Thrombotic microangiopathy following systemic AAV administration is dependent on anti-capsid antibodies

Abstract

BACKGROUNDSystemic administration of adeno-associated virus (AAV) can trigger life-threatening inflammatory responses, including thrombotic microangiopathy (TMA), acute kidney injury due to atypical hemolytic uremic syndrome-like complement activation, immune-mediated myocardial inflammation, and hepatic toxicity.METHODSWe describe the kinetics of immune activation following systemic AAV serotype 9 (AAV9) administration in 38 individuals following 2 distinct prophylactic immunomodulation regimens. Group 1 received corticosteroids and Group 2 received rituximab plus sirolimus in addition to steroids to prevent anti-AAV antibody formation.RESULTSGroup 1 participants had a rapid increase in immunoglobulin M (IgM) and IgG. Increase in D-dimer, decline in platelet count, and complement activation are indicative of TMA. All Group 1 participants demonstrated activation of both classical and alternative complement pathways, as indicated by depleted C4 and elevated soluble C5b-9, Ba, and Bb antigens. Group 2 patients did not have a significant change in IgM or IgG and had minimal complement activation.CONCLUSIONSThis study demonstrates that TMA in the setting of AAV gene therapy is antibody dependent (classical pathway) and amplified by the alternative complement pathway. Critical time points and interventions are identified to allow for management of immune-mediated events that impact the safety and efficacy of systemic gene therapy.

Keywords: Genetics; Immunology; Innate immunity.

Figure 1. IgG and IgM antibodies against AAV9.

Group 1 (dashed black lines and solid circles) received an i.v. dose of AAV9 without adjuvant immune modulation regimen (IMR). Group 2 (solid red lines and triangles) received IMR with rituximab and sirolimus before an intravenous dose of AAV9. IMR as an adjunctive therapy to AAV used in Group 2 prevented the formation of IgG and attenuated the formation of IgM. Data shown as mean ± SEM of Group 1 and Group 2 IgG and IgM baseline percentage change. P > 0.05 (nonsignificant), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001. Note: The split y axis is used to show the small response in Group2 compared with the 100-fold-higher antibody responses in Group 1.

Figure 2. Hematology.

Figure shows the percentage change from baseline for platelet count and D-dimer for Group 1 (dashed black lines and solid circles) and Group 2 (solid red lines and triangles). The data suggest that IMR as an adjunctive therapy to AAV used in Group 2 limits the depletion of platelets and the increase in D-dimer after AAV infusion. Data shown as mean ± SEM of Group 1 and Group 2 baseline percentage change for hematology. P > 0.05 (nonsignificant), *P ≤ 0.05, **P ≤ 0.01.

Figure 3. Complement system markers.

Figure shows C3, C4, C3a, C4a, Ba, Bb, C5a, and SC5b-9 for Group 1 (dashed black lines and solid circles) and Group 2 (solid red lines and triangles). Participants in Group 1 presented with activation of the complement system, as demonstrated by a reduction in C3 and C4, as well as increased C3a, C4, C5a, Ba, Bb, and SC5b-9. The activation of Ba and Bb indicates that both classical and alternative pathway are involved. On the other hand, Group 2 did not show an activation of the complement system, suggesting that IMR as an adjunctive therapy to AAV prevents the activation of the complement system. Data shown as mean ± SEM of Group 1 and Group 2 baseline percentage change for complement. P > 0.05 (nonsignificant), *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001.

Figure 4. Peripheral blood smear.

Sequence of peripheral blood smears after gene therapy administration in patient samples revealing increased schistocytes (blue circles), demonstrating evidence of endothelial damage, burr cells, polychromatic red blood cells, and thrombocytopenia, suggesting thrombotic microangiopathy. Blood smear of a patient with DMD (Group 1B) who received the investigational drug product in NCT03368742.

Figure 5. Immunological event timeline.

Events: (1) AAV dosing and capsid biodistribution; (2) platelet, C3, and C4 depletion; (3) aspartate aminotransferase (AST) and alanine aminotransferase (ALT) elevation; (4) D-dimer elevation; (5) IgM elevation; (6) SC5b-9, C5a, C3a, and C4a elevation; (7) Bb, Ba, and factor I elevation; and (8) IgG elevation. The curve for each parameter was created using the average values obtained in the corticosteroid monotherapy group. The curves of each parameter were overlaid on the graph to show the peaks and trough over time, creating a timeline of events. The amplitude of each curve was adjusted to fit all the curves in 1 graph. The ratio of the peaks’ amplitude was maintained from the original data.