Browsing by Subject "adeno-associated virus"
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Item Engineering inducible cell lines for recombinant Adeno-Associated Virus production(2021-08) Lee, ZionThe 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.Item TARGETING THE AGMATINERGIC SYSTEM USING AN AAV-BASED GENE THERAPY FOR THE TREATMENT OF CHRONIC PAIN(2019-12) Pflepsen, KelseyThe complex mechanisms underlying chronic pain and the challenges of current pharmacotherapy leave many chronic pain patients without adequate treatment due to negative physical and social side effects. Thus, there is a critical need to develop new pain management therapies with long-term effectiveness and minimal adverse effects. Using an adeno-associated virus (AAV) gene therapy to treat chronic pain has gained interest in the last twenty years because of several therapeutic advantages. AAV gene therapy allows for selective gene expression directly to sites of interest for chronic pain treatment with the potential for sustained expression following a single injection. In an effort to treat chronic pain using an AAV gene therapy, we have created a viral vector using recombinant adeno-associated virus which encodes the biosynthetic enzyme, human arginine decarboxylase (hADC). Arginine decarboxylase (ADC) is an endogenous enzyme that catalyzes the metabolism of L-arginine into agmatine. Agmatine is an endogenous small molecule that acts as a neurotransmitter and has been previously shown to modulate neuroplastic events by antagonizing the GluN2B subunit of the N-methyl-D-aspartate (NMDA) receptor. When agmatine is delivered centrally, a reduction in pain behavior following nerve injury is observed. The central hypothesis of this body of work is that overexpression of arginine decarboxylase using an adeno-associated viral vector construct results in long-term reduction of neuropathic pain due to the production of agmatine and subsequent antagonism of the GluN2B NMDA receptor by agmatine. The experiments presented in this dissertation build upon our agmatine-based pharmacotherapy for the treatment of chronic neuropathic pain. Specifically, this work furthers our AAV-based gene therapy by understanding intrathecal AAV kinetics, evaluating how therapeutic efficacy changes with age and with the use of cell-specific promoters, and interpreting associated behavioral implications.