Abstract
The inability to obtain sufficient numbers of transduced cells remains a limitation in gene therapy. One strategy to address this limitation is in vivo pharmacologic selection of transduced cells. We have previously shown that knockdown of HPRT using lentiviral delivered shRNA facilitates efficient selection of transduced murine hematopoietic progenitor cells (HPC) using 6-thioguanine (6TG). Herein, we now extend these studies to human HPC. We tested multiple shRNA constructs in human derived cell lines and identified the optimal shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 6TG treatment of mice resulted in increased percentages of transduced human CD45+ cells in the peripheral blood and in the spleen in particular, in both myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted in higher percentages of transduced human cells in the bone marrow, confirming selection from the progeny of long-term repopulating HPCs. However, the extent of selection of cells in the bone marrow at the doses of 6TG tested and the toxicity of higher doses, suggest that this strategy may be limited to selection of more committed progenitor cells. Together, these data suggest that human HPC can be programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based selection may not be robust enough for in vivo selection.
Highlights
In the past decade, several clinical trials have been carried out that have highlighted both the promise of gene therapy [1,2,3,4] and the potential harm from the uncontrolled integrations of the viral vectors used to deliver the therapeutic transgene [5,6]
Lentiviral vectors have been used in clinical trials, these studies used myeloablative conditioning and/or relatively high multiplicities of infection (MOIs) for transduction in order to achieve sufficient numbers of transduced cells [3,9], which may increase the risk for insertional mutagenesis
We have developed an alternative approach to hematopoietic progenitor cells (HPC) selection that relies on shRNA mediated knockdown of hypoxanthine phosphoribosyl transferase (HPRT), the enzyme required for metabolizing purine analogs like 6-thioguanine (6TG) into active agents [26]
Summary
Several clinical trials have been carried out that have highlighted both the promise of gene therapy [1,2,3,4] and the potential harm from the uncontrolled integrations of the viral vectors used to deliver the therapeutic transgene [5,6]. An alternative approach would be to use low MOI for transduction and use a selective agent for increasing the proportion of gene transduced cells, for those diseases in which the transgene does not provide a selective advantage. To this end, the multidrug resistance gene (MDR-1) [10,11,12,13], the dihydrofolate-reductase gene (DHFR) [14,15,16,17] and the O6-methylguanine-DNA-methyltransferase gene (MGMT) [18,19,20,21,22] have been tested extensively in this context. The carcinogenic potential of alkylating drugs represents a considerable risk for clinical applications of this approach [24,25]
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