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dc.contributor.authorWhitehead, Michael
dc.date.accessioned2022-04-05T16:50:10Z
dc.date.available2022-04-05T16:50:10Z
dc.date.submitted2021-09-15
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/335789
dc.description.abstractAdeno-associated virus (AAV) is a viral vector that can be used to deliver therapeutic genes to diseased cells in the eye. Whilst some consider the eye to be an immuneprivileged organ, recent reports have begun to suggest that injection of AAV may elicit local and systemic immune activation. The objective of this thesis was to further our understanding of the nature of the immune response to intraocularly-delivered AAV vectors, and to develop strategies to achieve repeated gene transfer (i.e. successful follow-on injections) to the inner retina. A literature review was conducted into the role that pre-existing and induced neutralising antibody responses may play in the context of ocular gene therapy. This concluded that a multifaceted/comprehensive approach may be required to circumvent anti-capsid humoral immunity and enable repeated gene transfer in humans. As such, two possible components of this strategy were tested in this thesis. First, a capsid mutagenesis strategy was tested in which certain capsid residues, termed phosphodegrons, were mutated to attenuate cytosolic degradation of AAV. This was to enable robust transduction of the murine retina at low vector dose and thereby circumvent the generation of neutralising antibodies that is associated with injection of high doses of AAV. In fact, it was found that phosphodegron mutant AAV serotype 2 (AAV2) resulted in higher levels of immune activation across all tested parameters vs. wild-type AAV2, including neutralising and total AAV binding antibody levels, microglia activation, Muller glia activation, CD4+ and CD8+ T-cell infiltration into the retina, splenic germinal center B-cell activation and class-switching, and activation of conventional dendritic cells. Mechanistically, it was shown that the capsids demonstrated reduced binding affinity to the AAV2 primary receptor, heparan sulphate proteoglycan, and as such it was suggested that reduced sequestration of virions in the heparan-rich inner limiting membrane may increase cellular infection levels in the retina, thereby increasing overall immune activation. The phosphodegron mutant capsids also demonstrated that a slight escape from anti-AAV2 neutralising serum, suggesting they may be more refractory to pre-existing neutralising antibodies in seropositive patients. Second, an assessment was made as to whether perioperative administration of immunosuppressive glucocorticoid steroids would circumvent immune responses to AAV. It was found that prednisolone was effective at reducing anti-AAV neutralising and total AAV binding antibody titres. This effect may have occurred via the inhibition of splenic germinal center B-cell reactions and class-switching of immunoglobulin antibody isotypes, and reducing follicular helper T-cell levels. Despite the reduction in NAb and TAb titres, it was found that perioperative prednisolone administration was insufficient to enable repeated gene transfer following a second eye injection of AAV2. Interestingly, repeated bilateral AAV2 injections may have resulted in a slight reduction in electrophysiological activity in the retina compared to mice who received two control PBS injections. In conclusion, this thesis highlighted novel aspects of phosphodegron mutant AAV2 immunobiology and identified a possible utility for the administration of prednisolone as a tool for enabling repeated gene transfer of AAV gene therapy vectors.
dc.rightsAll Rights Reserved
dc.rights.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.subjectAAV
dc.subjectGene therapy
dc.subjectEye
dc.subjectRetina
dc.subjectImmune response
dc.titleImmune responses to AAV gene therapy in the ocular compartment
dc.typeThesis
dc.type.qualificationlevelDoctoral
dc.type.qualificationnameDoctor of Philosophy (PhD)
dc.publisher.institutionUniversity of Cambridge
dc.date.updated2022-01-25T15:06:27Z
dc.identifier.doi10.17863/CAM.83225
rioxxterms.licenseref.urihttps://www.rioxx.net/licenses/all-rights-reserved/
dc.contributor.orcidWhitehead, Michael [0000-0002-5494-8302]
rioxxterms.typeThesis
dc.publisher.collegeQueens
pubs.funder-project-idMedical Research Council (1944187)
cam.supervisorYu Wai Man, Patrick
cam.supervisorMartin, Keith
cam.supervisor.orcidYu Wai Man, Patrick [0000-0001-7847-9320]
cam.supervisor.orcidMartin, Keith [0000-0002-9347-3661]
cam.depositDate2022-01-25
pubs.licence-identifierapollo-deposit-licence-2-1
pubs.licence-display-nameApollo Repository Deposit Licence Agreement


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