A GFP REPORTER SYSTEM TO ASSESS GENE-TRANSFER AND EXPRESSION IN HUMANHEMATOPOIETIC PROGENITOR CELLS

Hematopoietic stem cells are widely recognized as attractive targets f or gene therapy but current protocols to transduce these cells using r ecombinant retroviral vectors are inefficient. To evaluate optimizatio n of retroviral transduction of hematopoietic stem cells and stability of gene expression in their progeny, the green fluorescent protein (G FP) was explored as a reporter. We first improved sensitivity of detec tion > 100-fold over that achieved previously by using a novel retrovi ral vector (termed MGIN) expressing a high level of an enhanced GFP ge ne. Primitive human hematopoietic cells bearing the CD34 surface antig en and lacking lineage differentiation markers (CD34(+)Lin(-)) were tr ansduced with the MGIN vector using a clinically applicable supernatan t procedure. Under the conditions employed, > 75% of the target cells retained the CD34(+)Lin(-) primitive phenotype after 4-5 days in cultu re; of those greater than or equal to 25% expressed a high level of GF P detectable by both flow cytometric analysis and fluorescence microsc opy. When transduced cells were cultured in clonogenic progenitor assa ys, GFP fluorescence was readily detected in situ, indicating that GFP expression was stable and not detrimental to the differentiative pote ntial of the transduced CD34(+)Lin(-) cells. We conclude that GFP is e ffective as a vital marker to quantify retrovirus-mediated gene transf er into human hematopoietic and perhaps other types of stem/progenitor cells, and monitor gene expression during their subsequent cell linea ge determinations.