Novel Coagulation Factor VIII Gene Therapy in a Mouse Model of Hemophilia A by Lipid-Coated Fe3O4 Nanoparticles

Abstract

Hemophilia A is a bleeding disease caused by loss of coagulation factor VIII (FVIII) function. Although prophylactic FVIII infusion prevents abnormal bleeding, disability and joint damage in hemophilia patients are common. The cost of treatment is among the highest for a single disease, and the adverse effects of repeated infusion are still an issue that has not been addressed. In this study, we established a nonviral gene therapy strategy to treat FVIII knockout (FVIII KO) mice. A novel gene therapy approach was developed using dipalmitoylphosphatidylcholine formulated with iron oxide (DPPC-Fe₃O₄) to carry the B-domain-deleted (BDD)-FVIII plasmid, which was delivered into the FVIII KO mice via tail vein injection. Here, a liver-specific albumin promoter-driven BDD-FVIII plasmid was constructed, and the binding ability of circular DNA was confirmed to be more stable than that of linear DNA when combined with DPPC-Fe₃O₄ nanoparticles. The FVIII KO mice that received the DPPC-Fe₃O₄ plasmid complex were assessed by staining the ferric ion of DPPC-Fe₃O₄ nanoparticles with Prussian blue in liver tissue. The bleeding of the FVIII KO mice was improved in a few weeks, as shown by assessing the activated partial thromboplastin time (aPTT). Furthermore, no liver toxicity, thromboses, deaths, or persistent changes after nonviral gene therapy were found, as shown by serum liver indices and histopathology. The results suggest that this novel gene therapy can successfully improve hemostasis disorder in FVIII KO mice and might be a promising approach to treating hemophilia A patients in clinical settings.

Keywords: DPPC-Fe3O4; coagulation FVIII; gene therapy; hemophilia A mouse; nanoparticle.

Figure 1

Liver-specific promoter construction and promoter activity measurement. (A) Four different liver-specific promoters, Pα1-AT, Eα1-AT, EPα1-AT, and PmAlb, were generated and inserted into the pGL3-enhancer vector using the luciferase gene as a reporter for the promoter assay. (B) Detection of the firefly/Renilla ratio of each promoter in seven different cell lines (Hepa1-6, MEF, C2C12, CHO, A549, Caco-2, and Ca922) by dual-luciferase assays. Mock group: transfected cells as a background control. (C) PmAlb-BDD-FVIII-pCMV-EGFP plasmid map. Data are presented as the mean ± SD (n = 3), * p < 0.05 vs. the mock group, ** p < 0.01 vs. the mock group, *** p < 0.001 vs. the mock group.

Figure 2

Characteristics of DPPC-Fe₃O₄ nanoparticle binding with circular or linear plasmid DNA. (A) Transmission electron microscopy (TEM) examination of the nanoparticle morphology of DPPC-Fe₃O₄ binding with circular plasmid DNA (right; scale bar = 20 nm) and linear plasmid DNA (left; scale bar = 200 nm). (B) Dynamic light scattering (DLS) analysis of the nanoparticle sizes of DPPC-Fe₃O₄ alone, DPPC-Fe₃O₄ binding with circular plasmid, and DPPC-Fe₃O₄ binding with linear plasmid DNA. (C) DLS analysis of the polydispersity index (PDI) value of DPPC-Fe₃O₄ alone, DPPC-Fe₃O₄ binding with circular plasmid, and DPPC-Fe₃O₄ binding with linear plasmid DNA. (D) Electrophoretic gel mobility shift assay (EMSA) of different emulsification times and concentrations of DPPC-Fe₃O₄ (1273.6 ng, 636.8 ng, 318.4 ng, and 212.3 ng) binding with 1000 ng circular or linear plasmids. The DPPC-Fe₃O₄/DNA ratios (w/w) were defined as 1.2:1.0, 0.6:1.0, 0.3:1.0, and 0.2:1.0, respectively. Semiquantitative analysis of the binding ability (×100%) of DPPC-Fe₃O₄ with circular plasmid DNA (E) or linear plasmid DNA (F).

Figure 3

DLS analysis of nanoparticle characteristics in different dosages of DPPC-Fe₃O₄ binding with a constant amount of circular form plasmid DNA. (A) Average zeta potential (mV), (B) average size (nm), (C) polydispersity index (PDI) value, (D) profile of zeta potential distribution, and (E) profile of size distribution in different groups of circular plasmid DNA alone, DPPC-Fe₃O₄ alone, DPPC-Fe₃O₄-plasmid incomplete binding (200 ng DPPC-Fe₃O₄: 1000 ng circular DNA = 0.2:1.0; w/w), Fe₃O₄-plasmid complete binding (1000 ng DPPC-Fe₃O₄: 1000 ng circular DNA = 1.0:1.0; w/w), and Fe₃O₄-plasmid overdose binding (4000 ng DPPC-Fe₃O₄: 1000 ng circular DNA = 4.0:1.0; w/w). Data are presented as the mean ± SD (n = 3).

