Hematopoietic stem cells are widely recognized as attractconditions employed, Ͼ75% of the target cells retained the ive targets for gene therapy but current protocols to trans-CD34 + Lin − primitive phenotype after 4-5 days in culture; of duce these cells using recombinant retroviral vectors are those у25% expressed a high level of GFP detectable by inefficient. To evaluate optimization of retroviral transducboth flow cytometric analysis and fluorescence tion of hematopoietic stem cells and stability of gene microscopy. When transduced cells were cultured in clonexpression in their progeny, the green fluorescent protein ogenic progenitor assays, GFP fluorescence was readily (GFP) was explored as a reporter. We first improved sensidetected in situ, indicating that GFP expression was stable tivity of detection Ͼ100-fold over that achieved previously and not detrimental to the differentiative potential of the by using a novel retroviral vector (termed MGIN) expresstransduced CD34 + Lin − cells. We conclude that GFP is ing a high level of an enhanced GFP gene. Primitive effective as a vital marker to quantify retrovirus-mediated human hematopoietic cells bearing the CD34 surface gene transfer into human hematopoietic and perhaps antigen and lacking lineage differentiation markers other types of stem/progenitor cells, and monitor (CD34 + Lin − ) were transduced with the MGIN vector using gene expression during their subsequent cell lineage a clinically applicable supernatant procedure. Under the determinations.Keywords: gene therapy; gene transfer; gene expression; retroviral vectors; GFP; hematopoietic stem cells HSPC, the second problem associated with retroviral vecIntroduction tor-based gene therapy is low levels of sustained gene Hematopoietic stem and progenitor cells (HSPC) provide expression. Retroviral vectors derived from Moloney an attractive target for gene therapy because they have murine leukemia virus (MoMLV) are the most commonly the potential to continue producing progeny cells conused retroviral vectors in clinical trials. In a standard containing a therapeutic gene indefinitely. Hematological figuration, the gene of interest is placed under the trandiseases potentially benefiting from HSPC-based gene scriptional control of the viral long terminal repeat (LTR) therapy approaches include hereditary hemoglobinosince gene expression driven by LTR is generally higher pathies, immune deficiencies and disorders of phagocytic than by an internal promoter. 9,10 However, it has been cells, as well as other diseases such as acquired immunoreported that MLV LTR-mediated gene expression is fredeficiency syndrome and cancer. 1 Retroviral vectors, quently down-regulated during differentiation of which are being used in the majority of current clinical HSPC. 11,12 Because the LTR of the murine stem cell virus trials, are a primary choice as the vehicle for gene deliv-(MSCV) retroviral vector is permissive for expression in ery since they are capable of integrating into cellular murine HSPC, 8,13 we were interested...

The proliferative longevity of totipotent hematopoietic stem cells (THSC) is a limiting factor in normal hematopoiesis and in therapy by cell- or gene-replacement, but has not yet been ascertained. We have followed the long-term fate of individual clones of mouse THSC from fetal liver or adult bone marrow, after labeling in culture, followed by engraftment and serial transplantation in unirradiated W/Wv-C57BL/6 hosts. The ancestor cell of each clone and its mitotic progeny were uniquely identifiable retrospectively by the DNA integration pattern experimentally produced by replication-incompetent recombinant murine retroviruses. These viruses provided physiologically neutral markers. The marked clones proved to be derived from THSC, based on their contributions to a wide array of myeloid and lymphoid blood lineages in the hosts. The label also identified the target cells as the population displaying clonal succession. The various labeled stem cell clones proliferated for substantially different periods of time. The longest observed clone endured, after the original cell was marked, for at least 2 1/2 years--the equivalent of a mouse's lifetime. However, the results suggest that THSC clones are not all long-lived and that even the longest-lived ones may not be potentially immortal. Thus, the unpredictable lifespan of any given THSC clone indicates the desirability of introducing multiple clones in therapeutic transplants.

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