Generation of an isogenic iPSC-based model of Alexander’s Disease using genome editing
(2023) MOBN03 20221Degree Projects in Molecular Biology
- Abstract
- Alexander’s Disease is a progressive neurological disorder caused by a wide array of dominant, gain of function mutations in the GFAP gene. The disorder is characterized by the accumulation of GFAP in astrocytes, leading to the formation of protein inclusion bodies known as Rosenthal fibers. This results in a disruption of astrocytic function and the subsequent degeneration of white matter. However, a better understanding of the disease mechanisms is required to standardize therapy and to develop disease modifying treatments. Since the disease is caused by a wide array of mutations, it is important to study the phenotypic characteristics associated with specific mutations to collect and collate data that can define the disease more... (More)
- Alexander’s Disease is a progressive neurological disorder caused by a wide array of dominant, gain of function mutations in the GFAP gene. The disorder is characterized by the accumulation of GFAP in astrocytes, leading to the formation of protein inclusion bodies known as Rosenthal fibers. This results in a disruption of astrocytic function and the subsequent degeneration of white matter. However, a better understanding of the disease mechanisms is required to standardize therapy and to develop disease modifying treatments. Since the disease is caused by a wide array of mutations, it is important to study the phenotypic characteristics associated with specific mutations to collect and collate data that can define the disease more precisely. Use of iPSCs coupled with CRISPR-Cas9 genome editing to generate isogenic controls, in combination with protocols to generate astrocytes and neurons allows us to model the disease in vitro and work towards this goal. Here, progress has been made towards obtaining an isogenic control line for mutation K228E and isogenic controls have been generated for the mutation Q93P in the GFAP gene. (Less)
- Popular Abstract
- Using genetic scissors to model diseases in a dish
Alexander’s disease (AxD) is a very rare, gradual, and fatal brain disease. The disease can occur due to inherited or spontaneous changes, mutations, in the DNA. Understanding this disease at a cellular level requires looking into these genetic changes and making comparisons with healthy cells without the mutation. However, to specifically study these genetic differences, we need to eliminate the possibility of other background genetic differences that arise when comparing cells from different sources, as every individual has a unique genetic makeup. Using the latest genome editing scissors, the CRISRP-Cas9 toolbox, we can circumvent this issue.
AxD is caused by genetic mutations in... (More) - Using genetic scissors to model diseases in a dish
Alexander’s disease (AxD) is a very rare, gradual, and fatal brain disease. The disease can occur due to inherited or spontaneous changes, mutations, in the DNA. Understanding this disease at a cellular level requires looking into these genetic changes and making comparisons with healthy cells without the mutation. However, to specifically study these genetic differences, we need to eliminate the possibility of other background genetic differences that arise when comparing cells from different sources, as every individual has a unique genetic makeup. Using the latest genome editing scissors, the CRISRP-Cas9 toolbox, we can circumvent this issue.
AxD is caused by genetic mutations in a protein that is made by star-shaped cells in the brain called astrocytes. These cells belong to cell types involved in supporting nerve cell function. The disease has a distinct feature: the clumpling of specific cellular proteins in astrocytes. Other aspects include a lack of the insulating layer that is formed around neurons. This whitish layer, known as myelin, is made of proteins and fatty material and helps in faster transmission of electric impulses through the nerve cells.
Several different genetic mutations in the GFAP gene, encoding for a protein of the same name, can result in AxD. GFAP is involved in maintaining the structure of astrocytes. Attempts have been made to use animal models to replicate AxD conditions for research purposes. However, these models do not fully capture the complexity of the human brain and the disease in humans, making it necessary to model the disease in human-based systems. However, obtaining human tissue from patients with fatal, nervous system diseases such as AxD is difficult and highly invasive. The development of technology that allows us to revert any type of mature cells to stem cells (cells having the ability to form other cell types in an organism) has led to the use of human stem cells as a disease modelling system that circumvents the problems of the previous models. The use of stem cells also allows us to then generate cell types relevant to the disease.
In this project, we used the CRISPR genome editing tool to edit two specific AxD mutations in stem cells created from skin cells of two AxD patients to create genetically identical healthy controls, i.e., isogenic controls (see Image 1 for workflow). The genetic scissors specifically identify and cut the genome at the region of interest, which is then repaired by internal DNA repair systems. In this case, we supplied a DNA template to nudge the repair system to use this to repair the broken DNA. While we were able to achieve the desired editing with one of the AxD stem cell lines, we speculate that the instability of the DNA template prevents successful editing of the second AxD stem cell line. Thus, alternative strategies that use a more stable DNA template are to be explored to achieve the desired editing.
Further work includes assessing the controls obtained such as checking for possible off-target genetic changes made by the CRISPR tool, loss of stem cell-like properties due to editing, etc. Upon clearing these checkpoints, the cells can be used to produce nerve cells and astrocytes from these stem cells to study the observable traits of each of these AxD mutations in greater depth.
Master’s Degree Project in Molecular Biology 60 credits 2023
Department of Biology, Lund University
Advisor: Henrik Ahlenius, PhD
Department of Clinical Sciences, Lund University (Less)
Please use this url to cite or link to this publication:
http://lup.lub.lu.se/student-papers/record/9113591
- author
- Kalpana Ganesh, Kavya
- supervisor
- organization
- course
- MOBN03 20221
- year
- 2023
- type
- H2 - Master's Degree (Two Years)
- subject
- language
- English
- id
- 9113591
- date added to LUP
- 2023-04-18 14:18:08
- date last changed
- 2023-04-18 14:18:08
@misc{9113591,
abstract = {{Alexander’s Disease is a progressive neurological disorder caused by a wide array of dominant, gain of function mutations in the GFAP gene. The disorder is characterized by the accumulation of GFAP in astrocytes, leading to the formation of protein inclusion bodies known as Rosenthal fibers. This results in a disruption of astrocytic function and the subsequent degeneration of white matter. However, a better understanding of the disease mechanisms is required to standardize therapy and to develop disease modifying treatments. Since the disease is caused by a wide array of mutations, it is important to study the phenotypic characteristics associated with specific mutations to collect and collate data that can define the disease more precisely. Use of iPSCs coupled with CRISPR-Cas9 genome editing to generate isogenic controls, in combination with protocols to generate astrocytes and neurons allows us to model the disease in vitro and work towards this goal. Here, progress has been made towards obtaining an isogenic control line for mutation K228E and isogenic controls have been generated for the mutation Q93P in the GFAP gene.}},
author = {{Kalpana Ganesh, Kavya}},
language = {{eng}},
note = {{Student Paper}},
title = {{Generation of an isogenic iPSC-based model of Alexander’s Disease using genome editing}},
year = {{2023}},
}