Serial bone marrow transplantation reveals in vivo expression of the pCLPG retroviral vector

Abstract

Background: Gene 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.

Results: We 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.

Conclusions: These 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.

Figure 1

p53-dependent expression from pCLPGeGFP revealed in a hematopoietic cell line. (A) Schematic representation of the parental, non-modified gamma retroviral vector, pCLeGFP, which utilizes the native LTR to drive transgene expression. For pCLPGeGFP, the LTR was modified by the removal of the enhancer region and insertion of a p53-responsive element, as described previously [8,9]. (B) K562 cells were used to test expression of the pCLeGFP and pCLPGeGFP vectors in response to p53 activity (intensity of eGFP; au, arbitrary units). The data represent the mean and standard deviation of duplicate samples from 3 independent experiments.

Figure 2

Experimental design of serial transplantation. Three groups of animals were treated with this procedure (mock-transduced BMC; animals receiving pCLeGFP-transduced BMC; animals receiving BMC transduced with pCLPGeGFP). Analysis included detection of eGFP by flow cytometry, hematologic exams, collection of gDNA and posterior determination of provirus copy number and chimerism.

Figure 3

Observation of eGFP expression in donor BMC. Total BMC were collected from male donor mice, stimulated with cytokines and either mock transduced or transduced with pCLeGFP or pCLPGeGFP in the presence of Retronectin and analyzed by flow cytometry for eGFP expression 48-hours later (percentage of eGFP-positive cells and intensity of eGFP; au, arbitrary units).

Figure 4

Analysis of eGFP expression by flow cytometry in cells recovered from transplant recipients. Short term (two months after transplant) or long term (6 to 10 months after transplant) observation cohorts were sacrificed after primar, secondary or tertiary transplantation (1°, 2° or 3°) and recovered cells were analyzed by flow cytometry for eGFP expression (percentage of eGFP-positive cells and intensity of eGFP; au, arbitrary units). Bars represent the mean and standard deviation among samples from the same cohort (please see Table 1 for the number of animals in each group). For statistical analysis, the Student's t-test (paired, 1-tailed; *, p < 0.0005; **, p < 0.005; #, p < 0.05) was performed using Excel, comparing pCLeGFP and pCLPGeGFP cohorts for each condition.