Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors
Gary C. Pien, … , Jordan S. Orange, Katherine A. High
Gary C. Pien, … , Jordan S. Orange, Katherine A. High
Published May 11, 2009
Citation Information: J Clin Invest. 2009;119(6):1688-1695. https://doi.org/10.1172/JCI36891.
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Research Article Genetics

Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors

Abstract

Adeno-associated virus (AAV) vectors are effective gene delivery vehicles mediating long-lasting transgene expression. Data from a clinical trial of AAV2-mediated hepatic transfer of the Factor IX gene (F9) into hemophilia B subjects suggests that CTL responses against AAV capsid can eliminate transduced hepatocytes and prevent long-term F9 expression. However, the capacity of hepatocytes to present AAV capsid–derived antigens has not been formally demonstrated, nor whether transduction by AAV sensitizes hepatocytes for CTL-mediated destruction. To investigate the fate of capsids after transduction, we engineered a soluble TCR for the detection of capsid-derived peptide:MHC I (pMHC) complexes. TCR multimers exhibited antigen and HLA specificity and possessed high binding affinity for cognate pMHC complexes. With this reagent, capsid pMHC complexes were detectable by confocal microscopy following AAV-mediated transduction of human hepatocytes. Although antigen presentation was modest, it was sufficient to flag transduced cells for CTL-mediated lysis in an in vitro killing assay. Destruction of hepatocytes was inhibited by soluble TCR, demonstrating a possible application for this reagent in blocking undesirable CTL responses. Together, these studies provide a mechanism for the loss of transgene expression and transient elevations in aminotransferases following AAV-mediated hepatic gene transfer in humans and a potential therapeutic intervention to abrogate these limitations imposed by the host T cell response.

Authors

Gary C. Pien, Etiena Basner-Tschakarjan, Daniel J. Hui, Ashley N. Mentlik, Jonathan D. Finn, Nicole C. Hasbrouck, Shangzhen Zhou, Samuel L. Murphy, Marcela V. Maus, Federico Mingozzi, Jordan S. Orange, Katherine A. High

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Figure 3

Confocal microscopy of TCR multimer staining following AAV-mediated transduction.

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Confocal microscopy of TCR multimer staining following AAV-mediated tran...
(A) HHL5-B7 hepatocytes were cultured alone or in the presence of 10 μg/ml VPQYGYLTL capsid peptide or AAV2-F9 vector at 3 × 105 MOI. Cells were then stained with WGA (green) to visualize the plasma membrane, DAPI (blue) to identify the nucleus, and TCR multimer (red) to stain pMHC complexes. Cells were visualized using spinning disk confocal microscopy, and representative cells are shown. Arrows indicate representative areas of TCR and plasma membrane colocalization (yellow). Total original magnification, ×63. (B) Staining intensity of colocalized signal from individual cells was quantitatively measured using Volocity and graphed as average intensity per cell ± SD (n = 17–25 cells per group). After normalization as detailed in Methods, the colocalized intensity was not detectable (nd) on untreated cells. *P < 0.004 versus untreated cells.