Variegation of retroviral vector gene expression in myeloid cells

We have comparatively evaluated the efficiency of a series of retroviral ve ctors transducing the gp91-phox gene, whose defects are responsible for imp aired production of superoxide anion (O-2(-)) by phagocytic cells and lead to the X-linked form of chronic granulomatous disease (X-CGD). These vector s included four constructs based on the MoMuLV backbone and expressing gp91 -phox from the viral long terminal repeat (LTR) or from internal promoters, and one construct based on the myelotropic FMEV vector. Expression of the therapeutic gene from the MoMuLV LTR was unsatisfactory after transduction of the PLB985 X-CGD knockout cell line and of primary CD34(+) hematopoietic progenitors from X-CGD patients. The presence of either constitutive or in ducible internal promoters did not result in important improvements in the efficiency of O-2(-) production and lowered the titers of the viral prepara tions. In contrast, sustained levels of superoxide generation were obtained upon transduction with the FMEV vector To analyze the efficiency of trans- gene expression at the single cell level, over 150 cellular clones were gen erated from bulk cultures of PLB985 X-CGD cells transduced with this vector , each one representative of an individual transduction event. These clones revealed a markedly heterogeneous pattern of gp91-phox expression, ranging from complete silencing to full restoration of superoxide production. With in each clone, expression of the therapeutic gene correlated with the numbe r of expressing cells rather than with the average levels of expression fro m each cell, indicating that at the single cell level, the proviral promote r is regulated by a binary, on/off mechanism. Moreover, both transduced bul k and clonal cell populations displayed a tendency to a progressive extinct ion of expression over time, with a mechanism involving LTR methylation. Th e design of novel retroviral vectors escaping silencing is highly desirable for efficient gene therapy.