Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FVIII composed of the human heavy chain and the rat light chain

doi:10.1007/s11684-021-0844-7

Front. Med.

2022, Vol. 16

Issue (4) : 584-595 https://doi.org/10.1007/s11684-021-0844-7

RESEARCH ARTICLE

Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FVIII composed of the human heavy chain and the rat light chain

Jianhua Mao¹(

), Yun Wang^1,², Wei Zhang¹, Yan Shen³, Guowei Zhang⁴, Wenda Xi⁵, Qiang Wang¹, Zheng Ruan¹, Jin Wang², Xiaodong Xi¹(

)

¹. Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, Collaborative Innovation Center of Hematology, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
². Shanghai Institute of Hematology, State Key Laboratory for Medical Genomics and Department of Hematology, Collaborative Innovation Center of Systems Biomedicine, Pôle Sino-Français des Sciences du Vivant et Genomique, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
³. Research Center for Experimental Medicine, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
⁴. The School of Medicine, Hangzhou Normal University, Hangzhou 310036, China
⁵. Shanghai Institute of Hypertension, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China

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Abstract

Conventional therapies for hemophilia A (HA) are prophylactic or on-demand intravenous FVIII infusions. However, they are expensive and inconvenient to perform. Thus, better strategies for HA treatment must be developed. In this study, a recombinant FVIII cDNA encoding a human/rat hybrid FVIII with an enhanced procoagulant potential for adeno-associated virus (AAV)-delivered gene therapy was developed. Plasmids containing human FVIII heavy chain (hHC), human light chain (hLC), and rat light chain (rLC) were transfected into cells and hydrodynamically injected into HA mice. Purified AAV viruses were intravenously injected into HA mice at two doses. Results showed that the hHC+ rLC protein had a higher activity than the hHC+ hLC protein at comparable expression levels. The specific activity of hHC+ rLC was about 4- to 8-fold higher than that of their counterparts. Hydrodynamic injection experiments obtained consistent results. Notably, the HA mice undergoing the AAV-delivered hHC+ rLC treatment exhibited a visibly higher activity than those treated with hHC+ hLC, and the therapeutic effects lasted for up to 40 weeks. In conclusion, the application of the hybrid FVIII (hHC+ rLC) via an AAV-delivered gene therapy substantially improved the hemorrhagic diathesis of the HA mice. These data might be of help to the development of optimized FVIII expression cassette for HA gene therapy.

Keywords hemophilia A adeno-associated virus (AAV) human/rat hybrid factor VIII gene therapy dual chain strategy

Corresponding Author(s): Jianhua Mao,Xiaodong Xi

About author:

Tongcan Cui and Yizhe Hou contributed equally to this work.

Just Accepted Date: 19 August 2021 Online First Date: 14 January 2022 Issue Date: 02 September 2022

Cite this article:

Jianhua Mao,Yun Wang,Wei Zhang, et al. Long-term correction of hemorrhagic diathesis in hemophilia A mice by an AAV-delivered hybrid FVIII composed of the human heavy chain and the rat light chain[J]. Front. Med., 2022, 16(4): 584-595.

URL:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-021-0844-7
https://academic.hep.com.cn/fmd/EN/Y2022/V16/I4/584

Fig.1 Activity and antigen expression levels of hHC+ hLC and hHC+ rLC in transfected 293T cells. (A) FVIII activity of hHC+ hLC and hHC+ rLC. Expression plasmid of hHC driven by CB promoter was transfected together with hLC or rLC into 293T cells at a ratio of 1:1 (HC:LC), and the total amount of the plasmids was 1 mg/well. At 48 h after transfection, the medium was collected, and FVIII activity was detected via aPTT assay. Normal human plasma (NHP) was used as the standard, and initial activity was defined as one unit. (B) Detection of FVIII antigen by ELISA. FVIII antigen was measured with a specific antibody to hHC. NHP was used as the standard, and one unit was calculated as 0.2 mg. (C) Specific activity of FVIII calculated on the basis of ELISA. The specific activity of FVIII was calculated on the basis of the data from A and B. (D) Detection of FVIII expression by WB. Expression of FVIII in reduced and nonreduced states was detected by WB. The samples used were the transfected cell media of hHC+ hLC and hHC+ rLC. The intensity ratio of hHC+ hLC and hHC+ rLC is shown at the bottom. The intensity ratio of hHC+ hLC was calibrated as 1.0, and the relative intensity of hHC+ rLC to hHC+ hLC was 1.1. (E) The specific activity of FVIII calculated on the basis of WB. The specific activity of FVIII was calculated on the basis of the data from A and D. (n = 3, ***P<0.001.)

