Towards gene therapy for haemophilia B using primary human keratinocytes

Abstract

Haemophilia B might be permanently cured by gene therapy--the introduction of a correct copy of the factor IX gene into the somatic cells of a patient. Here, we have introduced a recombinant human factor IX cDNA into primary human keratinocytes by means of a defective retroviral vector. In tissue culture, transduced keratinocytes were found to secrete biologically active factor IX and after transplantation of these cells into nude mice, human factor IX was detected in the bloodstream in small quantities for one week. This is the first demonstration of a therapeutic protein reaching the bloodstream from transduced primary keratinocytes. This may have implications for the treatment of haemophilia B and other disorders.

Current approaches and perspectives in human keratinocyte-based gene therapies

“…These transgene products were generally short lived and were usually found in small amounts. 21,22 A substantial amount of work has been performed on transgenic mice in addition to these and other ex vivo studies. This technology has contributed to the development of relevant human cutaneous gene therapy strategies.…”

In vivo transduction of mouse epidermis with recombinant retroviral vectors: implications for cutaneous gene therapy

“…The apparent loss of ␤-gal activity in SENCAR and CD-1 mice could be due to a decrease or loss of the viral promoter activity, [19][20][21][22] or a loss of transduced cells either by a failure to transduced stem cells or by immunological responses developed against the transduced cells. To examine the first possibility, LZRN-transduced skin was harvested 2 or 4 weeks after transduction.…”

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

“…2 Transduced HaCaT cells were selected and maintained in 1 mg/ml G418 (Gibco BRL, Uxbridge, UK) for 2 weeks after establishing that a concentration of Ͼ0.8 mg/ml G418 was required to kill nontransduced HaCaT cells in 10 days. Factor IX antigen in conditioned medium from transduced HaCaT cells was quantified by enzymelinked immunosorbent assay (ELISA) 34 as a reporter for the internal promoter/enhancer activity.…”

“…Such a concept has been proven feasible with low levels of factor IX being delivered to mouse plasma for 7 days. 2 More recent improvements in grafting and vector design have increased this to 30-40 days. 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.…”

“…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.…”