Adenoviral vector-mediated gene transfer to primitive human hematopoietic progenitor cells: assessment of transduction and toxicity in long-term culture

Adenoviral gene transfer to primitive hematopoietic progenitor cells (HPCs) would be useful in gene therapy applications where transient, high-level t ransgene expression is required. In the present Investigations, we have use d an adenoviral vector expressing the green fluorescent protein (AdGFP) to quantify transduction of primitive HPCs and assess adenoviral-associated to xicity in long-term culture. Here we show that a cytokine cocktail protects mass populations of CD34(+) cells and primary colony forming unit-cultures (CFU-Cs) from toxicity, enabling transduction of up to 79% of CD34(+) cell s. Transduction of CFU-Cs primitive (HPCs) and more primitive HPCs was quan tified following fluorescence activated cell sorting for green flourescence protein expression, Our results demonstrate transduction of 45% of primary CFU-Cs, 33% of week-5 cobblestone area forming cells (CAFCs), and 18% of w eek-5 CFU-Cs. However, AdGFP infection inhibited proliferation of more prim itive cells. Although there was no apparent quantitative change in week-5 C AFCs, the clonogenic capacity of week-5 AdGFP-infected cells was reduced by 40% (P < .01) when compared with mock-infected cells. Adenoviral toxicity specifically affected the transduced subset of primitive HPCs. Transduction of primitive cells is therefore probably underestimated by week-5 CFU Cs a nd more accurately indicated by week-5 CAFCs. These studies provide the fir st functional and quantitative evidence of adenoviral transduction of primi tive HPCs. However, further investigations will be necessary to overcome ad enoviral toxicity toward primitive HPCs before adenoviral vectors can be co nsidered a safe option for gene therapy. (Blood. 2000;96:100-108) (C) 2000 by The American Society of Hematology.