A Study of the Expression of Functional Human Coagulation Factor IX in Keratinocytes Using a Nonviral Vector Regulated by K14 Promoter

Abstract

Ex vivo gene therapy requires a suitable bioreactor for production and delivery of the gene products into a target tissue, and keratinocyte is suitable model in this regard because of its potential for systemic release of proteins. To establish a keratinocyte-specific expression system, a mammalian-based expression plasmid equipped with a 2,240-bp fragment from the human keratin 14 (k14) gene enhancer/promoter region was constructed and used for the insertion of the human coagulation factor IX (hFIX)-cDNA downstream the K14-derived regulatory elements. The human epidermal keratinocytes isolated from neonatal foreskin were cultivated in keratinocyte serum-free media and transfected with the recombinant plasmid. The K14-promoter-driven expression of recombinant hFIX (rhFIX) was evaluated by performing coagulation test as well as enzyme-linked immunosorbent assay on the cultured media collected from the transfected cells at various stages. The rhFIX corresponding transcript and protein were confirmed by performing reverse transcription PCR as well as immunoblotting experiments, respectively. Based on the coagulation activities obtained from the conditioned media of nine isolated clones, the hFIX expression levels vary from 5% to 39% of normal human plasma. Expression levels of the hFIX obtained in this study are comparable to those reported for viral systems. The obtained data supported the potential of keratinocyte for the expression and secretion of biologically active rhFIX and underscore the importance of the examined cis sequences for enhancing gene expression in a mammalian expression system. Besides, it has provided means for further bioengineering strategies to improve the expression efficiency of the hFIX in keratinocytes and other mammalian host cells.

The design and optimization of RNA trans‐splicing molecules for skin cancer therapy

“…For systemic application, the targeting and killing of SLCO1B3 ‐expressing hepatocytes by the RTM can be prevented by, e.g. the usage of a skin specific K14 promoter (Staggers et al., 1995; Hosseini et al., 2010). An alternative route for a possible application might also constitute the local injection of the RTM into the epidermal tumor tissue.…”

Genetic modification of bone-marrow mesenchymal stem cells and hematopoietic cells with human coagulation factor IX-expressing plasmids

“…Several potential target cells were shown to be able to produce an active hFIX such as hepatocytes [6], myoblasts [7], keratinocytes [8,9], endothelial cells [10], bone-marrow stromal cells [11] and hematopoietic stem cells (HSCs) [12].…”

Bioengineering of differentiated hepatocytes withhuman factor IX-expressing plasmidsin vitro

“…6,7 For the somatic gene therapy of hemophilia B, different cell types have been evaluated. [8][9][10][11][12][13][14][15] In our previous study, bone-marrow mesenchymal stem cells were used to transfect with different hFIX-expressing plasmids. As these cells are non-hepatocyte cell types, the capacity of cells to produce fully active hFIX was limited at high expression rates.…”