Targeting a Pre-existing Anti-transgene T Cell Response for Effective Gene Therapy of MPS-I in the Mouse Model of the Disease

Induction of Anti-IDUA Immune Response in MPS-I Mice

To mimic in the preclinical model of the disease the effects of ERT in MPS-IH patients, recombinant human IDUA (rhIDUA) was intravenously (i.v.) injected once a week (0.58 μg/g) in idua^−/− (MPS-I) mice (ERT, n = 22). After five injections, IDUA-specific IgGs were detectable in 12 of 22 treated mice (ranging from 0.079 to 2.358 μg/mL) (Figure 1A), while T cells isolated from the spleen of ERT-treated mice did not proliferate in response to rhIDUA in vitro (data not shown). ERT-treated and control naive MPS-I mice were lethally irradiated and transplanted with bone marrow (BM)-derived autologous HSCs transduced in vitro with LV encoding for human IDUA (LV.IDUA), as previously described.¹⁵ IDUA-transduced HSCs engrafted with the same efficiency in ERT-treated and naive MPS-I mice (data not shown), indicating that the pre-existing anti-IDUA response induced by ERT in MPS-I mice does not impair efficacy of ex vivo GT. However, in the sera of MPS-IH patients receiving ERT, we measured a 1,000-fold higher concentration of anti-IDUA IgGs (Figure 1B) compared to that observed in ERT-treated MPS-I mice (Figure 1A): on average 213 μg/mL in patients versus 0.26 μg/mL in mice. These data suggest that the ERT model we used may not be suitable to dissect the impact of pre-existing anti-IDUA immunity on ex vivo HSC GT for MPS-I.

To promote a stronger pre-existing anti-IDUA immunity in MPS-I mice, we developed an experimentally induced anti-IDUA immunity model. Specifically, MPS-I mice were immunized (IDUA-IMM) with rhIDUA in incomplete Freund’s adjuvant (IFA) and developed anti-IDUA IgGs (23.3 ± 18.95 μg/mL, mean ± SEM; n = 21; Figure 2A). Ex vivo analyses revealed the presence of B and plasma cells actively secreting anti-IDUA IgGs in the spleen and BM of IDUA-IMM mice (5.8 ± 6.4 and 2.5 ± 4 spot-forming units [SFU]/3.5 × 10⁵ cells, mean ± SEM; n = 6, respectively; Figure 2B). Moreover, significantly high numbers of anti-IDUA CD8⁺ T cells secreting interferon (IFN)γ were detected in the spleen of IDUA-IMM mice (14 ± 5.81 SFU/10⁵ cells, mean ± SEM) (Figure 2C), and splenocytes from IDUA-IMM mice secreted IFN-γ, granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-4, and IL-5 upon in vitro stimulation with rhIDUA protein (Figure 2D).

In conclusion, immunization of MPS-I mice with rhIDUA in IFA homogeneously induces a strong anti-IDUA humoral immunity, which is associated with IDUA-specific T cell responses.

Pre-existing Anti-IDUA Immunity Prevents Engraftment of IDUA-Transduced HSCs in Immunized MPS-I Mice in an Antigen-Specific Manner

To evaluate the impact of pre-existing anti-IDUA immunity on ex vivo HSC GT for MPS-I, we transplanted naive and IDUA-IMM MPS-I mice with LV.IDUA-transduced HSCs (IDUA-GT) or LV.OVA-transduced HSCs (OVA-GT), and we monitored their engraftment (Figure 3A). IDUA-IMM IDUA-GT mice died in 3 weeks with the same kinetic of mice irradiated and not transplanted (Irrad Ctrl, n = 8) as a consequence of engraftment failure, as confirmed by vector copy number (VCN) quantification in BM (Figures 3B and 3C). Conversely, LV.IDUA-transduced HSCs efficiently engrafted in naive MPS-I mice, as previously shown.¹⁵ Interestingly, LV.OVA-transduced cells engrafted both in IDUA-IMM and naive MPS-I mice (Figures 3B and 3C), indicating that the clearance of LV.IDUA-transduced HSCs in IDUA-IMM mice occurred in an antigen-specific fashion. IDUA-specific IgGs were detected at a high concentration in the sera of IDUA-IMM mice before and after IDUA-GT and OVA-GT (Figure 3D). Moreover, 12 weeks post-transplantation, B cells actively secreting anti-IDUA IgGs (Figure 3E) and IDUA-specific CD8⁺ T cells (Figure 3F) were still detectable in the spleen of IDUA-IMM OVA-GT mice.