Figure 4

Assessment of PmAlb-BDD-FVIII-pCMV-EGFP plasmid DNA expression in normal mouse liver cells. (A) Representation of quantitative RT-PCR results of EGFP and hFVIII gene expression in FL83B cells after transfection with PmAlb-BDD-FVIII-pCMV-EGFP plasmid DNA for two days. Nontransfected FL83B cells were used as a negative control. A β-actin gene was used as an internal mRNA loading control. (B) Quantification of EGFP gene expression in FL83B cells transfected with (transfected group) or without (control group) PmAlb-BDD-FVIII-pCMV-EGFP plasmid DNA. (C) Quantification of human FVIII gene expression in FL83B cells transfected with (transfected group) or without (control group) PmAlb-BDD-FVIII-pCMV-EGFP plasmid DNA. (D) Flow cytometry analysis of EGFP and FVIII expression in FL83B cells after transfection of the PmAlb-BDD-FVIII plasmid DNA. Transfected cells were stained with anti-hFVIII antibody conjugated with Alexa Fluor^® 546 dye, and hFVIII and EGFP dual fluorescence-positive cells were analyzed by flow cytometry (right). Nontransfected FL83B cells were used as a negative control (left). (E) The percentage of both EGFP- and FVIII-positive cells in a total of 100,000 cells was quantified. Data are presented as the mean ± SD, *** p < 0.001 vs. the control group (two-tailed t test).

Figure 5

Assessment of coagulation restoration after DPPC-Fe₃O₄-PmAlb-BDD-FVIII gene therapy in mice with hemophilia A. (A) The aPTT test was performed at 24 h (n = 3), 72 h (n = 3), and 120 h (n = 4) after intravenous injection with the DPPC-Fe₃O₄-plasmid complex to evaluate the short-term therapeutic effect. Untreated FVIII knockout mice (n = 6) were used as a control for the disease group, and age-paired male C57BL/6J mice (n = 6) were used as a normal control group. (B) aPTT was tested weekly after intravenous injection with the DPPC-Fe₃O₄-plasmid complex to evaluate the long-term therapeutic effect. Data are presented as the mean ± SD, *** p < 0.001 vs. the untreated FVIII knockout mouse group (two-tailed t test).

Figure 6

Perls’ Prussian blue staining to observe the localization of DPPC-Fe₃O₄-plasmid nanoparticles in the liver sections of recipient mice with hemophilia A. (A) Mice were sacrificed at 24 h (n = 3) after delivery of the DPPC-Fe₃O₄-plasmid complex, and Perls’ Prussian blue-stained iron oxide was observed in liver sections (right). C57BL/6J mice (n = 6) were used as a normal control (left). Untreated FVIII knockout mice (n = 6) were used as a negative control (middle). Images were obtained at 400 x magnification, scale bar = 50 µm. (B) Representative images of the Prussian blue-stained iron clusters at different time points. The recipient mice were sacrificed at 24 h (n = 3), 72 h (n = 3), and 120 h (n = 3) after delivery of the DPPC-Fe₃O₄-plasmid complex, and the liver sections were stained with Perls’ Prussian blue. Images were obtained at 400× magnification, scale bar = 50 µm. (C) Quantification of iron cluster numbers at different time points. (D) Representative images of the iron clusters in the cell nucleus (black arrows) or cytoplasm (white arrows). Images were obtained at 1000× magnification, scale bar = 20 µm. (E) Quantification of the percentage of iron clusters in the cell nucleus (black bar) or cytoplasm (white bar) at different time points by counting 100 clusters randomly. Data are presented as the mean ± SD, * p < 0.05, *** p < 0.001 (two-tailed t test).

Figure 7

Verification of the side effects after delivery of the DPPC-Fe₃O₄-plasmid complex to the liver tissue of mice with hemophilia A. (A) The recipient mice were sacrificed at 24 h (n = 3), 72 h (n = 3), and 120 h (n = 3) after delivery of the DPPC-Fe₃O₄-plasmid complex, and the histopathological changes in H&E-stained liver sections were analyzed. C57BL/6 mice (n = 6) were used as a normal control, and FVIII knockout mice (n = 6) without DPPC-Fe₃O₄-plasmid complex injection were used as an untreated control. Images were obtained at 40× magnification, scale bar = 50 µm. Serum biochemical parameters for liver indices, including ALT (B), AST (C), and ALKP (D), were measured in the normal C57BL/6J control mice, FVIII knockout mice without treatment, and FVIII knockout mice treated with the DPPC-Fe₃O₄-plasmid complex at different time points (24 h, 72 h, and 120 h after gene therapy). The dashed lines show the normal range of each parameter in normal mice. Data are presented as the mean ± SD.