Date of Award


Degree Type


Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Cell & Molecular Biology

First Advisor

James M. Wilson


Impact of host factor on the adaptive immune response to AAV gene therapy

Scott Ashley

James Wilson

Adaptive immune responses to the transgene product remain an active area of concern for the gene therapy field. How host factors can influence the activation of the immune system is an important consideration in the development of gene therapy for different genetic disorders. One factor considered by many to shape the adaptive immune response is an individual’s genotype. Nonsense mutations were thought to result in an absence of tolerance to a replacement protein provided by gene therapy, due to a lack of antigen presentation during T cell development and negative selection. In this work we demonstrated that a class of nonsense mutations, premature termination codons (PTC), found in ornithine transcarbamylase deficiency (OTC) patients do not inhibit antigen presentation of C-terminal epitopes. We further found that these PTC containing OTC genes were able to induce anergy in a model of peripheral tolerance. These results change how we think about the relationship between the genotype and immune response, which indicate that individuals with PTC mutations may be less at risk of an adverse immune response attenuating the effects of gene therapy. We also identified vector factors that influence the adaptive immune response, activation of TLR9 and the tissues targeted for transduction and expression of the transgene. To investigate how inflammatory signaling might impact the outcome of adaptive immune responses; we use the transgenic OT-1 mouse model to interrogate how TLR9, the primary sensing molecule for vector DNA, can activate cytotoxic T cells against the transgene product OTC. These results confirm an important role for TLR9 induced inflammation being necessary for transgene specific T cell activation. To investigate the influence of TLR9 signaling and tissue targets on the humoral response, we used a mouse model of Mucopolysaccharidosis type I (MPS1) disease, and measured antibody generation to the secreted transgene product alpha-L-iduronidase (IDUA). We report that TLR9 signaling is also instrumental for the formation of anti-IDUA antibodies, as is expression of the transgene from the muscle. This work describes a novel process by which tolerance to a peptide located downstream of a PTC can be induced. This insight can help us better define the risks associated with an adaptive immune response based on an individual’s personal mutation. We also defined important vector factors which are important for activation of an adaptive immune response, and this knowledge could be exploited to generate safer gene therapy delivery methods.