To exclude that rejection of transduced cells occurred as a consequence of IFA-mediated activation of innate immunity, MPS-I mice immunized with IFA emulsified with PBS (IFA-PBS) were lethally irradiated and transplanted with LV.IDUA-transduced HSCs. Results showed that IDUA-transduced HSCs engrafted in IFA-PBS mice, but not in IDUA-IMM mice (Figure S1A). Moreover, anti-IDUA IgGs were absent in IFA-PBS, but not in IDUA-IMM, MPS-I mice 2 weeks after transplantation (Figure S1B), and no IDUA-specific CD8⁺ T cells were detected in the spleen of IFA-PBS mice 12 weeks after transplantation (data not shown).

These results demonstrate a detrimental effect of pre-existing anti-IDUA immunity on the engraftment of IDUA-transduced HSCs in MPS-I mice, and they show the persistence of anti-IDUA humoral and cellular responses in immunized MPS-I mice after irradiation and HSC transplantation.

Pre-existing Anti-IDUA IgGs Do Not Prevent Engraftment of IDUA-Transduced HSCs in Pre-immunized MPS-I Mice

To elucidate the role of anti-IDUA IgGs in the inhibition of IDUA-transduced HSC engraftment in IDUA-IMM mice, total IgGs isolated from the sera of MPS-I mice immunized with IDUA or OVA (IDUA-IMM or OVA-IMM) were transferred into naive MPS-I mice. IgG-treated and control naive MPS-I mice were then irradiated and transplanted with IDUA-transduced HSCs. All transplanted mice survived (Figure 4A). LV-transduced HSCs engrafted in IgG-treated MPS-I mice, and the VCN quantified in BM and peripheral blood cells was comparable among the groups, regardless of the antigen specificity of the injected IgGs (Figure 4B). Moreover, anti-IDUA and anti-OVA IgGs were detected in the sera of transplanted mice before and after GT (Figure 4C).

These findings demonstrate that the presence of pre-existing IDUA-specific IgGs neither impairs the engraftment of IDUA-transduced HSCs in BM nor mediates the selection of untransduced HSCs in the periphery.

Pre-existing IDUA-Specific CD8⁺ T Cells Impair Engraftment of IDUA-Transduced HSCs in Pre-immunized MPS-I Mice

To investigate the effects of anti-IDUA T cell responses on ex vivo HSC GT, LV.IDUA-transduced (CD45.1) and untransduced (CD45.2) HSCs were transplanted at a 1:1 ratio in IDUA-IMM or OVA-IMM mice (Figure S2A). The engraftment of untransduced HSCs allowed the survival of treated mice (Figure S2B) and the analysis of anti-IDUA T cell responses at endpoint in all groups. In IDUA-IMM, but not in OVA-IMM, mice, IDUA-transduced cells were selectively eliminated (Figure S2C). At 12 weeks post-transplantation in the spleen of IDUA-IMM mice, we identified a high frequency of anti-IDUA CD8⁺ T cells (127 ± 26 SFU/10⁵ cells, mean ± SEM; n = 7; Figure S2D), which was 10-fold higher than that detected in IDUA-IMM mice not transplanted or transplanted with OVA-transduced HSCs (Figures 2C and 3F, respectively). These data, together with the observation that, in IDUA-IMM mice, a significant percentage of CD8⁺ T cells persists in the peripheral blood after lethal irradiation (Figure S3), suggest that residual IDUA-specific CD8⁺ T cells may be re-activated and expanded after exposure to IDUA-overexpressing HSCs, thus potentially affecting the engraftment of ex vivo HSC GT in IDUA-IMM mice.

