Targeting transmembrane-domain-less MOG expression to platelets prevents disease development in experimental autoimmune encephalomyelitis

Abstract

Multiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system with no cure yet. Here, we report genetic engineering of hematopoietic stem cells (HSCs) to express myelin oligodendrocyte glycoprotein (MOG), specifically in platelets, as a means of intervention to induce immune tolerance in experimental autoimmune encephalomyelitis (EAE), the mouse model of MS. The platelet-specific αIIb promoter was used to drive either a full-length or truncated MOG expression cassette. Platelet-MOG expression was introduced by lentivirus transduction of HSCs followed by transplantation. MOG protein was detected on the cell surface of platelets only in full-length MOG-transduced recipients, but MOG was detected in transmembrane-domain-less MOG_1-157-transduced platelets intracellularly. We found that targeting MOG expression to platelets could prevent EAE development and attenuate disease severity, including the loss of bladder control in transduced recipients. Elimination of the transmembrane domains of MOG significantly enhanced the clinical efficacy in preventing the onset and development of the disease and induced CD4⁺Foxp3⁺ Treg cells in the EAE model. Together, our data demonstrated that targeting transmembrane domain-deleted MOG expression to platelets is an effective strategy to induce immune tolerance in EAE, which could be a promising approach for the treatment of patients with MS autoimmune disease.

Keywords: MOG (myelin oligodendrocyte glycoprotein); experimental autoimmune encephalomyelitis; gene therapy; immune tolerance induction; platelet-targeted.

Figure 1

Generation and evaluation of platelet-specific MOG expression lentiviral vectors. (A) Schematic diagram of MOG_TD, MOG_1-157, and MOG_FL expression cassettes. Each MOG expression cassette was placed under the control of the platelet-specific αIIb promoter. (B) MOG expression in a promegakaryocyte cell line, Dami cells. Lentiviral vectors harboring the 2bMOG_TD, 2bMOG_1-157, or 2bMOG_FL expression cassette were produced by transient transfection of HEK293 cells. Dami cells were transduced with lentiviruses. After 72 hours of culture, cells were stained with anti-MOG antibody with or without permeabilization and analyzed by flow cytometry. Representative figures from flow cytometry analysis are shown.

Figure 2

Targeting MOG expression to platelets did not affect the leukocyte profile. Sca-1⁺ HSCs/progenitors isolated from CD45.2 WT C57BL/6J donors were transduced with lentiviruses and transplanted into CD45.1 recipients preconditioned with 660 cGy total body irradiation. After HSCT and BM reconstitution, blood samples were collected from recipients at various time points, and leukocytes were stained for CD45.1, CD45.2, CD4, CD8, and B220. After staining, cells were analyzed by flow cytometry. Representative results from week 7 from one trial after HSCT are shown. (A) Schematic diagram of experimental design to generate 2bMOG_TD, 2bMOG_1-157, 2bMOG_FL, and 2bGFP recipients. (B) Representative dot plots from flow cytometry analysis of chimerism. (C) Chimerism in transduced recipients at various time points. (D) The chimerism in transduced recipients. (E) The percentage of CD4 T cells in transduced recipients. (F) The percentage of CD8 T cells in transduced recipients. (G) The percentage of B220 cells in transduced recipients. There were no significant differences in chimerism, CD4, CD8, or B cells between 2bMOG-transduced groups and the 2bGFP control group. (D-G) Each data point represents one mouse. Three replicate experiments were performed.

Figure 3

Platelet-MOG expression in 2bMOG-transduced recipients. Blood samples were collected from 2bMOG-transduced recipients after at least 3 weeks of BM reconstitution. Platelets were isolated, stained for CD41 and MOG with or without cell permeabilization, and analyzed by flow cytometry. 2bGFP was used as a control. (A) Representative dot plots from flow cytometry analysis by surface staining of MOG expression at 3 weeks after HSCT. (B) The percentages of MOG positive platelets in recipients by surface staining are shown. For individual mice analyzed more than once over the study, the average platelet MOG expression was calculated. (C) Representative dot plots from flow cytometry analysis by intracellular staining of MOG expression at 3 weeks after HSCT is shown. (D) The percentages of MOG positive platelets in indicated recipients by intracellular staining is shown. For individual mice analyzed more than once over the study, the average platelet MOG expression was calculated. (E) Representative mean fluorescent intensity (MFI) of intracellular MOG expression in transduced recipients from one trial at 3 weeks after HSCT is shown. MOG MFI was analyzed by flow cytometry analysis through intracellular staining. *P < 0.05; **P < 0.01; ****P < 0.0001. “n.s.” indicates no statistically significant difference between the two groups. (B, D, E) Each data point represents one mouse. Data were summarized from four trials. MFI, mean fluorescence intensity.

