Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction
Wenwei Shao, Lauriel F. Earley, Zheng Chai, Xiaojing Chen, Junjiang Sun, Ting He, Meng Deng, Matthew L. Hirsch, Jenny Ting, R. Jude Samulski, Chengwen Li
Wenwei Shao, Lauriel F. Earley, Zheng Chai, Xiaojing Chen, Junjiang Sun, Ting He, Meng Deng, Matthew L. Hirsch, Jenny Ting, R. Jude Samulski, Chengwen Li
View: Text | PDF
Research Article Immunology Therapeutics

Double-stranded RNA innate immune response activation from long-term adeno-associated virus vector transduction

Abstract

Data from clinical trials for hemophilia B using adeno-associated virus (AAV) vectors have demonstrated decreased transgenic coagulation factor IX (hFIX) expression 6–10 weeks after administration of a high vector dose. While it is likely that capsid-specific cytotoxic T lymphocytes eliminate vector-transduced hepatocytes, thereby resulting in decreased hFIX, this observation is not intuitively consistent with restored hFIX levels following prednisone application. Although the innate immune response is immediately activated following AAV vector infection via TLR pathways, no studies exist regarding the role of the innate immune response at later time points after AAV vector transduction. Herein, activation of the innate immune response in cell lines, primary human hepatocytes, and hepatocytes in a human chimeric mouse model was observed at later time points following AAV vector transduction. Mechanistic analysis demonstrated that the double-stranded RNA (dsRNA) sensor MDA5 was necessary for innate immune response activation and that transient knockdown of MDA5, or MAVS, decreased IFN-β expression while increasing transgene production in AAV-transduced cells. These results both highlight the role of the dsRNA-triggered innate immune response in therapeutic transgene expression at later time points following AAV transduction and facilitate the execution of effective strategies to block the dsRNA innate immune response in future clinical trials.

Authors

Wenwei Shao, Lauriel F. Earley, Zheng Chai, Xiaojing Chen, Junjiang Sun, Ting He, Meng Deng, Matthew L. Hirsch, Jenny Ting, R. Jude Samulski, Chengwen Li

×

Figure 4

dsRNA immune response is activated at a later time point after AAV transduction.

Options: View larger image (or click on image) Download as PowerPoint
dsRNA immune response is activated at a later time point after AAV trans...
HeLa cells were transduced with 5 × 103 particles of scAAV2/GFP per cell. The expression of MDA5 (A), RIG-I (B), and IFN-β (C) in HeLa cells was detected by Q-PCR at different time points after transduction. *P < 0.05, **P < 0.01, when compared with the PBS group, as measured by 2-way ANOVA. The data represent the mean ± SEM from 3 independent experiments. For each experiment, the PBS- or AAV-infected group contained 2 or 3 wells of cells. For Q-PCR data analysis, one sample from the PBS group was normalized to one in each time point of each experiment. (D) GFP expression level in HeLa cells was detected by flow cytometry at different time points after AAV2/GFP transduction. (E) MDA5 expression in HeLa cells in each group was detected by Western blot 8 days after scAAV2/GFP transduction. The relative level of MDA5 expression was calculated based on the intensity of β-actin protein from 3 independent experiments. ***P < 0.001, when compared with the PBS group, as measured by 2-tailed Student’s t test.