Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein

Abstract

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1␣ promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL͞6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2-10 g͞ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200 -400 ng͞ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

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

“…It has been revealed that the majority of the FVIII protein expressed from FVIII gene transfected cells is hydrolyzed to heavy and light chains in the Golgi apparatus during post-translational processing and then secreted as a functional FVIII heterodimer [13]. Based on this understanding, a dual-vector strategy for FVIII gene split transfer was developed for the study of hemophilia A gene therapy [3,4].…”

“…C57BL/6 mice were injected with 200 g of column-purified pCMV-C662HC and pCMV-C1828LC plasmids in 2 mL saline via the portal vein, as described by Burton et al [3]. At 48 h after injection of vectors, blood samples were collected by tail clipping in an anticoagulant at a final concentration of 0.38% (w/v) sodium citrate.…”

“…A Coatest chromogenic assay was used to detect transgene-produced human FVIII coagulation activity in mouse plasma, as described by Burton et al [3], with some modifications. Total FVIII activity detected in transgenic mouse plasma comprised both mouse and human-derived activity.…”

“…Adeno-associated virus (AAV) vectors are ideal gene delivery vehicles for the stable expression of transgenes, and their non-pathogenic, replication-defective characteristics, but proved difficult for the transfer of the FVIII gene because of their capacity constraint [2]. The separate delivery of FVIII heavy chain (HC) and light chain (LC) genes by a dual-vector is one strategy to circumvent the packaging limit of AAV vectors, but transgenic efficacy is adversely affected because of the inefficient secretion of the HC [3,4]. In addition, it was previously demonstrated that von Willebrand factor (vWF) is needed for the assembly of HC and LC to form the stable functional FVIII heterodimer, and helpful for its secretion in Chinese hamster ovary cells [5].…”

Gene therapy for hemophilia

“…Two strategies to circumvent this limitation have been explored. Firstly, the heavy and light chains of F.VIII can be split into separate vectors, and co-injection of these AAVs can induce the expression of biologically active F.VIII in circulation (260). However, the utility of this approach is limited by the fact that the F.VIII light chain can interact with the heavy chain within the cell, significantly enhancing secretion (261,262).…”