Capsid antigen presentation flags human hepatocytes for destruction after transduction by adeno-associated viral vectors
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
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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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 4

Cytotoxic activity against peptide-pulsed and AAV-transduced hepatocytes.

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Cytotoxic activity against peptide-pulsed and AAV-transduced hepatocytes...
In separate experiments, the human hepatocyte cell line HHL5 expressing HLA-A*0101 was cultured with (A) 10 μg/ml of SADNNNSEY capsid peptide, or the HLA-B*0702–expressing HHL5-B7 cell line was cultured with (B) 3 × 105 MOI AAV2-F9 vector or (C) 3 × 105 MOI AAV2-empty capsids devoid of genomes. HLA-matched human PBMCs were added at the indicated effector/target (E:T) ratios in a CTL cytotoxicity assay. The percentage of specific lysis was calculated from the release of intracellular lactate dehydrogenase. To determine the effects of antigen load on CTL cytotoxicity, HHL5-B7 target cells were incubated with the indicated concentrations of VPQYGYLTL capsid peptide (D) or MOI of AAV2-F9 (E) overnight, then effectors were added at an effector/target ratio of 10:1. Effector cells were derived as follows: (A–D) normal human donor PBMCs were independently expanded for 2 rounds with capsid peptide or AAV2-empty capsid and pooled for use as effector cells or (E) human PBMCs were expanded for 2 rounds with AAV2-empty capsid only.