Fig.2 Activity and antigen expression of hHC+ hLC and hHC+ rLC in transfected HepG2 cells. (A) Detection of FVIII activity. The expression plasmids of hHC, which were driven by the ApoE-hAAT promoter, were co-transfected into HepG2 cells with LC (hLC and rLC). The total amount of plasmids of each group was 1 mg, and the ratio of HC:LC was 1:1. The activity of FVIII of each group was detected at 48 h post-transfection via aPTT assay. Normal human plasma (NHP) was used as the standard, and initial activity was defined as one unit. (B) Detection of FVIII antigen by ELISA. The FVIII antigens of hHC+ hLC and hHC+ rLC were monitored by ELISA, and the samples used were the same as those utilized in aPTT assay. NHP was used as the standard, and one unit was calculated as 0.2 mg. (C) Detection of FVIII expression by WB. Expression of FVIII of hHC+ hLC and hHC+ rLC in reduced and nonreduced states was detected by WB. The intensity ratio of hHC+ hLC and hHC+ rLC is shown at the bottom. The intensity ratio of hHC+ hLC was calibrated as 1.0, and the relative intensity of hHC+ rLC to hHC+ hLC was 1.03. (D) Specific activity of FVIII calculated on the basis of ELISA. Specific activity of FVIII was calculated on the basis of the data from A and B. (E) Specific activity of FVIII calculated on the basis of WB. Specific activity of FVIII was calculated on the basis of the data from A and D. (n = 3, ***P<0.001.)

Fig.3 Detection of the stability of hHC+ hLC and hHC+ rLC in 293T and HepG2 cells. (A) The sample was the culture medium from the transfected 293T cells. It was incubated at 37?°C for 2, 6, 12, and 24 h. (B) The sample was the culture medium from the transfected HepG2 cells. It was incubated at 37?°C for 2, 4, 6, 8, 10, 12, and 24 h. At the indicated time points, the residual FVIII activities of the samples were measured by aPTT (n = 3). Relative activity was calculated according to the activity before incubation at 37?°C, which was defined as 1.0 AU (arbitrary unit).

Fig.4 Detection of FVIII activity, specific activity, TEG, and hemoglobin concentration in the hydrodynamically injected HA mice. (A) Detection of FVIII activity. Expression plasmids for hHC were hydrodynamically injected into HA mice together with hLC and rLC at a ratio of 1:1. A total of 150 mg plasmid diluted in 2 mL saline was injected into each HA mouse through the lateral tail vein. Plasma was collected at 48 h after injection. FVIII activity was detected by aPTT (*P<0.05, **P<0.01, ***P<0.001). Normal human plasma (NHP) was used as the standard. (B) The FVIII antigen was detected using hHC antibody and ELISA. NHP was employed as the standard. (C) Specific FVIII activity was calculated on the basis of the data from A and B (****P<0.0001). (D) Detection of TEG. Fresh citrated blood sample was harvested from the hydrodynamically injected HA mice at 54 h after injection and immediately used for TEG assay. (E) Detection of hemoglobin concentration. Citrated peripheral blood (eye-bleeding) was collected after 6 h from the mouse whose tail was half-clipped. Hb concentration was measured using an animal blood cell counter.

Fig.5 Detection of the activity and specific activity of hHC+ hLC and hHC+ rLC in AAV-mediated gene therapy in HA mice in vivo. (A) Detection of FVIII activity in the intermediate dose group. AAV viruses delivering hHC together with hLC and rLC at a ratio of 1:1 were injected into HA mice through the lateral tail vein at the intermediate dose group (8×10¹² vg/kg), and FVIII activity was measured using aPTT assays from 0 to 40 weeks (*P<0.05). Normal human plasma (NHP) was used as the standard. (B) Detection of FVIII activity in the high dose group (2×10¹³ vg/kg). The ratio and the injection method were the same as those in the intermediate dose group. FVIII activity was measured from 0 to 40 weeks (*P<0.05, ***P<0.001). (C) The FVIII antigen of the mice from the high dose group was detected using ELISA at 16 and 24 weeks. hHC antibody was used to detect FVIII expression, and NHP was employed as the standard. (D) Specific FVIII activity at 16 and 24 weeks was calculated on the basis of the data from B and C (*P<0.05).

Fig.6 Detection of liver function. (A) ALT was measured in HA mice administered with a high dose of hHC+ hLC and hHC+ rLC (n = 4). (B) AST was measured in HA mice administered with a high dose of hHC+ hLC and hHC+ rLC (n = 4). The reference range of ALT or AST is displayed at the top of the bar chart. The data were from the corresponding HA mice before AAV-delivered FVIII was administered.

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