Immunological responses to retinal gene therapy

Thesis

Dose-dependent inflammatory responses in adeno-associated virus (AAV)–mediated retinal gene therapy trials have restricted the use of high vector doses and impeded the ability to enhance therapeutic efficacy. Characterising the nature of this response, and devising methods to overcome it, are thus becoming increasingly important in the clinical application of gene therapies. In the experiments herein, I investigated the composition, timing, and magnitude of the cellular immune response to retinal gene therapy in mice using quantitative flow cytometric analysis. I observed a significant increase in retinal leukocytes from 14 days after subretinal injection of an AAV8 vector expressing green fluorescent protein (GFP). These consisted of a constant population of resident microglia and infiltrating peripheral leukocytes including macrophages, natural killer cells, CD4 and CD8 T cells, and natural killer T cells. An identical vector at an equivalent dose encoding a human therapeutic transgene elicited a diminished response compared to paired injections with a GFP vector. However, the proportions of the leukocyte populations remained similar, suggesting a common mechanism of immunity. In addition to the induction of a cell-mediated immune response, innate detection of AAV vectors may directly restrict transgene expression. Preliminary evidence suggested in vitro AAV transduction may be limited by the anti-viral nucleic acid sensors Toll-like receptor 9 and cyclic GMP AMP synthase, suggesting possible detection of transgenic AAV genomes. I also showed that the immunomodulatory drug hydroxychloroquine can significantly enhance AAV transduction in vitro, ex vivo, and in vivo, with indirect indication of the partial antagonism of TLR9. The lack of structural change in the retina following subretinal hydroxychloroquine delivery indicates that it could be a safe adjunct for improving retinal gene therapy efficacy.

Authors: Chandler, L

• DOI: 10.5287/ora-dxrraxq1d
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: AAV; retinal gene therapy; hydroxychloroquine; immunity; adeno-associated virus; inflammation
• Subjects: Immunology; Adeno-associated virus; Retina; Gene therapy