Directed Evolution of Novel AAV Variants for Enhanced Gene Targeting in Pluripotent Human Stem Cells and Investigation of Dopaminergic Neuron Differentiation

Directed Evolution of Novel AAV Variants for Enhanced Gene Targeting in Pluripotent Human Stem Cells and Investigation of Dopaminergic Neuron Differentiation

Funding Type: 
Tools and Technologies I
Grant Number: 
RT1-01021
Award Value: 
$918,000
Disease Focus: 
Parkinson's Disease
Neurological Disorders
Stem Cell Use: 
Embryonic Stem Cell
Cell Line Generation: 
iPS Cell
Status: 
Closed
Public Abstract: 
Statement of Benefit to California: 
Progress Report: 

Year 1

The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells. In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.

Year 2

The central goal of this is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. We have been using a novel directed evolution technology to improve the properties of a promising viral vehicle, and we are in the progress of progressively increasing gene delivery efficiency. In particular, we have isolated several viral vector variants with enhanced gene delivery to human embryonic stem cells. In parallel, we have a strong interest in understanding and elucidating mechanisms of human pluripotent stem cell differentiation into dopaminergic neurons, with implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We are conducting chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. We have generated an antibody to isolate this protein from cells and are in the process of pulling down DNA bound to this factor within cells undergoing dopaminergic specification. Once we have identified relevant target genes, we will use the new gene delivery technology to study their functional role in dopaminergic specification of human embryonic stem cells.

Year 3

The central goal of this project is to develop enhanced vehicles for gene delivery to human embryonic stem cells, both to modulate gene expression and to edit the cellular genome via homologous recombination. Harnessing a novel directed evolution technology we have developed to improve the properties of a promising viral vehicle, we have significantly increased its gene delivery efficiency to human embryonic and human induced pluripotent stem cells. Furthermore, this advance resulted in considerable improvements in the efficiency of gene targeting (i.e. editing) in the genomes of these cells. In parallel, we have a strong interest in understanding and elucidating mechanisms of luripotent stem cell differentiation into neurons, with for example implications for Parkinson's Disease. In particular, the transcription factor Lmx1a plays a role in this fate specification, but the underlying mechanisms are largely unknown. We attempted chromatin immunoprecipitation coupled with next generation DNA sequencing to identify the genes in the cellular genome that this factor regulates. Progress in this objective was ultimately hampered by the lack of a suitable antibody against Lmx1a. However, in parallel we have used an analogous approach to investigate mechanisms by which RNA transcription is regulated during the differentiation of embryonic stem cells into neurons, including motor neurons. These basic results can now be applied to enhance the efficiency of neuronal differentiation.

© 2013 California Institute for Regenerative Medicine