Browsing by Subject "synthetic biology"

Now showing 1 - 3 of 3
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    Development of Methods and Tools for Cell-Free Synthetic Biology Applications
    (2017-08) Sadler, Fredrik
    A primary advantage of bead surface display is the potential for highly controlled, multivalent display of immobilized protein. To realize this potential, we built a bead surface display platform with multivalency in mind. Starting with a display particle with dense functional groups, we systematically designed and synthesized a bead saturated in DNA and protein attachment sites utilizing chemoselective coupling mechanisms. With the potential of tens of millions of biomolecule attachment sites, we optimized the biological steps of genotype and phenotype immobilization empirically. Starting with a single gene copy per bead to mimic monoclonality, we amplified genotype onto the bead surface to a sufficient degree to express attachable protein and, yet sparse to leave most of the bead surface area exposed for protein attachment. We systematically rescued the in vitro transcription step to maximize protein expression from the immobilized context. Our methodology enables more insensitive and indirect detection mechanisms for rank-sort screening of protein libraries.
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    Engineering inducible cell lines for recombinant Adeno-Associated Virus production
    (2021-08) Lee, Zion
    The recombinant adeno-associated virus (rAAV) gene therapy field has experienced landmark regulatory approvals in recent years by demonstrating high efficacy in treating monogenic diseases. There is therefore an immediate and increasing need for highly productive manufacturing platforms to generate large quantities of rAAV vectors. Current rAAV synthesis methods require multiple-component plasmid transfections or viral infections, which have increasing costs, variability, and technical challenges at large scales. To address the growing need for robust methods to produce and characterize rAAV vectors, we used synthetic biology tools to gain control over viral gene expression dynamics. First, we engineered a rAAV infectious titer assay cell which contains the AAV and helper genes necessary to replicate transduced genomes. Once exposed to small molecule inducers and a rAAV vector, the assay cell line performed similarly to the standard adenovirus (Ad) coinfection method in determining the infectious titer of the vector preparation. Omitting the use of Ad greatly simplifies the potency assay and reduces the variability of input reagents. Building on that success, we further engineered cells that not only have replicative capacity, but also packaging capacity and a latent copy of a rAAV genome. Small molecule induction allowed expression of the necessary viral genes to synthesize infectious rAAV vectors. The variation of inducer ratios enabled manipulation of vector quality, in terms of the fraction of DNA-containing particles. These synthetic cellular technologies address the needs of a reliable potency bioassay and a scalable and robust production platform for rAAV manufacturing.
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    Systems Design and Synthetic Construction of Influenza Virus for Flu Vaccine Application
    (2021-11) Phan, Thu
    Influenza A virus (IAV) is the leading cause of annual flu epidemics, which inflicts about 250,000-500,000 deaths worldwide. The morbidity and mortality rate are much higher when a novel strain of IAV arises, resulting in flu pandemics. Vaccination has been the best prevention strategy for influenza. However, flu viruses constantly evolve and escape the established immunity, thus annual flu vaccination is required. Most current flu vaccine manufacturing platforms use multi-plasmid transfection to rescue seasonal seed viruses, the seed viruses are then used to infect either embryonic chicken eggs or cultured cells to produce viruses. Both production methods have high degrees of variability and produce viruses with a high content of non-infectious particles that reduce vaccine effectiveness. To address the need for more reliable and scalable processes, we applied systems biology and synthetic biology approaches to understand the kinetics of virus replication and to engineer cell lines that can control viral gene expression dynamics. First, we established a new data analysis pipeline using RNA sequencing to study segment-specific kinetics of all IAV RNA molecules. Using the pipeline, InVERT, to study the kinetics of IAV infection, revealed different phases of virus infection, and groups of genes whose kinetics are similar. This was the first-time IAV replication kinetics of all segments is reported. Building on that success, we then developed the second pipeline named InVERT II, which can further differentiate mRNA transcripts made by the viral replication enzyme RdRP from mRNA transcripts synthesized by host cells' RNA Polymerase II, to study the kinetics of virus rescue by transfection. With the understanding gained from the kinetics of virus infection and replication, we engineered the human cell line HEK 293T to express inducible components of IAV that not only have inducible replicative activity but also can package virus particles. This is the first proof of principle to show that mammalian cells can be engineered to produce complex negative-sense RNA viruses. Our integrative approach using both systems biology and synthetic biology has enabled the creation of a platform that could be further optimized for reliable, robust, and scalable flu vaccine manufacturing processes.