Effective Depletion of Pre-existing Anti-AAV Antibodies Requires Broad Immune Targeting

Abstract

Pre-existing antibodies (Abs) to AAV pose a critical challenge for the translation of gene therapies. No effective approach is available to overcome pre-existing Abs. Given the complexity of Ab production, overcoming pre-existing Abs will require broad immune targeting. We generated a mouse model of pre-existing AAV9 Abs to test multiple immunosuppressants, including bortezomib, rapamycin, and prednisolone, individually or in combination. We identified an effective approach combining rapamycin and prednisolone, reducing serum AAV9 Abs by 70%-80% at 4 weeks and 85%-93% at 8 weeks of treatment. The rapamycin plus prednisolone treatment resulted in significant decreases in the frequency of B cells, plasma cells, and IgG-secreting and AAV9-specific Ab-producing plasma cells in bone marrow. The rapamycin plus prednisolone treatment also significantly reduced frequencies of IgD^-IgG⁺ class-switched/FAS⁺CL7⁺ germinal center B cells, and of activated CD4⁺ T cells expressing PD1 and GL7, in spleen. These data suggest that rapamycin plus prednisolone has selective inhibitory effects on both T helper type 2 support of B cell activation in spleen and on bone marrow plasma cell survival, leading to effective AAV9 Abs depletion. This promising immunomodulation approach is highly translatable, and it poses minimal risk in the context of therapeutic benefits promised by gene therapy for severe monogenetic diseases, with a single or possibly a few treatments over a lifetime.

Keywords: AAV; gene therapy; pre-existing antibody.

Figure 1

Significant Reduction of Pre-existing Antibodies against AAV9 in Mice by Immunosuppression Regimens (A–D) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with selected immunosuppressants (IS), beginning at 4 weeks pi. Serum samples (n = 3–13/group) from IS-treated animals and non-IS controls were assayed for anti-AAV9-IgG at day 0, 4 weeks, and 8 weeks of IS treatment. (A) Treatment with Rap or Pred only is shown. (B) Dose response of Bort + Pred treatment is shown. (C) Dose response of Rap + Pred treatment is shown. (D) Rap + Pred: dose response and administration route of pred are shown. Pre-IS, prior to IS treatment; non-IS, non-IS controls; RH, high dose of rapamicin, i.p.; RL, low dose of rapamicin, i.p.; BH, high dose of bortezomib, i.p.; BL, low dose of bortezomib, i.p.; PH, high dose of prednisolone, i.p.; PL, low dose of prednisolone; PL(O), oral low dose of prednisolone. *p ≤ 0.05 versus pre-IS treatment; #p ≥ 0.05 versus pre-IS treatment; ˆp ≤ 0.05 versus non-IS; @n = 2.

Figure 2

Differential Impacts of Rap + Pred on Bone Marrow B Cells and Plasma Cells (A–D) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with rapamycin (R, 2 mg/kg, every other day) and prednisolone (P, 0.75 mg/kg, daily) via i.p. injection, beginning at 4 weeks post-immunization. Controls were matched naive and AAV9-immunized mice without IS treatment. At 8 weeks on IS treatment, bone marrow (BM) cells were assayed by flow cytometry for (A and B) B cells (B220⁺MHCII⁺) and (C and D) plasma cells (PC, B220⁻CD138⁺). Naive, non-immunized WT mice; non-IS, AAV9-immunized WT mice without IS treatment; R + P, AAV9-immunized mice treated with R + P.

Figure 3

Differential Impacts of Rap + Pred on Bone Marrow Antibody-Secreting Cells (A–E) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with rapamycin (R, 2 mg/kg, every other day) and prednisolone (P, 0.75 mg/kg, daily) via i.p. injection (R + P), beginning at 4 weeks post-immunization. Controls were matched naive and AAV9-immunized mice without IS treatment. Bone marrow (BM) cells were assayed at 8 weeks of IS treatment by ELISpot for (A, B, and D) IgG-secreting and (C and E) AAV9-Ab-secreting plasma cells (PCs). (B and C) p = 0.04 and p = 0.13 R + P versus non-IS. Naive, non-immunized WT mice; non-IS, non-IS-treated AAV9-immunized mice; R + P, AAV9-immunized mice treated with R + P.

Figure 4

Effects of Rap + Pred on Splenocytes (A–D) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with rapamycin (R, 2 mg/kg, every other day) and prednisolone (P, 0.75 mg/kg, daily) via i.p. injection, beginning at 4 weeks post-immunization. Controls were matched naive and AAV9-immunized mice without IS treatment. At 8 weeks on IS treatment, splenocytes were assayed by flow cytometry for CD3, CD4, and CD19 to determine (A) total splenocytes, (B) B cell frequencies, (C) T cell frequencies, and (D) frequencies of CD8⁺ and CD4⁺ T cell subsets. Naive, non-immunized WT mice; non-IS, AAV9-immunized WT mice without IS treatment; R + P, AAV9-immunized mice treated with R + P.

Figure 5

Effects of Rap + Pred on Splenic B Lymphocytes (A–F) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with rapamycin (R, 2 mg/kg, every other day) and prednisolone (P, 0.75 mg/kg, daily) via i.p. injection (R + P), beginning at 4 weeks post-immunization. Controls were matched naive and AAV9-immunized mice without IS treatment. At 8 weeks on IS treatment, splenocytes were assayed by flow cytometry for CD19, IgG, IgD, CD38, GL7, and CD95. (A and B) IgD⁻IgG⁺ class switch, (C and D) CD38^low/MHCII^high B cell frequencies, and (E and F) GL7⁺CD95⁺ B cell frequencies are shown. Naive, non-immunized WT mice; non-IS, AAV9-immunized WT mice without IS treatment; R + P, AAV9-immunized mice treated with R + P.

Figure 6

Effects of Rap + Pred on Splenic T Lymphocytes (A–F) WT mice were immunized with an i.p. injection of rAAV9 vector, and then they were treated with rapamycin (R, 2 mg/kg, every other day) and prednisolone (P, 0.75 mg/kg, daily) via i.p. injection, beginning at 4 weeks post-immunization. Controls were matched naive and AAV9-immunized mice without IS treatment. At 8 weeks on IS treatment, splenocytes were assayed by flow cytometry for CD4, PD1, GL7, and CXCR5. (A and B) CD4⁺PD1⁺ T cell frequency, (C and D) CD4⁺GL7⁺ T cell frequency, and (E and F) CD4⁺CXCR5⁺ T cell frequency are shown. Naive, non-immunized WT mice; non-IS, AAV9-immunized WT mice without IS treatment; R + P, AAV9-immunized mice treated with R + P.