title: The enhancing effects of the light chain on heavy chain secretion in split delivery of factor VIII gene.
abstract: Coagulation factor VIII (FVIII) is secreted as a heterodimer consisting of a heavy chain (HC) and a light chain (LC), which can be expressed independently and reassociate with recovery of biological activity. Because of the size limitation of adeno-associated virus (AAV) vectors, a strategy for delivering the HC and LC separately has been developed. However, the FVIII HC is secreted 10-100-fold less efficiently than the LC. In this study, we demonstrated that the F309S mutation and enhanced B-domain glycosylations alone are not sufficient to improve FVIII HC secretion, which suggested a role of the FVIII LC in regulating HC secretion. To characterize this role of the FVIII LC, we compared FVIII HC secretion with and without the LC via post-translational protein trans-splicing. As demonstrated in vitro, ligation of the LC to the HC significantly increased HC secretion. Such HC secretion increases were also confirmed in vivo by hydrodynamic injection of FVIII intein plasmids into hemophilia A mice. Moreover, similar enhancement of HC secretion can also be observed when the LC is supplied in trans, which is probably due to the spontaneous association of the HC and the LC in the secretion pathway. In sum, enhancing the secretion of the FVIII HC polypeptide may require the proper association of the FVIII LC polypeptide in cis or in trans. These results may be helpful in designing new strategies to improve FVIII gene delivery.
authors.affiliation: Department of Pediatrics, University of Pennsylvania Medical Center and The Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Lingxia
authors.initials: L
authors.lastname: Chen
authors.forename: Fuxiang
authors.initials: F
authors.lastname: Zhu
authors.forename: Juan
authors.initials: J
authors.lastname: Li
authors.forename: Hui
authors.initials: H
authors.lastname: Lu
authors.forename: Haiyan
authors.initials: H
authors.lastname: Jiang
authors.forename: Rita
authors.initials: R
authors.lastname: Sarkar
authors.forename: Valder R
authors.initials: VR
authors.lastname: Arruda
authors.forename: Jinhui
authors.initials: J
authors.lastname: Wang
authors.forename: Jennifer
authors.initials: J
authors.lastname: Zhao
authors.forename: Glenn F
authors.initials: GF
authors.lastname: Pierce
authors.forename: Qiulan
authors.initials: Q
authors.lastname: Ding
authors.forename: Xuefeng
authors.initials: X
authors.lastname: Wang
authors.forename: Hongli
authors.initials: H
authors.lastname: Wang
authors.forename: Steven W
authors.initials: SW
authors.lastname: Pipe
authors.forename: Xiang-Qin
authors.initials: XQ
authors.lastname: Liu
authors.forename: Xiao
authors.initials: X
authors.lastname: Xiao
authors.forename: Rodney M
authors.initials: RM
authors.lastname: Camire
authors.forename: Weidong
authors.initials: W
authors.lastname: Xiao
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D010957:Plasmids; D011499:Protein Processing, Post-Translational
chemical_list: D005169:Factor VIII