Immune response to ocular gene therapy with AAV8

DSpace Repository


Dateien:

URI: http://hdl.handle.net/10900/109624
http://nbn-resolving.de/urn:nbn:de:bsz:21-dspace-1096243
http://dx.doi.org/10.15496/publikation-51000
Dokumentart: Dissertation
Date: 2020-11-17
Language: English
Faculty: 4 Medizinische Fakultät
Department: Medizin
Advisor: Fischer, M. Dominik (Prof. Dr. Dr.)
Day of Oral Examination: 2020-11-09
DDC Classifikation: 610 - Medicine and health
Keywords: Gentherapie , Dependoviren , Immunreaktion , Auge , Netzhaut
Other Keywords: AAV
Immunantwort
Erbliche Netzhauterkrankung
License: Publishing license including print on demand
Order a printed copy: Print-on-Demand
Show full item record

Abstract:

1 Introduction Adeno-associated viral (AAV) vectors are commonly used in ocular gene therapy for inherited retinal degenerations. They are known for their excellent efficacy in transducing retinal cells and their benign immune profile. However, the observation that subretinal AAV can lead to ocular inflammation highlights the importance to re-evaluate the immunogenicity of AAV. 2 Methods and material Cynomolgus monkeys (Macaca fascicularis) were treated with subretinal or intravitreal injections of AAV serotype 8. The high dose group received 1x10^12 viral genomes (vg) and the low dose group 1x10^11 vg. The control group received injections with vehicle only (balanced salt solution). Of the 34 animals included in the study, 12 were sacrificed after 28 days and 22 were sacrificed after 90 days. During the observation period, blood was taken at fixed timepoints for ELISA of antibodies directed against AAV capsid epitopes. After the inlife phase of the study, eyes were fixed in 4% PFA, embedded and frozen at -80°C. 20µm cryosections were subsequently used for expression profiling and immunohistochemistry. These data were compared to clinical observations from three patients treated with the same vector subretinally. OCT images were taken and peripheral blood mononuclear cells were isolated from patient blood at defined timepoints to be analysed for AAV specific reactivity. 3 Results The ELISA study of NHP derived blood samples showed an increase in anti-AAV8 titer at day 30 in the intravitreal high dose group but not in the subretinal high or low dose group. No antibody formation was observed in the three patients. Immunohistochemistry showed an inflammation of the NHP retina with activation of IBA1+ microglia and infiltration of CD8+ T cells and CD20+ B cells at 28 days following surgery. Expression analysis of the retinal tissue showed an upregulation of genes related to the innate antiviral immune response and associated with the release of INF-. None of the three patients showed clinically significant inflammation, but one featured hyperreflective foci in OCT images one month after surgery, which disappeared following steroid treatment. PBMC analysis showed changes in the activation pattern of CD8+ T cells one month after surgery in 2 out of 3 patients. 4 Discussion The data presented in this study suggests the activation of the innate and adaptive immune response following AAV administration. This hypothesis is supported by the activation of markers of antiviral immune pathways (Toll like, RIG1 like and NOD like receptor pathways) and the increase of capsid specific antibodies as well as retinal infiltration of cells of the adaptive immune response. Future studies will have to determine the exact dynamics of the infiltrating immune cells and how this immune response can be effectively inhibited.

This item appears in the following Collection(s)