To confirm that IDUA-specific CD8⁺ T cells affect the engraftment of IDUA-transduced HSCs, we treated IDUA-IMM IDUA-GT mice with anti-CD8-depleting monoclonal antibody (mAb). Untreated IDUA-IMM and naive MPS-I mice transplanted with LV.IDUA-transduced HSCs served as positive and negative controls, respectively (Figure 5A). At the time of transplantation, CD8⁺ T cells were completely depleted from the peripheral blood of anti-CD8-treated IDUA-IMM mice (Figure 5B), and, to ensure continuous CD8⁺ T cell ablation, anti-CD8 mAb was weekly injected in mice after transplantation. Depletion of CD8⁺ T cells allowed the survival of IDUA-IMM IDUA-GT mice (Figure 5C) and the engraftment of IDUA-transduced HSCs (Figure 5D). Anti-IDUA IgGs, regardless of anti-CD8 treatment, were detected over time in the sera of IDUA-IMM IDUA-GT mice (Figure 5E), confirming that the presence of anti-IDUA IgGs does not impair the engraftment of IDUA-transduced HSCs.

These data show that pre-existing IDUA-specific CD8⁺ T cells prevent the engraftment of IDUA-transduced HSCs in pre-immunized MPS-I mice.

T Cell-Depleting Treatments Partially Rescue Engraftment of IDUA-Transduced HSCs in Pre-immunized MPS-I Mice

To eradicate pre-existing anti-IDUA CD8⁺ T cells, we tested two different T cell depleting agents: Fludarabine and anti-CD3 mAb. We first treated MPS-I mice with 600, 800, or 1,000 μg/g Fludarabine intraperitoneally to quantify the dose-dependent lympho-depletive effect. The highest tolerated dose, providing 50% depletion of circulating CD3 cells, was chosen for further investigations (Figure S4). IDUA-IMM mice were left untreated or treated with Fludarabine or anti-CD3 mAb and subsequently lethally irradiated and transplanted with IDUA-transduced HSCs. Naive MPS-I mice were irradiated and transplanted in parallel as positive controls of engraftment (Figure 6A). At the time of transplantation, a complete depletion of CD8⁺ T cells from the peripheral blood of IDUA-IMM mice was induced by anti-CD3 mAb, while treatment with Fludarabine partially depleted CD8⁺ T cells (Figure 6B). Accordingly, IDUA-transduced HSCs engrafted more efficiently in IDUA-IMM mice treated with anti-CD3 (6 of 7) than in Fludarabine-treated IDUA-IMM mice (2 of 8) (Figure 6C). At 12 weeks after transplantation, analysis of IDUA-specific CD8⁺ T cells in the spleen of treated mice demonstrated their persistence and expansion in those mice in which IDUA-transduced HSCs were completely eliminated (Figure 6D).

These observations confirm the critical role played by pre-existing anti-IDUA CD8⁺ T cells in preventing the engraftment of IDUA-transduced HSCs, and they demonstrate that, prior to ex vivo HSCT GT, a profound CD8⁺ T cell depletion is required to favor efficient engraftment of gene-corrected cells in IDUA-IMM mice.

