UC Berkeley

Gene therapy, the introduction of genetic material to drive a therapeutic effect in a cell, has gained traction as one of the most promising methods of treating human disease. Advances in genetic and proteomic data have improved our understanding of the underlying causes of inherited diseases – combined with the development of new tools and vectors for the delivery of genetic material, the field is poised to make great strides in developing novel approaches to not only treat the symptoms of disease but cure them entirely. Viral vectors have emerged as promising delivery vehicles for gene therapies, as their natural evolution has conferred the ability to infect human cells in a cell-type selective manner to deliver and express foreign DNA. The Adeno-Associated Virus (AAV) has demonstrated itself as one of the most promising viral delivery platforms to date. It has a low baseline level of immunogenicity, its protein capsid makeup confers cell-selective infectivity, and it is capable of stable long-term gene expression in nondividing cells. However, due to its small size it suffers from a limited packaging capacity – only 4.7 kilobase pairs of DNA can be delivered by a single viral capsid.This limited packaging capacity has driven the field to identify methods to increase the number of clinical indications that can be targeted by AAV-based gene therapy. One of the primary methods has been through the engineering of the promoter element that drives subsequent expression of the therapeutic transgene. Promoters vary drastically in size, cell specificity, and strength, but most endogenous cell-specific promoters are large and relatively weak; viral-derived promoters are strong but similarly large and lack cell selective expression profiles. These three areas – size, cell specificity, and strength – are the main areas of optimization in promoter engineering for AAV-based gene delivery. An ideal promoter is short in length to accommodate larger transgenes; cell-selective to avoid expression of therapeutic cargo in off-target cells, and has a strong expression profile to reduce the amount of viral material necessary to achieve a therapeutic effect. These three characteristics have been historically difficult to optimize in parallel. To address these limitations, we have developed a platform termed Expression-Linked Promoter Selection (ELiPS). This platform is capable of generating large (>107) libraries of short (200 – 400 bp) synthetic promoters that can be screened in a high-throughput manner to identify short cell-specific promoters that drive high levels of gene expression, particularly for their size. The ELiPS platform takes advantage of short (6-14 bp) transcription factor binding sites (TFBS) that recruit transcription factors, protein machinery critical to the expression of DNA. Where an endogenous promoter may contain dozens of TFBS across thousands of base pairs, ELiPS promoters are composed nearly entirely of these TFBS across 100 – 300 bp, resulting in high local concentrations of transcriptional machinery that drive high levels of gene expression. The selection of TFBS motifs can inform cell-specific expression – to date, we have performed screens of these synthetic ELiPS promoters and identified promoters active in human embryonic kidney 293T (HEK293T) cells, and both the human retina and lung in vitro. By repeating the modular TFBS elements in the ELiPS promoters, in 293T cells we have identified promoter sequences that drive high levels of gene expression in a cell-selective manner –though less than 25% of the size, the ELiPS promoters are comparable to the viral CAG promoter in AAV-based transduction and 1.3-fold stronger in plasmid-based transfection. Work is ongoing to apply the same modular improvements to ELiPS promoters identified in screens of the human retina and lung. These drastic reductions in promoter size while maintaining high levels of activity position the ELiPS platform as a promising method of identifying new promoters for gene therapy applications in AAV. Transcription factors are not only important elements for engineering novel promoters, but their expression is directly linked to cell fate. Their forced overexpression is capable of directly differentiating multipotent stem cells into a variety of somatic cell types. The study of the human brain in vitro has matured in recent years, resulting in three-dimensional organoids that contain the three most abundant cell types of the central nervous system – astrocyte, neurons, and oligodendrocytes. However, these models make tradeoffs – those that closely model organization and structure of the in vivo brain lack physiologically relevant numbers of oligodendrocytes, cells important for metabolic and structural support to neurons. Additionally, current organoid models that incorporate oligodendrocytes in higher numbers do not resemble the complex architecture of the brain. There is a strong opportunity in this space to produce more complex, physiologically relevant organoids that contain large numbers of oligodendrocytes. Previous work has shown it possible to force expression of the transcription factors Sox10 and Olig2 to generate oligodendrocytes from stem cells. Thus, another portion of this thesis aims to utilize the forced expression of transcription factors as a differentiation strategy to generate oligodendrocytes within a brain organoid, increasing their numbers while preserving self-organization that results in complex architecture. Through more physiologically relevant models of the human brain, we can study basic human development and begin to design new gene therapeutics to target neurodegenerative disorders.

Overall, this dissertation presents the use of transcription factors as key factors in engineering better gene delivery strategies and modeling the human brain. The development of ELiPS as a novel method for synthetic promoter engineering will increase our ability to target specific cell types for use in gene delivery, fundamental developmental biology, and beyond. Similarly, delivering transcription factors themselves as transgenic cargo is a promising method of manipulating human development to bring us closer to physiological accuracy in our in vitro study of the human brain.