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. 2023 Jun 12;29(1):74.
doi: 10.1186/s10020-023-00680-z.

Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A

Jie Gong  1   2 , Rui Yang  2 , Min Zhou  1 , Lung-Ji Chang  3   4
Affiliations

Improved intravenous lentiviral gene therapy based on endothelial-specific promoter-driven factor VIII expression for hemophilia A

Jie Gong et al. Mol Med. .

Abstract

Background: Hemophilia A (HA) is an X-linked monogenic disorder caused by deficiency of the factor VIII (FVIII) gene in the intrinsic coagulation cascade. The current protein replacement therapy (PRT) of HA has many limitations including short term effectiveness, high cost, and life-time treatment requirement. Gene therapy has become a promising treatment for HA. Orthotopic functional FVIII biosynthesis is critical to its coagulation activities.

Methods: To investigate targeted FVIII expression, we developed a series of advanced lentiviral vectors (LVs) carrying either a universal promoter (EF1α) or a variety of tissue-specific promoters, including endothelial-specific (VEC), endothelial and epithelial-specific (KDR), and megakaryocyte-specific (Gp and ITGA) promoters.

Results: To examine tissue specificity, the expression of a B-domain deleted human F8 (F8BDD) gene was tested in human endothelial and megakaryocytic cell lines. Functional assays demonstrated FVIII activities of LV-VEC-F8BDD and LV-ITGA-F8BDD in the therapeutic range in transduced endothelial and megakaryocytic cells, respectively. In F8 knockout mice (F8 KO mice, F8null mice), intravenous (iv) injection of LVs illustrated different degrees of phenotypic correction as well as anti-FVIII immune response for the different vectors. The iv delivery of LV-VEC-F8BDD and LV-Gp-F8BDD achieved 80% and 15% therapeutic FVIII activities over 180 days, respectively. Different from the other LV constructs, the LV-VEC-F8BDD displayed a low FVIII inhibitory response in the treated F8null mice.

Conclusions: The LV-VEC-F8BDD exhibited high LV packaging and delivery efficiencies, with endothelial specificity and low immunogenicity in the F8null mice, thus has a great potential for clinical applications.