Concomitant Activation of Innate Response Promotes IDUA-Specific T Cells in MPS-I Mice Receiving ERT

ERT treatment is poorly immunogenic in MPS-I mice (Figure 1A), however, it is known that simultaneous stimulation of the innate immune system by danger signals is needed to promote adaptive immunity against exogenous proteins. We therefore administered ERT to MPS-I mice either intramuscularly (i.m.) to generate a tissue damage or i.v. in combination with poly(I:C) to promote innate immunity activation through TLR3 stimulation (ERT-poly(I:C)) and simulate an undergoing viral infection. IDUA-specific IgGs and CD8⁺ T cells were induced in both i.m. and ERT-poly(I:C) mice, but not, as expected, in the ERT control group (Figures S5A and S5B). Since MPS-I patients under ERT may undergo viral infections, we performed ex vivo HSC GT in ERT-poly(I:C) mice. Results showed that engraftment of IDUA-transduced HSCs was impaired in 4 of 6 ERT-poly(I:C)-treated mice (Figure 7A), and a high number of anti-IDUA CD8⁺ T cells was detected in the spleen of those 4 ERT-poly(I:C)-treated mice in which IDUA-transduced HSCs were completely eliminated (Figure 7B). On the other hand, detectable levels of IDUA enzymatic activity were quantified in sera of the 2 mice from the ERT-poly(I:C) group in which transduced HSCs engrafted (Figure 7C).

These findings demonstrate that anti-IDUA T cell responses could be triggered in MPS-I mice under ERT treatment in the presence of a pro-inflammatory stimulus.

Mice

The idua^−/− mice (C57BL/6 background)⁵⁹ were imported in our animal facility as a kind gift of professor J.M. Heard and crossed with C57BL/6 mice from Charles River. Progeny was intercrossed to obtain an inbred strain. Mice were genotyped to identify idua^−/− mice (MPS-I), and idua^+/− and idua^+/+ siblings were used as BM donors. C57BL/6-Ly5.1 mice were purchased from Charles River. Experimental mice were randomized according to the gender.

Anti-IDUA IgG Concentration in Human Samples

Peripheral blood was obtained from MPS-IH patients and healthy subjects in accordance with local committee approval (TIGET05 and TIGET09) and with the Declaration of Helsinki. Plasma was collected after centrifugation of EDTA blood samples, and anti-IDUA IgG concentration was determined by ELISA. Briefly, 0.2 μg rhIDUA (Aldurazyme, Genzyme) was coated onto the wells of a 96-well microtiter plate (Thermo Fisher Scientific). After 12–16 h of incubation at 4°C, the plate was blocked for 2 h at room temperature (RT) with PBS-5% BSA (Roche), and diluted plasma samples were plated. After an additional 2 h at RT, biotinylated anti-human immunoglobulin (Thermo Fisher Scientific) was added to the plate for 2 h at RT, followed by 45-min incubation with Streptavidin horseradish peroxidase (HRP) conjugate. A substrate solution containing o-Phenylenediamine dihydrochloride (OPD) was prepared according to the manufacturer’s instructions (Sigma-Aldrich, P4664), and the colorimetric reaction was stopped after 6–10 min by the addition of sulfuric acid (stop solution, R&D Systems). Absorbance was read at 492-nm wavelength using SkaltRE for Multiskan go version 3.2 (Thermo Scientific). IgG concentration was determined by comparison to a serial dilution of a known-concentration solution of human IgGs (Sigma-Aldrich), run in parallel.

ERT and Immunization Protocols

rhIDUA (Aldurazyme, Genzyme) was diluted in PBS and weekly injected i.v. in 4-week-old MPS-I mice. Mice received 5 injections. Doses were adjusted according to body weight (0.58 μg/g).

PBS, rhIDUA, or OVA protein was emulsified with IFA (Sigma-Aldrich) and subcutaneously injected nearby the inguinal lymph nodes of 4-week-old MPS-I mice. 50 μL emulsion was administered to each side (total 29 μg protein/mouse).

To mimic viral infection, 4-week-old MPS-I mice received ERT as described above. Second and third doses were administered together with 50 μg/mouse poly(I:C) (tlrl-pic, InvivoGen).

