Human induced pluripotent stem cell (HIPSCS) based Huntington’s disease (HD) neurons and their genetic correction using personalized RNA-guided designer nucleases (CAS9) mediated genome editing

• Autorzy: Golonko A. , Pollak A. , Rydzanicz M. , Ploski R. , Lisowski P.
• Języki publikacji: EN

Abstrakty

EN

INTRODUCTION: Huntington’s Disease (HD) is an neurodegenerative disorder, caused by a mutation in the CAG repeat tract of the huntingtin gene HTT, characterized by a progressive loss of neurons in the striatum and phenotypic features that are common in other neurodegenerative diseases. AIM(S): The aim was to 1) test personalized, precise genome editing technology based on high fidelity Cas9 variants in somatic gene therapy of trinucleotide repeat degenerative disorder such as HD, 2) and to test whether the N-terminal truncated protein is able to support normal neuronal development, 3) dissect the impact of the mutation on neuronal development. METHOD(S): Patient specific hiPSCs were generated using an integration-free method. hiPSC were edited by improved fidelity Cas9‑sgRNA expression vectors located upstream and downstream of the CAG repeats in Exon 1, HDR repair templates with different numbers of CAG repeats. Gene edited iPS clones were characterized for the potential modification at predicted off‑target sites. The lines were subjected for the functional studies with high-content screening. RESULTS: hiPSCs editing resulted into different products that underwent the non-homologous end joining (NHEJ), precisely corrected clones by homologous recombination (HR) and NHEJ mediated excision of the Q/P repeat region by reannealing of the DSB resulted into an in-frame Htt coding region lacking the N‑terminal Q/P repeat. CONCLUSIONS: 1) HD and corrected isogenic hiPSCs can be differentiated into excitable, synaptically active neurons; 2) Study demonstrates associated phenotypic abnormalities; 3) Personalized high fidelity Cas9 variants showed an improved specificity profile, suitable for somatic gene therapy; 4) Study shows the importance of isogenic controls for modeling and personalized gene therapy using patient specific hiPSCs. FINANCIAL SUPPORT: Acknowledgements: National Science Centre (Poland) Grant No. 2016/22/M/NZ2/00548 to PL.

• Wydawca: -
• Czasopismo: Acta Neurobiologiae Experimentalis
• Rocznik: 2017
• Tom: 77
• Numer: Suppl.1
• Opis fizyczny: p.135-136

Twórcy

autor

Golonko A.

• Department of Biotechnology, Faculty of Civil and Environmental Engineering, Bialystok University of Technology, Bialystok, Poland

autor

Pollak A.

• Department of Genetics, Institute of Physiology and Pathology of Hearing, Warsaw, Poland

autor

Rydzanicz M.

• Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland

autor

Ploski R.

• Department of Medical Genetics, Warsaw Medical University, Warsaw, Poland

autor

Lisowski P.

• Department of Molecular Biology, Institute of Genetics and Animal Breeding Polish Academy of Sciences, Jastrzebiec, Magdalenka, Poland
• iPS Cell-Based Disease Modeling Group, Max-Delbruck Center for Molecular Medicine (MDC) in the Helmholtz Association, Berlin, Germany