Figure 4

Platelet-specific MOG_FL, not MOG_TD, expression ameliorated EAE disease severity. After lentivirus transduction of HSCs followed by HSCT and at least 3 months of BM reconstitution, mice were challenged with MOG_35-55 peptide emulsified in CFA along with intraperitoneal injection of pertussis toxin on days 0 and 2. Animals were monitored, and clinical scores were recorded during the study period of day 5-31 after EAE induction. (A) Clinical scores in 2bMOG_FL-, 2bMOG_TD-, and 2bGFP-transduced recipients through the study period after EAE induction. N = 10 in each group. (B) The percentage of paralysis-free transduced recipients after EAE induction during the study period of 5-31 days. Mice with a clinical score ≥ 2.5 were defined as having paralysis. N = 10 in each group. (C) Clinical scores in transduced recipients at day 17 after EAE induction. (D) Cumulative disease scores in transduced recipients during the study period. (E) Surface expression of MOG in transduced recipients. (F) Intracellular expression of MOG in transduced recipients. (G) PCR analysis of MOG proviral DNA in leukocytes from transduced recipients. DNA was purified from peripheral blood leukocytes at 3 weeks after HSCT and the MOG expression cassette was amplified by PCR using primers designed for each construct. WT mouse FVIII was used as an internal control for DNA integrity. (H) Quantitative real-time PCR analysis was used to determine the average copy number of LTR in 2bMOG_TD, 2bMOG_FL, 2bGFP-transduced recipients. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. “n.s.” indicates no statistically significant difference between the two groups. (C–F, H) Each data point represents one mouse. Data were summarized from two trials.

Figure 5

Eliminating MOG transmembrane domains (MOG_1-157) enhanced clinical efficacy in immune tolerance induction in EAE. After lentivirus transduction of HSCs followed by HSCT and at least 3 months of BM reconstitution, EAE was induced. Clinical scores and body weights were monitored daily during the study period of 5-31 days after EAE induction. Loss of bladder control (urinary incontinence) during days 5-20 was assessed by visual observation of wetness on the animal’s fur on the caudal abdomen. (A) The average daily EAE score of 2bMOG_1-157-, 2bMOG_FL-, 2bGFP-transduced recipients over time are shown. (B) Body weights of 2bMOG_1-157-, 2bMOG_FL-, 2bGFP-transduced recipients over time are shown. (C) The percentages of transduced recipients that were paralysis free after EAE induction are shown. (D) The cumulative scores of 2bMOG_1-157, 2bMOG_FL, and 2bGFP recipients up to 31 days after EAE induction are shown. (E) The EAE score of 2bMOG_1-157-, 2bMOG_FL-, and 2bGFP-transduced recipients at day 17 after EAE induction are shown. (F) Body weights of the 2bMOG_1-157, 2bMOG_FL, and 2bGFP recipients at day 17 after EAE induction are shown. (G) Days of bladder control loss in transduced recipients after EAE induction during the study period are shown. (H) Surface expression of MOG in transduced recipients is shown. (I) Intracellular expression of MOG in transduced recipients is shown. *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. “n.s.” indicates no statistically significant difference between the two groups. (D-H, I) Each data point represents one mouse. Data were summarized from three trials.

Figure 6

Platelet-targeted MOG_1-157 expression lead to Treg accumulations and suppressed CD8 T cell recall responses to MOG_35-55 stimulation. Leukocytes from peripheral blood at 11 weeks after transplantation of transduced HSCs before EAE induction and 20 days after EAE induction were stained for CD4, CD25, and Foxp3 and analyzed by flow cytometry. One to two months after EAE induction, splenocytes from transduced recipients were isolated, labeled with Violet CellTracer, and cultured with various doses (0, 2, 10, 50, and 100 μg/ml) of MOG_35-55 peptide for 3 days. Cells were harvested and stained for CD4 and CD8. Zombie Red™ staining was used to exclude dead cells. After staining, cells were analyzed by flow cytometry. (A) Representative dot plots of flow cytometry analysis of Treg cells in transduced recipients after EAE induction are shown. (B) The percentages of Foxp3⁺ Treg cells in transduced recipients after EAE induction are shown. (C) The percentages of CD25⁺Foxp3⁺ Treg cells in transduced recipients after EAE induction are shown. (D) The percentages of Foxp3⁺ Treg cells in transduced recipients before EAE induction are shown. (E) The percentages of CD25⁺Foxp3⁺ Treg cells in transduced recipients before EAE induction are shown. (F) The workflow of the T cell proliferation assay is shown. (G) The stimulation index of CD4 T cell proliferation in each group cultured with various concentrations of MOG_35-55 is shown. (H) The stimulation index of CD8 T cell proliferation in indicated groups with various concentrations of MOG_35-55 is shown. The stimulation index (SI) was calculated as follows: SI = (the percentage of proliferating daughter cells in MOG_35-55-treated wells)/(the percentage of proliferating daughter cells in control wells with 0 μg/ml of MOG_35-55). Two-way ANOVA was used to compare T cell stimulation indexes among groups. *P < 0.05; **P < 0.01. (B–E, G, H) Each data point represents one mouse. Data were summarized from two trials.