Efficient transduction of human hematopoietic repopulating cells generating stable engraftment of transgene-expressing cells in NOD/SCID mice

Abstract

In an attempt to develop efficient procedures of human hematopoietic gene therapy, retrovirally transduced CD34+ cord blood cells were transplanted into NOD/SCID mice to evaluate the repopulating potential of transduced grafts. Samples were prestimulated on Retronectin-coated dishes and infected with gibbon ape leukemia virus (GALV)-pseudotyped FMEV vectors encoding the enhanced green fluorescent protein (EGFP). Periodic analyses of bone marrow (BM) from transplanted recipients revealed a sustained engraftment of human hematopoietic cells expressing the EGFP transgene. On average, 33.6% of human CD45+ cells expressed the transgene 90 to120 days after transplantation. Moreover, 11.9% of total NOD/SCID BM consisted of human CD45+ cells expressing the EGFP transgene at this time. The transplantation of purified EGFP+ cells increased the proportion of CD45+ cells positive for EGFP expression to 57.7% at 90 to 120 days after transplantation. At this time, 18.9% and 4.3% of NOD/SCID BM consisted of CD45+/EGFP+ and CD34+/EGFP+ cells, respectively. Interestingly, the transplantation of EGFP− cells purified at 24 hours after infection also generated a significant engraftment of CD45+/EGFP+ and CD34+/EGFP+ cells, suggesting that a number of transduced repopulating cells did not express the transgene at that time. Molecular analysis of NOD/SCID BM confirmed the high levels of engraftment of human transduced cells deduced from FACS analysis. Finally, the analysis of the provirus insertion sites by conventional Southern blotting indicated that the human hematopoiesis in the NOD/SCID BM was predominantly oligoclonal.

A Preclinical Model for the Analysis of Genetically Modified Human SkinIn Vivo

“…Similar results have been reported in the hematopoietic system, another rapidly renewing tissue. In these studies, the clonal make-up of transduced human hematopoiesis-engrafting NOD/SCID mice was demonstrated to occur also at the expense of a discrete number of clones (Barquinero et al, 2000;Güenechea et al, 2001). The loss of polyclonality and emergence of a small number of clones strongly suggest in vivo selection for epidermal stem cells with extended growth capacity.…”

In Vivo Dynamics of Human Stem Cell Repopulation in NOD/SCID Mice

“…In the remaining mice there was an overall increase in EGFP‐expressing cells. Recently, Barquinero et al also observed a decline in EGFP expression in a small cohort of mice that they attributed to gene silencing 19. Second, assessment of the proportion of human cells containing integrated provirus revealed the same patterns of repopulation kinetics as with EGFP expression.…”

“…Our original studies indicated that SRCs from cord blood (CB) are difficult to transduce with retroviral vectors (only 50% of transplanted mice contained 0.1% to 3% transduced human cells) 13. Subsequently, we and others have optimized the transduction procedure both for transduction efficiency and the maintenance and/or expansion of functional repopulating cells 14–19. Human bone marrow‐derived SRCs are still transduced with low efficiency under these conditions, which points to major differences in CB and BM‐derived SRCs.…”

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“…Cord blood CD34+ cells have been transduced successfully with a range of viral vectors bearing a variety of markers (Schilz et al, 1998;Conneally et al, 1998;Miyoshi et al, 1999;Woods et al, 2000;Guenechea et al, 2000;Barquinero et al, 2000;Abonour et al, 2000;Salmon et al, 2000). Primitive cord blood cells are favourable as they have greater transduction efficiency in comparison to similar cells from the bone marrow.…”