Anti-IDUA or Anti-OVA IgG Quantification and Isolation

IDUA or OVA-specific IgGs were quantified in the sera of mice by ELISA. rhIDUA or OVA (Sigma-Aldrich) was used to coat microtiter plates (0.2 μg/well in 0.1 M carbonate buffer [pH 9.6]). After 12–16 h of incubation at 4°C, the plate was blocked for 2 h at RT with PBS-1% BSA (Roche). Diluted sera were added, and, after an additional 2 h at RT, anti-IDUA or anti-OVA antibodies were detected with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Sigma-Aldrich). Plates were reacted with H₂O₂ and OPD and read at 492-nm wavelength using SkaltRE for Multiskan go version 3.2 (Thermo Scientific). IgG concentration was determined by comparison to a serial dilution of a known-concentration solution of murine IgGs (554721, BD Biosciences), run in parallel.

Sera from IDUA-IMM and OVA-IMM MPS-I mice were collected and used for IgG purification. Pre-packed spin columns from GE Healthcare (Ab Spin Trap, 28-4083-47) were used according to the manufacturer’s instructions. Purified IgGs were quantified by ELISA as described above and i.v. injected into naive mice.

Cytokine Production

Spleens from experimental mice were collected and processed into a single cell suspension. 0.7 × 10⁶ splenocytes were plated in triplicate in 96-well plates in the presence of 10 μg/mL rhIDUA in complete RPMI 1640 (Lonza, Switzerland) with 10% fetal bovine serum (FBS) (Euroclone, ECS0180L), 100 U/ml penicillin/streptomycin (Lonza, 17-602E), 2 mM L-glutamine (Lonza, 17-605E), Minimum Essential Medium Non-Essential Amino Acids (MEM NEAA) (Gibco, 11140-035), 1 mM Sodium Pyruvate (Gibco, 11360-039), 50 nM 2-Mercaptoethanol (Gibco, 31350-010). After 96 h of culture, supernatants were collected and tested for cytokine production with Bio-Plex Pro Mouse Cytokine Th1/Th2 assay (Bio-Rad), according to the manufacturer’s instructions.

ELISPOT Assays

CD8⁺ T cells were magnetically isolated from the spleen of experimental mice (Miltenyi Biotec, 130-104-075). 10⁵ CD8⁺ T cells were plated in triplicate in ELISPOT plates (Millipore, Bedford, MA) pre-coated with anti-IFN-γ capture mAb (2.5 μg/mL; BD Pharmingen, R46A2) in the presence of IL-2 (50 U/mL; BD Pharmingen) and 10⁵ irradiated (6,000 rad) untransduced or LV.IDUA-transduced autologous EL-4 cells. After 42 h of incubation at 37°C and 5% CO₂, plates were washed and IFN-γ-producing cells were detected by biotin-conjugated anti-IFN-γ mAb (0.5 μg/mL; BD Pharmingen, XMG 1.2). Streptavidin-HRP conjugate (Roche) was added. Total splenocytes or total BM (0.35 × 10⁶ cells/well) was plated in complete RPMI in triplicate in ELISPOT plates pre-coated with rhIDUA (2 μg/well). After 24 h of incubation at 37°C and 5% CO₂, plates were washed and anti-IDUA IgG-secreting cells were detected with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Sigma A2554). All plates were reacted with H₂O₂ and 3-Amino-9-ethylcarbazole (Sigma, A6926). Spots were counted by ImmunoSpot reader (Cellular Technology).

Ex Vivo HSC GT

BM from 6-week-old idua^+/− and idua^+/+ mice was harvested by flushing femurs. HSCs were purified for lineage⁻ selection using the Lineage Cell Depletion Kit (Miltenyi Biotec, 130-090-858), according to the manufacturer’s instructions, and they were transduced at an MOI of 100 with IDUA- or OVA-encoding LV (LV.IDUA, LV.OVA). All LVs were produced by transient four-plasmid transfection of HEK293T cells, as previously reported.⁶⁰

