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
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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

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Figure 8

dsRNA response in human hepatocytes from xenografted mice after scAAV8/hFIX-opt transduction.

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dsRNA response in human hepatocytes from xenografted mice after scAAV8/h...
(A) Two xenografted mice with human hepatocytes were injected with 3 × 1011 particles of scAAV8/hFIX-opt. The expression of MDA5, RIG-I, and IFN-β in human hepatocytes in mice was detected by Q-PCR at 8 weeks after AAV transduction. Three to four pieces of xenografted liver were chosen randomly for RNA extraction. MDA5 protein in the liver was detected by Western blot after 8 weeks. The band intensity was measured to show the relative MDA5 expression based on β-actin; the data were from 4 separate experiments. *P < 0.05, **P < 0.01, when compared with the control group; data for multiple comparisons were compared using 2-way ANOVA, and data for single comparisons were evaluated by the 2-tailed Student’s t test. (B) Two xenografted mice with human hepatocytes from another donor were injected with a dose of scAAV8/hFIX-opt. The expression of MDA5, RIG-I, and IFN-β in human hepatocytes in mice was detected by Q-PCR at 4 and 8 weeks after AAV transduction. *P < 0.05, **P < 0.01, ***P < 0.001, when compared with the control group, as measured by 2-way ANOVA. (C) MDA5 protein in livers from the second experiment was detected by Western blot, and the relative expression level of MDA5 was calculated based on β-actin intensity; the data were from 4 separate experiments. *P < 0.05, when compared with the control group, as measured by 2-way ANOVA.