Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity
Yuanyuan Xiao, … , Phillip W.L. Tai, Guangping Gao
Yuanyuan Xiao, … , Phillip W.L. Tai, Guangping Gao
Published May 21, 2019
Citation Information: JCI Insight. 2019;4(13):e99052. https://doi.org/10.1172/jci.insight.99052.
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Research Article Immunology Therapeutics

Circumventing cellular immunity by miR142-mediated regulation sufficiently supports rAAV-delivered OVA expression without activating humoral immunity

Abstract

Recombinant adeno-associated virus–mediated (rAAV-mediated) gene delivery can efficiently target muscle tissues to serve as “biofactories” for secreted proteins in prophylactic and therapeutic scenarios. Nevertheless, efficient rAAV-mediated gene delivery is often limited by host immune responses against the transgene product. The development of strategies to prevent antitransgene immunity is therefore crucial. The use of endogenous microRNA-mediated (miRNA-mediated) regulation to detarget transgene expression from antigen-presenting cells (APCs) has shown promise for reducing immunogenicity. However, the mechanisms underlying miRNA-mediated modulation of antitransgene immunity by APC detargeting are not fully understood. Using the highly immunogenic ovalbumin (OVA) protein as a proxy for foreign antigens, we show that rAAV vectors containing miR142-binding sites efficiently repress costimulatory signals in DCs, significantly blunt the cytotoxic T cell response, allow for sustained transgene expression in skeletal myoblasts, and attenuate clearance of transduced muscle cells in mice. Furthermore, the blunting of humoral immunity against circulating OVA correlates with detargeting of OVA expression from APCs. This demonstrates that incorporating APC-specific miRNA-binding sites into rAAV vectors provides an effective strategy for reducing transgene-specific immune response. This approach holds promise for clinical applications where the safe and efficient delivery of a prophylactic or therapeutic protein is desired.

Authors

Yuanyuan Xiao, Manish Muhuri, Shaoyong Li, Wanru Qin, Guangchao Xu, Li Luo, Jia Li, Alexander J. Letizia, Sean K. Wang, Ying Kai Chan, Chunmei Wang, Sebastian P. Fuchs, Dan Wang, Qin Su, M. Abu Nahid, George M. Church, Michael Farzan, Li Yang, Yuquan Wei, Ronald C. Desrosiers, Christian Mueller, Phillip W.L. Tai, Guangping Gao

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Figure 5

Transgene detargeting of DCs with miR142BS reduces CD8+ infiltrates and OVA-specific tissue clearance.

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Transgene detargeting of DCs with miR142BS reduces CD8+ infiltrates and ...
(A and B) In vivo CTL assay of vector-treated animals. (A) C57BL/6 male mice, 6 weeks old, were i.m. injected with PBS, rAAV1.OVA, or rAAV1.OVA.miR142BS (1 × 1011 GCs/mouse). Seventeen days after injection, target cells from the spleens of the same strain of treatment-naive mice were either labeled with a high concentration of CFSE (CFSEhi, 5 μM) and pulsed with SIINFEKL peptide (OT-1, 1 μg/mL) or labeled with a low concentration of CFSE (CFSElo, 0.5 μM) and not pulsed. (B) Populations of target cells were mixed (1:1) and cotransferred intravenously (4 × 107 cells) to PBS- or AAV1-treated mice. Six hours later, splenocytes were isolated and analyzed by flow cytometry to determine the percentage of remaining target cells. Bar graphs represent mean ± SD (n = 5). ***P < 0.001, unpaired t test. (C) Serum OVA levels in AAV1.OVA- or AAV1.OVA.miR142BS-treated mice were measured by ELISA (mean ± SD, n = 5). ***P < 0.001, unpaired t test. (D) C57BL/6 male mice, 6 weeks old, were i.m. injected with rAAV1.OVA, rAAV1.OVA.miR142BS, or AAV1 empty vector (1 × 1011 GCs/mouse, n = 5). Two weeks after injection, muscles were harvested for H&E (left, original magnification: ×10) and CD8 staining (right; green, anti-CD8; blue, DAPI; original magnification: ×40). Scale bars: 250 μm (H&E images), 50 μm (fluorescence images). (E) Quantitation of CD8+ T cells counted in 4 fields at original magnification of ×20. *P < 0.05, unpaired t test (n = 5). (F) AAV1.OVA.miR142BS-treated C57BL/6 mice show reduced tumor killing after E.G7-OVA lymphoma cell inoculation. C57BL/6 male mice, 6 weeks old, were injected i.m. with AAV1.OVA or AAV1.OVA.miR142BS (1 × 1011 GCs/mouse). Two weeks after injection, the OVA-expressing E.G7 cells were subcutaneously inoculated (2 × 106 cells/mouse, n = 4). Ten days after inoculation, tumors were harvested and weighed. *P < 0.05, **P < 0.01; 1-way ANOVA followed by Tukey’s test. (G) Serum levels of Th1 and Th2 cytokines in mice treated with AAV1 vectors. C57BL/6 male mice, 6 weeks old, were i.m. injected with PBS, rAAV1.OVA, or rAAV1.OVA.miR142BS (1 × 1011 GCs/mouse). Two weeks later, serum levels of the cytokines IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-12, and GM-CSF were determined by multiple Bio-Plex analysis. Bar graphs represent mean ± SD (n = 3).