targeting of the parental hiPSC line.Samples collected at day 16 of differentiation were analysed by qRT-PCR for the expression of HAND1 (mesoderm),GATA6 (endoderm) and PAX6 (ectoderm). C) Immunofluorescence analysis of LDLR (E101K/E101K) hiPSCs after EBdifferentiation: endoderm (α-fetoprotein; AFP), mesoderm (α-smooth muscle actin; α-SMA) and ectoderm (β-tublinlllb; TUJ1). Nuclei are stained with DAPI (blue).

Figure 1: A) Horizon pipeline. A hiPSC line known to differentiate well to liver cell types was transfected with plasmidsexpressing Cas9 nuclease, a validated gRNA and an 200bp oligo donor. Single cell clones were derived and screened forthe incorporation of the mutation of interest. The genotype of the resultant LDLR iPS cell line was verified by Sangersequencing and ddPCR. B) Schematic representation of the oligo donor used for the introduction of the E101K KImutation. C) Sanger analysis showed that close to half of the clones were modified by CRISPR. D) Sanger sequencingdata indicates the correct incorporation of the mutation E101K (codon GAG>AAG).

Transfection

Project & nuclease design, reagent production

Single Cell Dilution

Genotyping

Bank

Validation and QC

hiPSC Project

AFP/DAPI

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HEPATOCYTE DIRECTED DIFFERENTIATION OF LDLR-EDITED hiPSC CLONES

FH MODELLING – VALIDATION OF LDLR-EDITED hiPSC CLONES

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Figure 4: A) Phase contrast image of hiPSC stock, B) at endoderm stage, C) at hepatic specification stage and D) ofdifferentiated hepatocyte. Magnification of A), B) and C) is 40x. Magnification of D) is 100x. B) Quantitative PCR analysisof Alpha-fetoprotein (AFP) and Albumin (ALB) for the WT and targeted lines differentiated into hepatocytes. This datashows that the hiPSC-differentiated hepatocytes express similar levels of ALB to Primary Human Hepatocytes (PHH)and levels of AFP lower than the human liver cancer cell line (HepG2). It also demonstrate that targeted LDLRhomozygous and heterozygous lines differentiated in hepatocytes exhibit normal expression in comparison to the non-targeted WT hepatocytes.

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Figure 5: A) Fluorescently labelled LDL (Dil LDL) uptake by LDLR WT hepatocyte incubated with 0µg/ml of Dil LDL (topimage) or 10µg/ml of Dil LDL (bottom image) showing uptake of Dil LDL when added. B) Quantitative analysis offluorometric assay showing uptake of Dil LDL by WT and targeted LDLR hIPSC-hepatocytes in comparison with PHH. Thisdata shows higher Dil LDL uptake in LDLR WT compared to LDLR heterozygous and homozygous lines indicating reducedreceptor function in LDLR diseased lines.

0µg/ml Dil LDL

10µg/ml Dil LDL

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MODELLING FAMILIAL HYPERCHOLESTEROLEMIA USING HUMAN ISOGENIC INDUCED PLURIPOTENT STEM CELLSAlejandro Armesilla-Diaz1, Filipa Soares2, Rodrigo Santos1, Kalpesh Jhaveri2, Christine L Schofield1, Christopher E Lowe1, Marcus Yeo2

1 - Horizon Discovery Group Plc, Cambridge Research Park, Waterbeach, 2 - DefiniGEN Ltd, Babraham Research Campus, Cambridge, UK. Cambridge, UK

# clones % modified clones % KI/KI % KI/WT

346 47.5 4.4 2.7

DIRECTED DIFFERENTIATION – OptiDIFF PLATFORM

OptiDIFF is a technology platform for the production of key commercial cell types from humaninduced pluripotent stem cells (hiPSC).

Definigen’s OptiDIFF platform can generate cell lines from a population-relevant set of patientdonors with diverse genetic disorders. These cells offer substantive predictive performance benefitsover the immortalised cell lines and animal models currently used in preclinical Studies.

Human iPSC circumvent many of the ethical and commercial barriers associated with embryonicstem cells as they are derived from adult cells that have been reprogrammed to the stem cell state.Stem cells have two major properties:1) they can form any cell type of the body.2) they can self-renew indefinitely, producing a limitless supply of cells.

SUMMARY

The collaboration between Horizon Discovery and DefiniGen offers a range of unique, gene-engineeredhiPS (human induced pluripotent stem)-based cell lines for use in research and drug discovery

CRISPR gene editing in hiPSCs does not affect pluripotency and differentiation

Successful generation of genetically validated isogenic hepatocytes carrying E101K LDLR mutation for FHdisease modelling

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Figure 3: Def-HEP hepatocyte cells are produced from hiPSC using the OptiDIFF platform. The cells are generated usinghiPSC technology and the resulting cells display many of the functional characteristics of primary human cells includingalbumin production and glycogen storage.

SMAa/DAPI Tuj1/DAPI

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For more information, contact Dr Alejandro Armesilla-DiazAlex.Armesilla@horizondiscovery.com