Generation of New Isogenic Models of Huntington's Disease Using CRISPR-Cas9 Technology.

Magdalena Dabrowska,Agnieszka Fiszer,Emilia Kozlowska,Marta Olejniczak,Agata Ciolak

doi:10.3390/ijms21051854

Abstract

Huntington’s disease (HD) is a fatal neurodegenerative disorder caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT). Despite its monogenic nature, HD pathogenesis is still not fully understood, and no effective therapy is available to patients. The development of new techniques such as genome engineering has generated new opportunities in the field of disease modeling and enabled the generation of isogenic models with the same genetic background. These models are very valuable for studying the pathogenesis of a disease and for drug screening. Here, we report the generation of a series of homozygous HEK 293T cell lines with different numbers of CAG repeats at the HTT locus and demonstrate their usefulness for testing therapeutic reagents. In addition, using the CRISPR-Cas9 system, we corrected the mutation in HD human induced pluripotent stem cells and generated a knock-out of the HTT gene, thus providing a comprehensive set of isogenic cell lines for HD investigation.

Highlights

Huntington’s disease (HD) is an incurable and progressive neurodegenerative disease caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT) [1]
To generate a model that is useful for studying the influence of repeat tract length on various aspects of the pathogenesis and therapy of HD, we generated HEK 293T cell lines with different numbers of CAG repeats at the HTT locus
We demonstrate the generation of new models of HD based on HEK 293T and human iPSCs (hiPSCs)

Summary

Introduction

Huntington’s disease (HD) is an incurable and progressive neurodegenerative disease caused by the expansion of CAG repeats in exon 1 of the huntingtin gene (HTT) [1]. Different cellular models are being applied to study the pathogenesis of the disease and to test therapeutic approaches for HD. These models include HEK 293T cells with exogenous expression of mutant HTT or a fragment thereof, patient-derived fibroblasts, human and rodent induced pluripotent stem cells (iPSCs), neural stem cells (NSCs) and postmitotic neurons [7]. The influence of the genetic background on the disease phenotype is increasingly being proven in HD, especially in light of the identification of genetic modifiers that affect the age of onset of HD in genome-wide association studies (GWAS) [8]. Subtle differences in DNA may influence phenomena such as somatic instability or disease onset

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Conclusion