Circulating human factor IX produced in keratin promoter transgenic mice: a feasibility study for gene therapy of haemophilia B

Abstract

It has previously been suggested that keratinocytes might provide a suitable target cell for delivery of factor IX to the systemic circulation for gene therapy of haemophilia B. Here, an investigation of the use of cellular gene promoters specific for keratinocytes was undertaken to examine whether factor IX could be passed from the epidermis to the systemic circulation. Utilizing two bovine cytokeratin gene promoters, BKIII and BKVI, three lines of transgenic mice were generated with targeted expression of human factor IX in the epidermis. All three transgenic mouse lines secreted epidermally derived human factor IX into the blood system. Most effective factor IX expression (46 ng/ml steady-state levels of circulating human factor IX) was obtained utilizing the BKVI gene promoter, the human homologue of K10, which is expressed exclusively in differentiated keratinocytes, localized distal to the basement membrane. This report demonstrates, for the first time, that human factor IX can be efficiently synthesized and secreted from keratinocytes in situ, and can cross the epidermal basement membrane to reach the systemic circulation. The transgenic mouse model will provide a good in vivo system with which to optimize the efficiency of different keratin gene promoter constructs for delivery of therapeutic gene products to the serum, especially for those promoters, such as K10, which are not effectively expressed in vitro.

Transgenic studies with a keratin promoter-driven growth hormone transgene: Prospects for gene therapy

“…These animals died within several weeks after birth, making it impossible to assess the long-term potential for keratinocyte-derived K14-TNF-␣ to sustain high serum levels of the transgene product. The bovine K10 promoter, expressed in terminally differentiating keratinocytes, has also been used to produce and secrete high levels of circulatory factors in transgenic mice (51,52).…”

Nonviral transfer of genes to pig primary keratinocytes. Induction of angiogenesis by composite grafts of modified keratinocytes overexpressing VEGF driven by a keratin promoter

“…24,25 Our study is the first to demonstrate that targeting to keratinocytes, using lipid-mediated transfection of plasmid constructs containing endogenous promoters as well as the expansion and subsequent grafting of these cells for gene therapy, is a feasible approach. Expression driven by regulatory sequences of genes coding for structural proteins such as keratins, involucrin or fillagrin ensures precise stratum localization and abundant production of the therapeutic protein.…”

“…These and other transgenic data reinforce the concept that keratinocytes may act as a source of cytokines and/or therapeutic proteins that reach the underlying dermis or the bloodstream to exert biological action. 24,25 Ex vivo targeting of keratinocytes for gene therapy has been performed almost exclusively using retroviral vectors. [26][27][28][29][30][31] In spite of several advantages of this approach, in vivo maintenance of gene expression driven by the viral long terminal repeat (LTR) promoters has proved difficult for reasons not yet entirely understood.…”

Differentiation-specific enhancer activity in transduced keratinocytes: a model for epidermal gene therapy

“…The acellular matrix was fixed in 4% glutaraldehyde for 1 h, washed extensively in phosphate-buffered saline followed by HaCaT medium overnight. The matrix was inverted and 5 × 10 5 MRC5 fibroblasts 35 were seeded on to the bottom surface, followed by 5 × 10 5 HaCaT cells on to the upper surface of the reverted matrix the following day. After 3 days in culture at the air-liquid interface, the 'hat-like' component of the transplantation chamber (Renner) was placed over the ring and the entire organotypic skin structure was embedded within a full thickness incision on the back of an 8-to 10-week-old male MF1 nu/nu mouse (Harlan Olac, Bicester, UK) on to granulation tissue pre-formed by the temporary implantation of a 13-mm glass disc (Renner) 2 weeks before skin grafting.…”

“…3,4 The levels of protein detected in plasma after transduced cells are transplanted to animals are much lower than predictions based upon the high levels of gene expression observed in the same cells in culture. 2 This might be due to cell death or might be caused by a problem of transport to the plasma, although transgenic studies have shown that when factor IX 5 and human growth hormone 6 are placed under the transcriptional control of keratinocyte-specific regulatory elements, protein is detected in the plasma. Control experiments show that this is likely to be due to lymphatic drainage of the skin.…”