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Immunoadsorption enables successful rAAV5-mediated repeated hepatic gene delivery in nonhuman primates

David Salas, Karin L. Kwikkers, Nerea Zabaleta, Andrea Bazo, Harald Petry, Sander J. van Deventer, Gloria Gonzalez Aseguinolaza and Valerie Ferreira

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Reduction of anti-AAV5 NAB levels after IA procedure. (A). Levels of anti-AAV5 NAB (IC50) levels are reported at baseline (day 0), after rAAV5-hSEAP administration (day 49, NHPs 1-6), after IA (post IA, NHPs 4-6) and after rAAV5-hFIX readministration (day 77, NHPs 1-6). (B). Fold-decrease in anti-AAV5 NAB (IC50) levels measured in plasma samples taken after every cycle of the IA procedure.

  • Figure 2.

    Dual hSEAP and hFIX transgene protein expression only in NHPs following IA. NHPs received a primary administration of rAAV5-hSEAP (day 0) and a secondary readministration of rAAV5-hFIX (day 49) with or without prior IA treatment (IA). (A) hSEAP protein expression was stable throughout the experiment and not impacted by the IA procedure. (B) hFIX protein was expressed successfully after IA. As a reference for hFIX levels, 2 NHPs that were administered with 3 × 1013 gc/kg rAAV5-hFIX were included. (C) The averages of the areas under the curves (AUC) ± standard deviation calculated for the hFIX expression over time showed an hFIX increase for the group submitted to IA (NHPs 4-6) when compared with the group without IA (NHPs 1-3). Furthermore, a decrease of 2.9 times was observed for the NHPs submitted to IA (NHPs 4-6) compared with the control animals (NHPs controls 1 and 2). RLU, relative light unit.

  • Figure 3.

    Increase of hFIX-vector DNA in liver following IA. Levels of hSEAP- and hFIX-vector DNA were monitored by qPCR in the liver tissue at euthanization. NHPs received a primary administration of rAAV5-hSEAP (day 0) and a second administration of rAAV5-hFIX (day 49) with or without pretreatment with IA (IA). Per animal, 30 different liver samples were analyzed. The hSEAP- (A) and hFIX-vector (B) DNA levels are plotted per animal with indication of the medians. (C-D) Medians and range per group are reported. Although animal groups with or without an IA procedure had similar levels of hSEAP-vector DNA, an increase of 27.86 times in genome copy number of hFIX-vector DNA was measured in the animal group that underwent the IA. The levels of hFIX-vector DNA measured in the control group, primary administered with rAAV5-hFIX at the same dose of 3 × 1013 gc/kg, were 4.62 times higher than the group that underwent IA.

  • Figure 4.

    Correlation between anti-AAV5 NABs and total antibodies. (A) Total anti-AAV5 antibodies were measured by ELISA on plasma samples collected before, during, and after the IA procedure. A significant decrease (*0.0266) was realized between the OD450 before and after the IA procedure using a Friedman test and a Dunn multiple comparison test. (B) A correlation was established by exponential regression between the levels of total and anti-AAV5 NABs just before and throughout the IA procedure (pre IA, cycle 1, 2, 3).

  • Figure 5.

    Anti-AAV5 NAB threshold compatible with readministration. The efficacy of rAAV5-hFIX liver transduction at readministration was determined as a function of anti-AAV5 NAB titers. Post-rAAV5-hFIX readministration levels of hFIX protein at day 56 were plotted against the anti-AAV5 NAB IC50 levels just before the rAAV5-hFIX readministration. The data for 14 NHPs are reported in the graph; NHPs 1 to 6 (closed symbols) and NHPs 7 to 14 (open symbols). The vertical dotted line represents the threshold of efficacy determined at an IC50 of 8250. The horizontal area represents the minimal therapeutic range of 150 to 250 ng/mL corresponding to 3% to 5% of 5 mg/mL.