Engineered AAV vector minimizes in vivo targeting of transduced hepatocytes by capsid-specific CD8+ T cells

Abstract

Recent clinical trials have shown that evasion of CD8(+) T-cell responses against viral capsid is critical for successful liver-directed gene therapy with adeno-associated viral (AAV) vectors for hemophilia. Preclinical models to test whether use of alternate serotypes or capsid variants could avoid this deleterious response have been lacking. Here, the ability of CD8(+) T cells ("cap-CD8," specific for a capsid epitope presented by human B*0702 or murine H2-L(d) molecules) to target AAV-infected hepatocytes was investigated. In a murine model based on adoptive transfer of ex vivo expanded cap-CD8, AAV2-transduced livers showed CD8(+) T-cell infiltrates, transaminitis, significant reduction in factor IX transgene expression, and loss of transduced hepatocytes. AAV8 gene transfer resulted in prolonged susceptibility to cap-CD8, consistent with recent clinical findings. In contrast, using an AAV2(Y-F) mutant capsid, which is known to be less degraded by proteasomes, preserved transgene expression and largely avoided hepatotoxicity. In vitro assays confirmed reduced major histocompatibility complex class I presentation of this capsid and killing of human or murine hepatocytes compared with AAV2. In conclusion, AAV capsids can be engineered to substantially reduce the risk of destruction by cytotoxic T lymphocytes, whereas use of alternative serotypes per se does not circumvent this obstacle.

Figures

Figure 1

Effects of AAV capsid-specific CD8+ T cells on systemic liver-derived hF.IX expression in vivo. (A) Diagram of in vivo model and experimental outline: In vitro expanded capsid-specific, Ld-restricted CD8+ T cells (cap-CD8) isolated from BALB/c mice were adoptively transferred by tail vein injection into Rag-1−/− BALB/c mice 24 hours after administration of AAV-ApoE/hAAT-hF.IX vector (1 × 1011 vg per mouse; n = 4 per experimental group). As negative control cells, CD8+ T cells specific for the influenza dominant epitope (con-CD8) were generated in BALB/c mice and in vitro expanded in parallel. Mice received cap-CD8, con-CD8, or no cells as indicated in each panel. Following T- cell transfer, mice were treated with LPS on days 0, 1, and 2 to provide an additional activation signal. Systemic hF.IX levels were measured as a function of time after administration of the following vectors: AAV2 (B), AAV2 (without LPS administration) (C), AAV2 in absence or presence of proteasome inhibitor bortezomib (PI) (D), or AAV2(Y-F) in absence or presence of PI (E). Fold differences of hF.IX levels for mice treated with con-CD8 and cap-CD8 are indicated (except for panel B, which is no CD8 compared with cap-CD8). Data are average ± standard deviation (SD) for each time point with n = 5 per experimental group. *P < .05; **P < .01 using Student t test for each time point. (F) Systemic hF.IX levels as a function of time after AAV2(Y-F) vector administration to Rag-1−/− BALB/c mice that received cap-CD8 cells at the indicated time points or no cells (control; n = 4 per group).

Figure 2

Liver toxicity caused by AAV capsid-specific CD8+ T cells. Circulating liver enzyme (AST and ALT) levels in Rag-1 −/− BALB/c mice were measured in serum collected 7 days after adoptive transfer of CD8+ T cells (as outlined in Figure 4A: AAV capsid-specific, cap-CD8; control cells, con-CD8; or no cells; n = 4 per experimental group). (A) AAV2. (B) AAV2(Y-F). Data are average ± SD. *P < .05; **P < .01 using Student t test (comparison between mice treated with cap-CD8 and con-CD8). (C) Liver cryosections were generated for the same day-7 time point. Antibody stains show CD8+ T cells (green) and hF.IX+ hepatocytes (red). Representative examples at the indicated original magnification are shown for mice having received cap-CD8 cells and the following vector capsid: AAV2 (upper panels); AAV2(Y-F) (lower left panel); day 28 after AAV2 vector administration (lower right panel). Arrows depict CD8+ cells. Original magnifications are as indicated for each panel.

Figure 3

Quantification of hF.IX+ hepatocytes 1 month after adoptive transfer of cap-CD8 or con-CD8 cells. Mice had been transduced with AAV2-hF.IX (A) or AAV2(Y-F)-hF.IX (B) vector. Percent decrease of hF.IX levels from cap-CD8 cells is indicated. Data from individual mice (n = 3-4 per group, with 10 random low-power fields for each liver) as well as averages are graphed. P values for significant differences are indicated. Representative hF.IX stains (red) are shown for AAV2 (C) and AAV2(Y-F) (D).

