Abstract
BackgroundGene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied. Our group has developed a retroviral vector where transgene expression is controlled by p53 with the intention of harnessing the dynamic and inducible nature of this tumor suppressor and transcription factor. We present here a test of in vivo expression provided by the p53-responsive vector, pCLPG. For this, we used a model of serial transplantation of transduced bone marrow cells.ResultsWe observed, by flow cytometry, that the eGFP transgene was expressed at higher levels when the pCLPG vector was used as compared to the parental pCL retrovirus, where expression is directed by the native MoMLV LTR. Expression from the pCLPG vector was longer lasting, but did decay along with each sequential transplant. The detection of eGFP-positive cells containing either vector was successful only in the bone marrow compartment and was not observed in peripheral blood, spleen or thymus.ConclusionsThese findings indicate that the p53-responsive pCLPG retrovirus did offer expression in vivo and at a level that surpassed the non-modified, parental pCL vector. Our results indicate that the pCLPG platform may provide some advantages when applied in the hematopoietic system.
Highlights
Gene therapy in the hematopoietic system remains promising, though certain aspects of vector design, such as transcriptional control elements, continue to be studied
Since retroviral vectors are best suited for ex vivo gene transfer and one of their typical uses in clinical trials has been in the hematopoietic system, we wished to test the pCLPG vector in such a model
The number of provirus detected in the genomic DNA isolated from the bone marrow cells (BMC) recovered from the transplanted animals ranged from 0.02-0.04 for the pCLeGFP group and 0.03-0.07 for the pCLPGeGFP group. We interpret this result as an indication that vector silencing was not observed in the BMC since the number of eGFP-positive cells closely matched the proportion of cells carrying provirus
Summary
P53-responsiveness of the pCLPG vector in the context of a hematopoietic cell A tissue culture assay was performed in order to determine if the expected p53-dependence of the pCLPG vector would be preserved in hematopoietic cells. Serial transplantation of transduced bone marrow In order to assess the expression of the p53-responsive pCLPG vector in vivo, a model of serial bone marrow transplantation was used For this procedure, as shown, total bone marrow cells (BMC) were collected from male donor mice previously injected with 5fluorouracil (5 FU). The number of provirus detected in the genomic DNA (gDNA) isolated from the BMC recovered from the transplanted animals ranged from 0.02-0.04 for the pCLeGFP group and 0.03-0.07 for the pCLPGeGFP group We interpret this result as an indication that vector silencing was not observed in the BMC since the number of eGFP-positive cells closely matched the proportion of cells carrying provirus. 5-aza may suggest that silencing of vector expression was not a significant issue in these assays
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