Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

Zhu, Fang; Liu, Ze-Long; Jiang, Miao; Qu, Hui-Ge; Chi, Xiaoyan

doi:10.1007/s11427-012-4333-8

Cited by 3 publications

(2 citation statements)

References 21 publications

(24 reference statements)

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“…This linkage between heavy and light chains did not affect FVIII activity while prolonging its half-life following thrombin activation. Coinjection of two vectors encoding the FVIII heavy and light chain with Cys mutations into mice through the portal vein increased plasma coagulation activity, suggesting that the secretion of disulfide crosslinked heterodimeric FVIII had increased [18]. Fusing intein sequences to the FVIII heavy and light chains, followed by dual-vector transfection into cultured cells, increased the secretion of spliced FVIII protein and activity indicating that the inter-chain disulfide bond improved protein trans-splicing [5].…”

Section: Discussionmentioning

confidence: 99%

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Zhu

Liu

Wang

et al. 2013

Sci. China Life Sci.

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Protein trans-splicing based dual-vector factor VIII (FVIII) gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide (442±151 ng mL 1 vs. 305±103 ng mL 1 ) and coagulation activi- Sci, 2013Sci, , 56: 262 -267, doi: 10.1007 Protein trans-splicing based dual-vector cotransfer of FVIII heavy and light chain genes is effective in overcoming the capacity limit of adeno-associate virus (AAV) vectors. This strategy uses split intein, a protein splicing element, linked to the FVIII heavy and light chains, resulting in functionally peptide-joined intact FVIII protein, with intein fragments removed post-translationally [1]. As an inter-peptide transsplicing reaction, its efficiency depends on the strength of the molecular interactions, although splicing information located in the intein molecules and protein splicing occur spontaneously without any auxiliary factors or energy consumption [2]. In our recent study, in which the FVIII gene was transfected into cultured cells using the dual-vector, the spliced FVIII protein, with coagulation activity recovered, was observed, but these cells contained unspliced precursors of intein fused heavy and light chain polypeptides [1]. These nonfunctionally unspliced protein precursors may be immunogenic. The amount of spliced FVIII protein depends on its splicing efficiency; this, in turn, determines the coagulation activity and the efficacy of gene therapy. Another dual-vector gene delivery strategy based on intracellular

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Section: Discussionmentioning

confidence: 99%

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Zhu

Liu

Wang

et al. 2013

Sci. China Life Sci.

Self Cite

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“…However, the main drawback of this approach is the apparent chain imbalance, which derives from less efficient secretion of the heavy chain than the light one. This results in the production of higher amounts of inactive protein compared with full‐length F8 (Burton et al , 1999 ; Scallan et al , 2003 ; Chen et al , 2009 ; Zhu et al , 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Liver gene therapy with intein‐mediated F8 trans ‐splicing corrects mouse haemophilia A

et al. 2022

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Liver gene therapy with adeno‐associated viral (AAV) vectors is under clinical investigation for haemophilia A (HemA), the most common inherited X‐linked bleeding disorder. Major limitations are the large size of the F8 transgene, which makes packaging in a single AAV vector a challenge, as well as the development of circulating anti‐F8 antibodies which neutralise F8 activity. Taking advantage of split‐intein‐mediated protein trans‐splicing, we divided the coding sequence of the large and highly secreted F8‐N6 variant in two separate AAV‐intein vectors whose co‐administration to HemA mice results in the expression of therapeutic levels of F8 over time. This occurred without eliciting circulating anti‐F8 antibodies unlike animals treated with the single oversized AAV‐F8 vector under clinical development. Therefore, liver gene therapy with AAV‐F8‐N6 intein should be considered as a potential therapeutic strategy for HemA.

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Design of AAV Vectors for Delivery of Large or Multiple Transgenes

Zhao

Duan

Lai

2019

Methods in Molecular Biology

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Enhanced plasma factor VIII activity in mice via cysteine mutation using dual vectors

Cited by 3 publications

References 21 publications

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Liver gene therapy with intein‐mediated F8 trans ‐splicing corrects mouse haemophilia A

Design of AAV Vectors for Delivery of Large or Multiple Transgenes

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