Modifying immune responses to adeno-associated virus vectors by capsid engineering

Abstract

De novo immune responses are considered major challenges in gene therapy. With the aim to lower innate immune responses directly in cells targeted by adeno-associated virus (AAV) vectors, we equipped the vector capsid with a peptide known to interfere with Toll-like receptor signaling. Specifically, we genetically inserted in each of the 60 AAV2 capsid subunits the myeloid differentiation primary response 88 (MyD88)-derived peptide RDVLPGT, known to block MyD88 dimerization. Inserting the peptide neither interfered with capsid assembly nor with vector production yield. The novel capsid variant, AAV2.MB453, showed superior transduction efficiency compared to AAV2 in human monocyte-derived dendritic cells and in primary human hepatocyte cultures. In line with our hypothesis, AAV2.MB453 and AAV2 differed regarding innate immune response activation in primary human cells, particularly for type I interferons. Furthermore, mice treated with AAV2.MB453 showed significantly reduced CD8⁺ T cell responses against the transgene product for different administration routes and against the capsid following intramuscular administration. Moreover, humoral responses against the capsid were mitigated as indicated by delayed IgG2a antibody formation and an increased NAb50. To conclude, insertion of the MyD88-derived peptide into the AAV2 capsid improved early steps of host-vector interaction and reduced innate and adaptive immune responses.

Keywords: AAV vectors; MyD88; Toll-like receptor; adaptive immune responses; capsid engineering; cytokine profile; enhanced transgene expression; gene therapy; innate immune responses.

Graphical abstract

Figure 1

Modeling of the MyD88 peptide-presenting AAV2.MB453 capsid The MyD88-derived peptide was inserted at I-453 flanked by five alanine residues (A). Structure prediction of the AAARDVLPGTAA peptide in the common VP3 region at I-453 (B). Three VP3 capsid subunits are depicted (white, dark gray, and light gray). The highest capsid peak formed by the β-turn of the GH loop with capsid position I-453 is illustrated in green and the inserted peptide is shown in orange. Close-up highlights the helical structures formed by the inserted MyD88 peptide (B). The interaction of the MyD88 peptide inserted at I-453 to a TIR domain of MyD88 (light brown) is shown in (C). Close-up illustrates binding of the inserted peptide (orange) to residues (light brown) of a MyD88 TIR domain (C).

Figure 2

Transduction of moDCs and impact on expression of immune response-related genes Human moDCs were incubated with indicated vectors at a GOI of 2.5 × 10⁴. To determine the level of vector transgene expression (A), cells were analyzed via flow cytometry 48 h p.t. Mean with individual donors is depicted in (A) for n = 5 donors with technical triplicates. Transduction levels were normalized (AAV2 set as 1) and transduction is shown as fold change (B). Data are presented as mean with individual donors for n = 5 donors. Mann-Whitney U test was applied to calculate statistical significance. ∗p < 0.05. For multigene analysis, cells were harvested 8 h p.t., RNA isolated and transcribed into cDNA and probed by Profiler PCR Array detecting the expression level of 84 genes involved in innate and adaptive immune responses. In (C), the expression levels of key innate immune mediators compared to mock control are depicted as fold change for treatment with AAV2, AAV2.MB453, and AAV2.VSSTSPR. Figure S3 displays all genes analyzed and samples that are expressed below the threshold (Ct value 40). Data presented are from n = 1 moDC donor (donor a). The gene expression pattern in AAV2.MB453-treated cells was compared with the AAV2 sample and presented as fold change (D). Relative upregulation (in AAV2.MB453 sample compared with AAV2 sample) is highlighted in red and relative downregulation (in AAV2.MB453 sample compared with AAV2 sample) is illustrated in blue. Only genes that are upregulated or downregulated more than 2-fold are shown (D).

Figure 3

Vector genomes and transgene expression in liver after intravenous AAV injection Male BALB/c mice were injected intravenously with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes. At 24 h p.i., 5 d p.i., or 10 d p.i. mice were sacrificed. For each time point, two independent experiments were performed with in total seven mice per group. Liver tissue was collected, and single cells isolated. DNA was isolated from single cells and analyzed with qPCR using eGFP-specific primers. The amount of vector genomes was normalized to AAV2 per experiment to consider variations between experiments for 24 h p.i. (A), 5 d p.i. (B), and 10 d p.i. (C). In addition, RNA was isolated from single cells and transcribed into cDNA analyzed with qPCR using eGFP-specific primers. The transgene expression was normalized to AAV2 per experiment to balance variations between experiments for 24 h p.i. (D), 5 d p.i. (E), and 10 d p.i. (F). The transgene expression efficiency is presented as ratio of transgene expression (cDNA) to vector genomes for 24 h p.i. (G), 5 d p.i. (H), and 10 d p.i. (I) and normalized to AAV2. Data are presented as mean ± SEM for n = 5 to 7 mice. Mann-Whitney U test was applied to calculate statistical significance. ∗p < 0.05.

Figure 4

AAV2-binding IgG2a antibodies in serum after intravenous injection Male BALB/c mice were injected intravenously with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes (same mice as in Figure 3). At 24 h p.i. (A), 5 d p.i. (B), or 10 d p.i. (C) mice were sacrificed, blood taken, and serum collected. Serum was analyzed regarding AAV2-binding antibodies of the IgG2a subtype by ELISA. Purified IgG2a was used to calculate concentrations. As mentioned above, for each time point, two independent experiments were performed with in total seven mice per group. In addition, female BALB/c mice were injected intravenously with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes. At 8 d p.i. (D, E) or 20 d p.i. (D, F), blood was taken and serum was collected. Serum was analyzed with ELISA as described above. Data are presented as mean ± SEM for n = 6–7 mice. Mann-Whitney U test was applied to calculate statistical significance. ∗p < 0.05; ∗∗p < 0.01.