Keywords: Endothelial-specific; Factor VIII; Gene therapy; Hemophilia A; Lentiviral vector.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
The NHP/TYF F8BDD LV system with an universal promoter and tissue-specific promoters for ECs and megakaryocytes. A Schematic illustration of self-inactivating LVs encoding the partially sequence-optimized human F8BDD gene. B Schematic illustration of the LV-F8BDD under the control of different promoters as depicted
Fig. 2
Fig. 2
Lentiviral expression in endothelial, megakaryocyte, myeloid and lymphoid cells. A, B The titration of LV-mWasabi (n = 4) (A) and LV-F8BDD (n = 3 to 7) (B) constructs containing the five promoters (EF1α, VEC, KDR, Gp and ITGA) illustrating the packaging efficiencies. C GFP expression in transduced ECs, megakaryocytes, myeloid and lymphoid cells detected under a fluorescent microscope. The left panels represent green fluorescent signals and the right panels were under bright field, at 20 × magnification. D Quantitative analysis based on mean fluorescence intensity (MFI) by flow cytometry showing GFP expression in ECs, megakaryocytes, myeloid and lymphoid cells (n = 3). The differences in characteristics between groups were analyzed using the one way Welch ANOVA tests with Games-Howell post hoc tests (A, D), and Kruskal–Wallis tests (B); *p < 0.05, **p < 0.01, ***p < 0.001, n.s., no significant difference
Fig. 3
Fig. 3
In vitro analyses of LV-F8BDD expression under different promoters in ECs and megakaryocytes. A Illustration of transduction efficiencies (VCN/cell) of the different LV promoter constructs (n = 3). B, C mRNA levels as percentages (%) of F8/GAPDH mRNA in ECs (B) and megakaryocytes (C) determined by gel electrophoresis (left) and RT-qPCR (right) (n = 3). D Protein concentrates detected using a human FVIII ELISA kit in ECs and megakaryocytes (n = 3). E FVIII activities determined by FVIII: C chromogenic assay in ECs and megakaryocytes (n = 3). The differences in characteristics between groups were analyzed using the one way Welch ANOVA tests with Games-Howell post hoc tests (A–C, E) and Kruskal–Wallis tests (D); *p < 0.05, **p < 0.01, ****p < 0.0001, n.s., no significant difference
Fig. 4
Fig. 4
Enhanced iv LV-mWasabi gene transfer in mice after non-myeloablative immune suppression. A Illustration of tail vein injection of LV-mWasabi into WT mice and LV-F8BDD into F8null mice pretreated with non-myeloablative radiation (6 Gy). The WT mice received tail vein iv injection of LV-mWasabi under the different promoters, EF1α, VEC, Gp and ITGA, at 1 × 107 TU per mouse or 100 μL PBS per mouse as mock control. The blood was collected on Day 7, 15, 30, 45, 60, 120 and 180 after injection. B–D LV-GFP expression analysis by flow cytometry in BM, liver and spleen on day 30. The BM cells (B) were analyzed using antibodies for the different lineage-specific markers including CD34 for hematopoietic stem/progenitor cells, CD11b for monocytes/macrophages or DCs, Ly-6G for granulocytes and F4/80 for mature macrophages. In addition, the BM, liver (C) and spleen cells (D) were analyzed using Abs to CD41, a megakaryotic marker and CD31, an early endothelial marker
Fig. 5
Fig. 5
Prolonged FVIII functional and phenotype correction in F8null mice after tail vein injection LV-VEC-F8BDD. The F8null mice were treated with non-myeloablative radiation and given an iv injection of LV-F8BDD under the control of EF1α, VEC, Gp and ITGA promoters (1 × 107 TU per animal) or PBS (100 μL per animal) mock control. A The kinetics of FVIII activities in plasma examined by the chromogenic assay on days 7, 15, 30, 45, 60, 120 and 180 (n = 3). B The FVIII acticities in 1 × 109 platelets based on the chromogenic assay at day 60 (n = 3). C The tail bleeding time analysis recorded at day 120. The time required to stop bleeding into the collection tubes containing saline solution was recorded and plotted (n = 3). D The percentage survival curves of mice after LV injection for up to 180 days. The tail clipping experiment was carried out at day 120. The differences in characteristics between groups were analyzed using the one way Welch ANOVA tests with Turkey post hoc tests (A, C) or Games-Howell post hoc tests (A and B); *p < 0.05, **p < 0.01, ****p < 0.0001
Fig. 6
Fig. 6
The kinetics of VCN and FVIII inhibitor formation in LV-F8BDD iv injected F8null mice. A The VCNs in blood cells were detected by genomic DNA qPCR in LV-treated F8null mice over time on days 7, 15, 30, 45, 60, 120 and 180 (n = 3). B The VCNs in the mouse organs including heart, lung, liver, spleen and kidney of the LV-treated F8null mice on day 120 (n = 3). C Determination of anti-FVIII IgG levels by ELISA in the LV-treated F8null mice. The plasma of LV-treated mice was collected 60 days after iv injection and diluted at 1:200 to determine anti-FVIII IgG levels (n = 3). D Analysis of inhibitor titer kinetics using plasma from LV-trated F8null mice over time on days 7, 15, 30, 45, 60, 120 and 180 (n = 3). The inhibitor titer was determined based on a modified Bethesda unit (BU) assay; * D15 and D120 of VEC vs. Gp, * D60 of EF1α vs. VEC, * D60, D120 and D180 of VEC vs. ITGA, ** D120 and D180 of EF1α vs. VEC, * D180 of EF1α vs. Gp. The differences in characteristics between groups were analyzed using the one way Welch ANOVA tests with Turkey post hoc tests (D) or Games-Howell post hoc tests (B–D); *p < 0.05, **p < 0.01, ***p < 0.001
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