Periodic Reporting for period 1 - MiniBRAIN (Investigating the pathogenic mechanisms underlying TUBB2B-related brain malformations using induced pluripotent stem cells and cerebral organoids.)
Periodo di rendicontazione: 2019-03-01 al 2021-02-28
Tubulin proteins (encoded by a family of highly-conserved tubulin genes) play important structural and mechanical roles within cells, including those involved in embryonic brain development. In recent years, mutations affecting a family of tubulin genes expressed highly during embryonic brain development have been associated with rare but clinically severe neurodevelopmental disorders characterized by structural malformations of the cerebral cortex. The aim of this fellowship was to tackle an important question in developmental neuroscience: what are the underlying mechanisms of tubulin gene-related disease that give rise to different malformations of the developing brain? To do this, we generated stem cells from patient skin or blood and subsequently produced cerebral organoids. Cerebral organoids are self-organising 3D neuronal cultures which offer a window into the early stages of human brain develop and therefore provide rare insights into the cellular mechanisms underlying disease.
Malformations of the cerebral cortex comprise a spectrum of severe congenital brain disorders, which cause lifelong suffering to patients and families. Affected individuals often present with drug-resistant seizures, profound cognitive impairment and reduced life expectancy. No curative therapies exist. Cortical malformations are rare but, taken together, form a great burden on health care and society (with an estimated incidence of 1 in 2500 newborns). A better understanding of the molecular mechanisms underlying these cortical malformations may suggest new opportunities for prevention and treatment. Moreover, unravelling disease pathways may give us insights into poorly understood mechanisms of normal brain development.
The overall aim of this project was to use state-of-the-art cerebral organoid neuronal cultures to better understand the mechanisms of tubulin-related disorders of brain development. To achieve this, this fellowship had three main objectives: 1) To reprogram patient skin fibroblasts to a stem cell state and to use gene-editing techniques to ‘correct’ patient mutations. This would enable us to study the tubulin mutation-specific effects on brain development. 2) To investigate abnormal cellular phenotypes of tubulin gene mutations in 2D neuronal cultures and, 3) to characterise the effects of these gene mutations on the development of cerebral organoids.
Potential impacts may include a better understanding of the molecular and cellular pathways underlying the tubulinopathies, as well as other genetic disorders associated with structural malformations of brain development. This understanding may pave the way for potential therapeutic interventions in the future. Additionally, the pipeline of stem cell reprogramming, gene-editing and neuronal culturing established as part of this individual fellowship will provide opportunities to study the role of further genes in brain development, as well as a broader range of neurological disorders, some of which with greater burdens on society (e.g. drug-resistant epilepsies and autism spectrum disorder). The outcomes of this fellowship with continue to impact the clinical and research domains and the progress made as part of this fellowship has been used to secure further funding to allow for a continuation of this research.
Malformations of the cerebral cortex comprise a spectrum of severe congenital brain disorders, which cause lifelong suffering to patients and families. Affected individuals often present with drug-resistant seizures, profound cognitive impairment and reduced life expectancy. No curative therapies exist. Cortical malformations are rare but, taken together, form a great burden on health care and society (with an estimated incidence of 1 in 2500 newborns). A better understanding of the molecular mechanisms underlying these cortical malformations may suggest new opportunities for prevention and treatment. Moreover, unravelling disease pathways may give us insights into poorly understood mechanisms of normal brain development.
The overall aim of this project was to use state-of-the-art cerebral organoid neuronal cultures to better understand the mechanisms of tubulin-related disorders of brain development. To achieve this, this fellowship had three main objectives: 1) To reprogram patient skin fibroblasts to a stem cell state and to use gene-editing techniques to ‘correct’ patient mutations. This would enable us to study the tubulin mutation-specific effects on brain development. 2) To investigate abnormal cellular phenotypes of tubulin gene mutations in 2D neuronal cultures and, 3) to characterise the effects of these gene mutations on the development of cerebral organoids.
Potential impacts may include a better understanding of the molecular and cellular pathways underlying the tubulinopathies, as well as other genetic disorders associated with structural malformations of brain development. This understanding may pave the way for potential therapeutic interventions in the future. Additionally, the pipeline of stem cell reprogramming, gene-editing and neuronal culturing established as part of this individual fellowship will provide opportunities to study the role of further genes in brain development, as well as a broader range of neurological disorders, some of which with greater burdens on society (e.g. drug-resistant epilepsies and autism spectrum disorder). The outcomes of this fellowship with continue to impact the clinical and research domains and the progress made as part of this fellowship has been used to secure further funding to allow for a continuation of this research.
Through a network of international collaborators, a collection of skin fibroblasts was obtained from a wide sample of individuals carrying variants in a number of tubulin genes. These were all successfully reprogrammed to a stem cell state. Upon completion, gene editing techniques were used to enable ‘correction’ of gene mutations back to normal ‘wild-type’ sequence. This was particularly challenging due to the high levels of genomic sequence similarity between the tubulin gene family. After extensive optimization however, an efficient gene editing strategy was established. This was critical to the long-term objectives of the project, as it allows mutation-specific effects of brain development to be investigated, rather than using a generic control cell line which would have a significantly different genetic background.
Alongside the stages of cell reprogramming and gene editing, I additionally used this time to acquire the skills relating to 2D and 3D neuronal culturing and analysis. Now that appropriate gene-edited controls have been generated, I will continue the process of analysing the effects of gene mutations on neuronal production, migration and positioning involved in cerebral cortex development.
Alongside the stages of cell reprogramming and gene editing, I additionally used this time to acquire the skills relating to 2D and 3D neuronal culturing and analysis. Now that appropriate gene-edited controls have been generated, I will continue the process of analysing the effects of gene mutations on neuronal production, migration and positioning involved in cerebral cortex development.
Potential impacts may include a better understanding of the molecular and cellular pathways underlying the tubulinopathies, as well as other genetic disorders associated with structural malformations of brain development. This understanding may pave the way for potential therapeutic interventions in the future. Additionally, the pipeline of stem cell reprogramming, gene-editing and neuronal culturing established as part of this individual fellowship will provide opportunities to study the role of further genes in brain development, as well as a broader range of neurological disorders, some of which with greater burdens on society (e.g. drug-resistant epilepsies and autism spectrum disorder). The outcomes of this fellowship with continue to impact the clinical and research domains and the progress made as part of this fellowship has been used to secure further funding to allow for a continuation of this research.