Cell-Mediated Immunity to NAGLU Transgene Following Intracerebral Gene Therapy in Children With Mucopolysaccharidosis Type IIIB Syndrome

Abstract

Mucopolysaccharidosis type IIIB syndrome (Sanfilippo disease) is a rare autosomic recessif disorder caused by mutations in the α-N-acetylglucosaminidase (NAGLU) gene coding for a lysosomal enzyme, leading to neurodegeneration and progressive deterioration of cognitive abilities in affected children. To supply the missing enzyme, several recent human gene therapy trials relied on the deposit of adeno-associated virus (AAV) vectors directly into the brain. We reported safety and efficacy of an intracerebral therapy in a phase 1/2 clinical trial (https://ichgcp.net/clinical-trials-registry/NCT03300453), with a recombinant AAV serotype 2/5 (rAAV2/5) coding human NAGLU in four children with MPS IIIB syndrome receiving immunosuppression. It was reported that AAV-mediated gene therapies might elicit a strong host immune response resulting in decreased transgene expression. To address this issue, we performed a comprehensive analysis of cellular immunity and cytokine patterns generated against the therapeutic enzyme in the four treated children over 5.5 years of follow-up. We report the emergence of memory and polyfunctional CD4+ and CD8+ T lymphocytes sensitized to the transgene soon after the start of therapy, and appearing in peripheral blood in waves throughout the follow-up. However, this response had no apparent impact on CNS transgene expression, which remained stable 66 months after surgery, possibly a consequence of the long-term immunosuppressive treatment. We also report that gene therapy did not trigger neuroinflammation, evaluated through the expression of cytokines and chemokines in patients' CSF. Milder disease progression in the youngest patient was found associated with low level and less differentiated circulating NAGLU-specific T cells, together with the lack of proinflammatory cytokines in the CSF. Findings in this study support a systematic and comprehensive immunomonitoring approach for understanding the impact immune reactions might have on treatment safety and efficacy of gene therapies.

Keywords: AAV; CNS; MPS IIIB; NAGLU; T cells; cellular immunity; cytokines.

Conflict of interest statement

MT has received consulting fees from UniQure. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Gougeon, Poirier-Beaudouin, Ausseil, Zérah, Artaud, Heard, Deiva and Tardieu.

Figures

Graphical Abstract

Figure 1

Time course distribution of lymphocyte subsets and expression of activation markers. (A) Lymphocyte subset enumeration was performed at indicated time points on whole blood by polychromatic flowcytometry. Cell subsets were characterized on gated CD45+ cells through the expression of CD3 (CD3+ T cells), coexpression of CD3 and CD4 (CD4+ T cells) or CD3 and CD8 (CD8+ T cells), CD3neg CD19+ (B cells) and CD3neg CD16+ (NK cells). Individual values of each patient (P1, P2, P3, P4) are shown. (B) The expression of the activation markers HLA-DR and CD38 on indicated T cell subsets was assessed on whole blood by polychromatic flowcytometry at indicated time points after gene therapy. Individual values of each patient (P1, P2, P3, P4) are shown.

Figure 2

Time course distribution and activation state of memory/effector CD4+ and CD8+ T cells. (A) Whole blood distribution of naïve/memory/effector subsets in CD4+ and CD8+ T cells. The percentage of cell subsets within CD4+ and CD8+ T cells are shown at indicated time points. (B) HLA-DR expression in indicated subsets within CD4+ and CD8+ T cells. (C) CD38 expression in indicated subsets within CD4+ and CD8+ T cells. N, naïve, CM, Central Memory; EM, Effector Memory; TD, Terminally Differentiated, identified through CD45RA/CCR7 expression. Mean value ± SD are shown. Statistical comparisons for each time point vs baseline are indicated. *0.01 <p< 0.05, **0.001<p< 0.01, ***0.0001<p<0.001.

Figure 3

Ex-vivo NAGLU-specific proliferative response.(A) Representative flow charts showing the proliferation of CD4+ and CD8+ T cells on gated CD3+ T cells in response to NAGLU (1 μg/ml), NAGLU peptide (1 μg/ml), SEB (1 μg/ml), or unstimulated (medium), assessed with the CSFE assay. Freshly isolated PBMCs were stained with CFSE and stimulated for 4 days with the indicated antigens. Percentage of proliferating CFSElow cells is indicated in the quadrants. (B) Time course evolution of NAGLU catalytic activity, assessed in concentrated CSF as described in “Patients and Methods”. (C) The ex-vivo detection of NAGLU-specific proliferating CD4+ and CD8+ T cells was assessed at baseline (BL) and 1, 3, 6, 12, 30, 48 and 66 months after surgery using the CFSE assay on freshly isolated PBMCs, stimulated for 4 days with the indicated concentrations of NAGLU or the peptide. The percentage of CFSElow proliferating cells within CD4+ and CD8+ T cells, obtained after removing the background in non-stimulated cultures, is indicated for each patient over the longitudinal follow-up. (D) Overlay of patients’ CD4+ and CD8+ T cell proliferating responses to NAGLU and peptide at 1 μg/ml.

Figure 4

Time course evolution of ex-vivo NAGLU-specific cytokine expressing CD4+ and CD8+ T cells. Whole blood was stimulated for 6 h at indicated concentrations of NAGLU or the peptide, and specific CD4+ and CD8+ T-cell responses were measured using an intracellular cytokine staining assay. For each patient, the percentage of NAGLU-specific CD69+ CD4+ or CD8+ T cells expressing TNF-α, IFN-γ or IL-2 is plotted at baseline (BL) and 1, 3, 6, 12, 30, 48 and 66 months after surgery.

Figure 5

Functional profiles of NAGLU-specific circulating CD4+ and CD8+ T cells. (A) Overlay of ex-vivo proliferation and TNF-α, IFN-γ or IL-2 expression by CD4+ and CD8+ T cells after exposure of patients’ PBMCs to NAGLU (0.5 μg/ml) or peptide (1 μg/ml) measured at baseline (BL) and 1, 3, 6, 12, 30, 48 and 66 months after surgery. Mean percentage of NAGLU-specific CFSElow cells or cytokine-expressing cells within CD4+ or CD8+ T cells is plotted for each time point. (B) CD4+ and CD8+ T-cell polyfunctionality analysis after whole blood stimulation for 6 h with NAGLU (0.5 μg/ml) or peptide (1 μg/ml). Bar charts represent the proportion of NAGLU-specific CD4+ or CD8+ T-cells displaying each particular cytokine or combination of cytokines at indicated time points of the follow-up. Data are presented as mean ± SD of specific responses from the four patients.

Figure 6

Baseline CSF and plasma cytokine/chemokine profiles. Boxplot diagrams of cytokine (A) and chemokine (B) concentrations in CSF and plasma from the four patients at baseline, assessed by multianalyte profiling of 27 cytokines and chemokines. Red star highlights cytokines or chemokines for which the values are markedly higher than those for healthy children (29). Data are presented as mean ± SD of cytokine/chemokine concentrations.

Figure 7

Impact of gene therapy on neuroinflammation. Heat map representing the broad array of cytokines (A) and chemokines (B) assessed by multianalyte profiling of 27 metabolites in patients’ CSF and plasma, at indicated time-points. The colored scale bar shows the range of concentrations expressed in pg/ml.