Figure 5

Neutralization assay Serum analyzed for IgG2a that was collected on day 20 (Figure 4F) was probed for neutralization assays. In (A), different serum dilutions (1:150, 1:300, 1:600, 1:1,200, 1:2,400), and in (B) equal amounts of IgG2a antibodies (as calculated before, Figure 4F) were incubated with AAV2 at GOI of 1 × 10³ for 1 h before infection of HEK293 cells. As control, cells were incubated with the same GOI of AAV2 in the absence of serum (“no serum” set as 100). Transgene-expressing cells were analyzed via flow cytometry. Data are presented as mean ± SEM for n = 6–7 mice. Green line indicates NAb50 value.

Figure 6

CD8⁺ T cell responses to transgene product and AAV2 capsid after intravenous AAV injection Female BALB/c mice were injected intravenously with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes. At 10 d p.i., mice were sacrificed, spleens harvested, and splenocytes isolated. Splenocytes were stimulated with immunodominant epitopes for eGFP (HYLSTQSAL) and AAV2 capsid (QYGSVSTNL + PQYGYLTL), respectively, and antigen-specific, IFN-y-secreting T cells were counted. T cells reactive to eGFP (A) and to AAV2 capsid (B) are shown per 1 × 10⁶ splenocytes. N = 7 mice per group. In an additional experiment, female BALB/c mice were injected intravenously with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes (same mice as in Figures 4D–4F). These mice were sacrificed at 58 d p.i., spleens harvested, and splenocytes isolated. Splenocytes were stimulated with peptides PQYGYLTL and QYGSVSTNL (capsid) or eGFP peptide HYLSTQSAL, respectively, and antigen-specific, IFN-y-secreting T cells were counted. T cells reactive to eGFP (C) and to AAV2 capsid (D) are shown per 1 × 10⁶ splenocytes. Male BALB/c mice were injected intravenously with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes (same mice as in Figure 4C). At 10 d p.i., mice were sacrificed, spleens harvested, and splenocytes isolated. Splenocytes were stimulated with immunodominant epitopes for eGFP (HYLSTQSAL) and AAV2 capsid (PQYGYLTL), respectively, and antigen-specific, IFN-y-secreting T cells were counted. T cells reactive to eGFP (E) and to AAV2 capsid (F) are shown per 1 × 10⁶ splenocytes. As mentioned above, two independent experiments were performed with in total seven mice per group (E and F). Data are presented as mean ± SEM for n = 7 mice. Mann-Whitney U test was applied to calculate statistical significance. ∗p < 0.05; ∗∗p < 0.01.

Figure 7

AAV2 capsid-binding IgG2a antibodies in serum after intramuscular injection and neutralization assay Female BALB/c mice were injected intramuscularly with 1 × 10¹⁰ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes. At 7 d p.i. (A, B) and 23 d p.i. (A, C) blood was taken and serum was collected. Serum was analyzed regarding AAV2 capsid-binding antibodies of the IgG2a subtype by ELISA. Purified IgG2a was used to calculate concentrations. Data are presented as mean ± SEM for n = 7 mice. Female BALB/c mice were injected intravenously with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes. At 7 d p.i. (D, E) and 23 d p.i. (D, F) blood was taken and serum was collected. Serum was analyzed with ELISA as described above. Data are presented as mean ± SEM for n = 4 mice. Mann-Whitney U test was applied to calculate statistical significance. ∗p < 0.05. Serum analyzed for IgG2a that was collected on day 23 (C) was probed in a neutralization assay (G). Different serum dilutions (1:150, 1:300, 1:600, 1:1,200, 1:2,400) were incubated with AAV2 at GOI of 1 × 10³ for 1 h before infection of HEK293 cells. As control, cells were incubated with the same GOI of AAV2 in the absence of serum (“no serum” set as 100). Transgene-expressing cells were analyzed via flow cytometry (G). Data are presented as mean ± SEM for n = 6–7 mice. Green line indicates NAb50 value.

Figure 8

CD8⁺ T cell responses toward the transgene product and the capsid after intramuscular AAV injection Female BALB/c mice were injected intramuscularly with 1 × 10¹⁰ vg/mouse or 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes. At 14 d p.i. (A, B) blood was taken and blood cells stimulated with a peptide for the immunodominant epitope of eGFP (HYLSTQSAL) and antigen-specific, IFN-y-secreting T cells were counted for low vector dose (A) and high vector dose (B). Male BALB/c mice were injected intramuscularly with 1 × 10¹¹ vg/mouse of either AAV2 or AAV2.MB453 encoding eGFP as transgenes. At 7 d p.i. (C, E), blood was taken and blood cells stimulated with a peptide for the immunodominant epitope of eGFP (HYLSTQSAL) or with peptides for AAV2 capsid epitopes (PQYGYLTL + QYGSVSTNL) antigen-specific, IFN-y-secreting T cells were counted for eGFP (C) and capsid (E). At 14 d p.i. mice were sacrificed, spleens harvested, and splenocytes isolated. Splenocytes were stimulated with peptides for eGFP (HYLSTQSAL; D) or AAV2 capsid (PQYGYLTL + QYGSVSTNL) (F) and antigen-specific, IFN-y-secreting T cells were counted. Data are presented as mean ± SEM for n = 4–7 mice. Mann-Whitney U test was applied to calculate statistical significance. ∗p < 0.05.