MPS-I mice received total body irradiation (935 cGy split in two doses performed at least 2 h apart) and were i.v. injected with LV-transduced cells (10⁶ cells/mouse) on day 0. IDUA-IMM mice received irradiation and transplantation 4 weeks after immunization. At weekly intervals, mice were bled and the engraftment of donor cells was determined by flow cytometry. In some experiments, IDUA-IMM MPS-I mice were i.v. injected with depleting anti-CD8 Ab (clone YTS169.4, BioXcell, 250 μg/mouse) 1 day before transplantation of transduced cells. After transplantation, mice were weekly i.v. injected with anti-CD8 Ab (clone 53-6.7, BioXcell, 250 μg/mouse). In some experiments, IDUA-IMM MPS-1 mice were i.v. injected with anti-CD3 depleting Ab (clone 145-2C11, BioXcell, 100 μg/mouse) 4 days before transplantation of transduced cells. After transplantation, mice were weekly i.v. injected with anti-CD3 Ab (25 μg/mouse) for 7 weeks. In some experiments IDUA-IMM MPS-I mice were intraperitoneally injected with a total amount of 1,000 μg/g Fludarabine Phosphate (Accord); the total amount of drug was divided into 3 administrations performed on days −4, −3, and −2 before transplantation.

Engraftment Quantification

VCN was measured on BM and peripheral blood cells to quantify engraftment of transduced HSCs. Genomic DNA was isolated with QIAamp DNA Blood Mini Kit (QIAGEN, 51106), according to the manufacturer’s instructions. Vector integrations were quantified by QX200 Droplet Digital PCR System (Bio-Rad), according to the manufacturer’s instructions. Primers and probes were designed on the vector sequence and on control sequence used for normalization (mouse Sema3a gene).

Flow Cytometry

Peripheral blood was harvested by temporal vein puncture and mixed with NaCitrate (5:1 ratio). 30 μL blood was incubated with 25 μL of a calibrated suspension of fluorescent beads (CountBright Absolute Counting Beads, Thermo Fisher Scientific, C36950) and stained with the following Abs: purified anti-CD16/CD32 (2.4G2), fluorescein isothiocyanate (FITC)-conjugated anti-CD3 (17A2), BD Horizon Brilliant Violet 786 (BV786)-conjugated anti-CD4 (RM4-5), Pacific Blue-conjugated anti-CD8 (53-6.7), R-phycoerythrin (PE)-conjugated anti-CD11b (M1/70), allophycocyanin-conjugated anti-CD19 (1D3), and allophycocyanin-Cy7-conjugated anti-B220 (RA3-62B) (all from BD Biosciences) for 30 min at 4°C. Red blood cells lysis was performed with BD Phosflow (BD Biosciences, 558049), according to the manufacturer’s instructions. Labeled cells were washed two times with PBS 1% FBS and acquired with a BD LSRFortessa analyzer, and results were analyzed with FlowJo 10 software. T cell populations were identified after exclusion of the CD11b⁺ CD19⁺ and B220⁺ cells. The absolute number of cells per microliter was obtained as the ratio of beads events to cells events.

IDUA Enzymatic Activity

Enzyme activity was measured fluorometrically⁶¹ following the incubation of 10 μL serum for 1 h at 37°C with 10 μL substrate (2 mM 4-methylumbelliferyl α-L-iduronide, Glycosynth) in 0.1 M Na formate buffer (pH 3.2). Fluorescence of the 4-methylumbelliferone released was measured after the addition of 1 mL 0.5 M carbonate buffer (pH 10.7). Fluorescence was read at 365 nm (excitation) and 448 nm (emission) on a PerkinElmer fluorometer.

Statistics

All statistical analysis was performed using non-parametric Mann-Whitney U test (two-tailed); p values below 0.05 were considered significant.

Study Approval

All the mice were maintained in specific-pathogen-free (SPF) conditions, and the procedures involving animals were designed and performed with the approval of the Animal Care and Use Committee of the San Raffaele Hospital (IACUC #830) and communicated to the Ministry of Health and local authorities according to Italian law. Peripheral blood was obtained from MPS-IH patients and healthy subjects upon informed consent signature in accordance with the Ethical Committee of San Raffaele Scientific Institute (protocols TIGET05 and TIGET 09).