Variegation of retroviral vector gene expression in myeloid cells.
We have comparatively evaluated the efficiency of a series of retroviral vectors transducing the gp91-phox gene, whose defects are responsible for impaired production of superoxide anion (O2-) by phagocytic cells and lead to the X-linked form of chronic granulomatous disease (X-CGD). These vectors 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 inducible internal promoters did not result in important improvements in the efficiency of O2- production and lowered the titers of the viral preparations. In contrast, sustained levels of superoxide generation were obtained upon transduction with the FMEV vector. To analyze the efficiency of transgene expression at the single cell level, over 150 cellular clones were generated 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. Within each clone, expression of the therapeutic gene correlated with the number of expressing cells rather than with the average levels of expression from each cell, indicating that at the single cell level, the proviral promoter is regulated by a binary, on/off mechanism. Moreover, both transduced bulk and clonal cell populations displayed a tendency to a progressive extinction of expression over time, with a mechanism involving LTR methylation. The design of novel retroviral vectors escaping silencing is highly desirable for efficient gene therapy.
Zentilin
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L
Molecular Medicine Laboratory, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy.
Qin
G
G
Tafuro
S
S
Dinauer
M C
MC
Baum
C
C
Giacca
M
M
D015870:Gene Expression; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006105:Granulomatous Disease, Chronic / Q000201:enzymology / Q000235:genetics* / Q000821:virology; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism; D011533:Proviruses / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D019076:Transgenes / Q000235:genetics
D019255:NADPH Oxidases