Generation of stem cell models of ITPR1-related neurological disorders using a novel genome engineering approach

Thesis

Mutations in the ITPR1 gene cause different forms of neurological disease in humans. Large heterozygous deletions result in adult-onset SCA15, heterozygous missense mutations cause infantile-onset SCA29 or Gillespie syndrome, and homozygous loss-of-function mutations cause autosomal recessive Gillespie syndrome. However, the mechanisms by which ITPR1 mutations lead to motor and cognitive symptoms are still poorly understood. Stem cell models hold promise for advancing mechanistic research and drug development, but their generation has been notoriously challenging despite the recent advances in genome engineering technology.

This thesis describes the development of a CRISPR/Cas9 system designed to introduce ITPR1 mutations into human stem cells using positive-negative antibiotic selection. The aim was to develop a system that could not only generate stem cell models of different ITPR1 mutations but also be easily adapted for any other genes or mutations of interest.

The Cassette Insertion-Replacement CRISPR Editing System (CIRCES) was designed as two stages of CRISPR/Cas9-induced homology-directed repair. Sequential application of positive and negative selection was planned to remove the overwhelming majority of wild-type cells, thereby increasing the frequency of correctly edited cells. Constructs were generated to introduce SCA29 and Gillespie syndrome mutations, with individual components of the system initially tested in HEK293T cells before further optimisation in H9 human embryonic stem cells.

Integration of the selection cassette was achieved in all three ITPR1 target exons. Analysis of clonally-isolated colonies revealed cassette insertion was always identified in combination with an in trans small insertion and/or deletion, suggestive of biallelic loss of function. While no heterozygous missense point mutations were generated with CIRCES, seven clones with biallelic loss-of-function mutations were confirmed to be ITPR1 knockouts, thereby reproducing the genetic defect found in autosomal recessive Gillespie syndrome. Loss of ITPR1 was not compensated for by upregulation of other IP3 receptors and neuronal differentiation induced significant downregulation of IP3 receptors in both knockouts and controls. Despite ITPR1 knockout, intracellular calcium dynamics were unchanged.

The results presented in this thesis suggest total loss of ITPR1 is tolerated in human embryonic stem cells and during early neuronal development. The methods used were able to generate several stem cell lines that can be used as cellular models of Gillespie syndrome, which should be useful tools in the study of ITPR1-related diseases and calcium signalling. Adaptation of this approach to different targets will facilitate generation of cellular models for other human diseases.

Authors: Parolin Schnekenberg, R

• DOI: 10.5287/ora-zbwwd07gn
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: stem cell model; ITPR1; Ataxia; SCA29; SCA15; intellectual disability; CRISPR; gene editing; Gillespie Syndrome
• Subjects: Intellectual disability; CRISPR (Genetics); Neurodevelopmental treatment; Embryonic stem cells; Cerebellar ataxia; Gene editing