Mikos, Antonios G2019-05-172019-08-012018-082018-06-04August 201Lee, Esther Joy. "Adeno-Associated Virus-Based Gene Delivery Approaches for Tissue Engineering." (2018) Diss., Rice University. <a href="https://hdl.handle.net/1911/105807">https://hdl.handle.net/1911/105807</a>.https://hdl.handle.net/1911/105807Tissue engineering constitutes non-trivial processes that can be promoted through combinations of cells, materials and bioactive factors. The formation of tissues and organs demands spatiotemporal precision that cannot, however, be achieved through sole reliance on microenvironment-based strategies. Gene delivery therefore offers a valuable avenue, whereby direct modifications can be made to dynamic systems such as cell populations. The work presented in this thesis explores several strategies for improving control with the intricacies of tissue engineering applications in mind and additionally leverages elements from synthetic biology and synthetic virology. Optogenetic tools based on light-sensitive proteins offer an exquisite option for fine-tuning expression levels with minimal interference and at low cost. Adeno-associated virus (AAV) is employed as the candidate gene delivery vehicle due to its ability to infect many cell types and good safety profile in humans, which bode well for clinical applications. The body of work is grouped into three parts. First, the design and construction of a consolidated red/far red light activatable gene regulatory circuit was pursued with the intent to tune levels of bone morphogenetic protein-2 (BMP-2) expression. The assembly of a large construct had its share of obstacles that eventually led to a downsized strategy, and a number of lessons were accrued from the duration of this endeavor. Second, an all-in-one AAV platform based activatable by blue light was developed and characterized in a series of light induction experiments. Finally, reverse transduction of three AAV serotypes ranging in degree of infectivity in HeLa cells was explored to determine if this strategy could lead to improved transduction efficiency. Parallel comparisons of standard and reverse infection approaches revealed that transduction efficiency could be improved for less amenable serotypes with the latter protocol. We then investigated whether increased transduction efficiency could be explained by higher cellular uptake of AAV. However, internalization assay results could not provide a full explanation for this phenomenon.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.AAVgene deliverytissue engineeringThesis2019-05-17