Assessment of pCLpG retroviral vector expression in vivo utilizing serial transplantation of transduced bone marrow cells.

Author(s)

Paula Fratini

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2009-03-05

Examining board members

Bryan Eric Strauss; Lygia da Veiga Pereira Carramaschi; Roger Chammas; Elizabeth Angelica Leme Martins; Luciana dos Reis Vasques

• Advisor: Bryan Eric Strauss

Abstract

The retroviral vector is a widely used gene transfer tool in both laboratory assays and clinical trials. Our laboratory developed a new vector, called pCLPG, with viral expression under the command of p53, a tumor suppressor and inducible activator of transcription, with the aim of establishing a vector with high level expression. The level of expression offered by the pCLPG system was superior to the non-modified vector in cell culture assays. In this project, our objective was to characterize the expression of the pCLPG vector in vivo utilizing an animal model where bone marrow cells (BMC) from C57BL/6 mice are transduced and then transplanted in recipient animals that have been previously irradiated in order to abolish the hematopoietic system. With the aim of observing sustained transgene expression in vivo, we standardized serial BMC transplantation, transduction with retroviral vectors and analyzed the eGFP reporter gene by flow cytometry and real time PCR, and also studied other tissues, such as spleen, thymus and peripheral blood. We also performed hematologic analyses in the transplanted animals in to observe possible adverse events related to the presence of the retrovirus.These assays did not reveal a significant difference between the performances of the parental pCLeGFP vector and pCLPGeGFP. Both the number of eGFP-positive cells and the intensity of reporter gene expression diminished during the serial transplant process. Expression was observed in 3-4%, 2-3% or 2-3% of cells recovered from bone marrow of the primary, secondary or terciary recipients of BMC transduced with the pCLeGFP vector, but not in peripheral blood, thymus or spleen. Similarly, eGFP-positive cells (6-7%, 4-4.5% or 3-3.5%) were observed after serial transplantation only in the bone marrow of animals that received BMC transduced with the pCLPGeGFP vector. However, peripheral blood was recovered from recipients and treated with 5-azacytidine, inducing the expression of eGFP from both vectors in approximately 4% of these cells, implying that viral silencing may have been related with methylation. This study demonstrated that the modifications in the promoter of the pCLPG vector were not sufficient to avoid silencing of viral expression in this model. (AU)