Date of Award

2024

Document Type

Dissertation

Degree Name

Philosophy (Ph.D)

Department

Pharmaceutical Sciences

First Advisor

Ashley Thomas Martino

Abstract

Adeno-Associated Virus (AAV) is extensively utilized in various clinical trials and approved gene therapies. Despite its widespread use, the immune response to AAV vectors remains a significant limitation. Extensive research has been conducted to mitigate adaptive immune challenges, such as pre-existing antibodies and inhibiting the adaptive immune response. This study, however, centers on the innate immune response elicited by AAV, exploring the modulatory effects of insulin and DMA. Initially, we examined the impact of DMA and insulin on the human liver cell line Hep3B and the human macrophage cell line U937 by culturing together in transwell inserts. Following stimulation with AAV1 and a TLR9 agonist for 2 hours, Hep3B cells exhibited increased levels of TNF-α, IL-6, and IL-12, while U937 cells showed heightened IL-1β, IL-6, and INF-β. These elevations were effectively mitigated by either DMA or insulin. At subsequent 6-hour and 24-hour intervals, neither U937 nor Hep3B cells responded to AAV and TLR9 agonist stimulation. We further extended our analysis in vivo using C57BL/6 mice. AAV and TLR9 agonists were administered intramuscularly, while insulin and DMA were delivered intraperitoneally. At 2 hours post-administration, there was a notable increase in IL-6 expression in vivo, accompanied by approximately 2.5-fold increases in interferon-α and interferon-β. These increases were significantly attenuated by the administration of insulin or DMA. By the 6-hour mark, gene expression levels stimulated by AAV and TLR9 agonists had normalized, and no additional suppressive effects were observed from insulin and DMA. Concurrently, immunohistochemical analysis was conducted to assess CD11b positive macrophage recruitment. At 2 hours, no groups exhibited M1/70 positive macrophage recruitment. However, at 6 hours, the group stimulated with AAV and TLR9 agonists showed macrophage recruitment, whereas groups co-administered with insulin or DMA did not, suggesting a potential blockade or delay in recruitment by these agents. In summary, our findings indicate that insulin and DMA can effectively suppress the innate immune response triggered by AAV. This suppression could provide a strategy to prevent the activation of adaptive immunity. However, further investigation is required to fully elucidate the underlying mechanisms of this suppression.

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