Topical colchicine selection of keratinocytes transduced with the multidrug resistance gene (MDR1) can sustain and enhance transgene expression in vivo

In gene therapy, a clinically relevant therapeutic effect requires
long-term expression of the desired gene at a level sufficient to
correct or at least alleviate the underlying gene defect. One
approach to achieve persistent as well as high-level transgene
expression in a significant percentage of target cells would be to
select cells expressing both the desired transgene and a linked
selectable gene such as the human multi-drug resistance (MDR1)
gene-in a bicistronic vector. Because of its accessibility, the
skin is a very attractive target tissue to select genetically
modified cells, allowing topical application of a selecting agent,
thus minimizing potential toxic side effects. Among the potential
selecting drugs, agents that block cell division, such as
colchicine, are of particular interest because the use of
anti-mitotic drugs takes advantage of the rapid keratinocyte ( KC)
turnover in the epidermis and the need for continued proliferation
to substitute the KC lost due to selection. Before assessing the
therapeutic benefit of such an approach, several key questions need
to be answered in preclinical models: ( 1) Does topical colchicine
application achieve the desired in vivo effect by blocking KC
mitosis without eliciting unwanted toxic side effects? ( 2) Are
MDR-transduced (MDR+) human KC still able to proliferate and
differentiate when treated with colchicine? ( 3) Can MDR+ KC be
enriched by topical selection? ( 4) Does topical selection result
in persistent transgene expression by selecting KC stem cells
expressing MDR? To answer these questions and to test the
feasibility of such an approach both an in vitro skin equivalent
and an in vivo human skin graft model were developed in which MDR+
KC were treated with different dosages of colchicine. Quantitative
and qualitative analyses of MDR expression in human KC showed that
topical colchicine treatment selects high-level transgene
expression in a high percentage of KC. Moreover, determination of
transgene copy numbers demonstrated that MDR+ KC progenitor cells
were enriched by topical selection resulting in long-term
expression of the transgene in the skin. Thus, in summary, these
models demonstrate that topical selection of MDR+ KC is a safe
approach to efficiently enhance long-term gene expression in the
skin and holds future promise for clinical gene therapy
applications. Copyright (C) 2004 S. Karger AG, Basel.