Figure 4

In vitro killing of AAV-pulsed murine hepatocytes by capsid-specific CD8+ T cells. (A) Percent death of target cells (adult BALB/c hepatocyte cell line H2.35), pulsed with either AAV2 or AAV2(Y-F), as a function of MOI (at effector to target ratio of 80:1). (B) Percent surviving hF.IX+ H2.35 hepatocytes (as determined by flow cytometry after completion of in vitro killing assay) pulsed with either AAV2 or AAV2(Y-F), as a function of MOI (at effector to target ratio of 80:1). (C) Percent hF.IX+ H2.35 hepatocytes transduced with either AAV2 or AAV2(Y-F) (in the absence of effector cells) as a function of MOI. Data are average ± SD for quadruplicate measurements. Panels are representative examples of at least 2 experiments. Statistically significant differences between AAV2 and AAV2(Y-F) for a specific target:effector ratio: *P < .05; **P < .01, unpaired, 2-tailed t test. (D-E) Effects of proteasomal inhibition on liver target cell death. Shown are percent dead H2.35 target cells that had been pulsed with either AAV2 or AAV2(Y-F) at MOI of 104 vg per cell and treated with proteasomal inhibitor (PI). Liver cells were treated with 100 nM bortezomib (D) or 300 nM MG-132 (E). Controls include mock-transduced target cells and cells that were not treated with PI. Assays were again performed in quadruplicate.

Figure 5

Capsid antigen presentation and killing assay in vitro in AAV-transduced human hepatocytes. HHL5 human hepatocytes were transduced in vitro at increasing MOI with AAV2 or AAV2(Y-F) vectors. (A) Levels of antigen presentation measured with Jurma-VPQ reporter cells 24 hours after vector transduction. Jurma-VPQ cells were added in culture overnight at a ratio of 10:1 reporter:target. RLU, relative light units. (B) CTL assay. HHL5 target cells were transduced overnight and cocultured for 4 hours with effector cells derived by peripheral blood mononuclear cells at an effector:target ratio of 10:1. Percent cytotoxicity was measured relative to a maximum lactate dehydrogenase release (tritonX-treated targets) after background subtraction. All results are reported as average ± standard error of the mean. *P < .05, unpaired, 2-tailed t test. (C) The peptide VPQYGYLTL is shown bound to H2-Ld based on the crystal structure of SPLDSLWWI bound to H2-Ld in Protein Data Bank code 3TJH. VPQYGYLTL is shown as yellow sticks for carbon, blue for nitrogen, and red for oxygen. The peptide VPQYGYLTL is shown bound to HLA-B*-07:02 as modeled from the crystal structure of HLA-B8 from PDB code 3SPV (95.7% identical to HLA-B*07:02:01). The molecular surfaces of H2-Ld and HLA-B*07:02:01 are shown as orange for carbon, blue for nitrogen, and red for oxygen.

Figure 6

Cell entry and nuclear colocalization of AAV vectors. The human hepatocyte cell line HHL5 was incubated with AAV vectors at an MOI of 105 in a tissue culture incubator and assayed at 1, 5, and 24 hours for virus nuclear colocalization on an ImageStream imaging flow cytometer. (A) Virus intracellular localization. Five hours after the time course started, virtually all virions were internalized into hepatocytes. (B) Colocalization of fluorescently labeled viral particles and nuclei, shown as percent of total viral particles. Results from 1 representative experiment. All experiments were repeated and analyzed at least twice.

Figure 7

Effects of capsid-specific CD8+ T cells on hF.IX expression from an AAV8 vector. In vitro expanded capsid-specific, Ld-restricted CD8+ T cells (cap-CD8) isolated from BALB/c mice were adoptively transferred by tail vein injection into Rag-1 −/− BALB/c mice after administration of AAV8-ApoE/hAAT-hF.IX vector (1 × 1011 vg per mouse; n = 4 per experimental group). Cells were given 1 day (A-B) or 1 day, 7 days, or 14 days (C-D) after vector administration. (A,C) Systemic hF.IX expression. (B) Percent hF.IX+ hepatocytes 4 weeks after gene transfer. Statistically significant differences to the control group: *P < .05; **P < .01; ***P < .001. (D) Percent hF.IX+ hepatocytes 6 weeks after gene transfer. (E) Immunostains for hF.IX and CD8+ T cells 1 and 6 weeks after cap-CD8 T-cell administration to mice that had received AAV8 vector 7 days prior. Analyses were performed as for Figure 1.