Comparison of bicistronic retroviral vectors containing internal ribosome entry sites (IRES) using expression of human interleukin-12 (IL-12) as a readout.
BACKGROUND
Many gene therapy applications require the co-ordinated delivery of more than one reading frame. We wished to systematically compare IRES in the context of a retroviral vector to determine which was the most effective for protein production and viral titre. To do this we monitored expression of IL-12, as co-ordinated expression of both p35 and p40 subunits is required for production of the active heterodimer.


METHODS
Retroviral vectors were constructed to express human IL-12 in which an IRES initiates translation of the p40 subunit, with the IRES optimally aligned to the initiation codon of p40. Vectors containing an IRES from either polio virus (PV), encephalomyocarditis virus (EMCV), foot and mouth disease virus (FMDV) or murine leukaemia virus (MLV) were compared with a vector expressing IL-12 as a single protein (Flexi-12; in which the two IL-12 subunits are linked by a peptide).


RESULTS
All vectors produced high titre virus and directed synthesis of IL-12 in target cells. The bicistronic vectors containing the IRES from EMCV and PV were the most effective in infected 3T3 cells, producing up to 40 ng IL-12/10(6) cells/48 h, similar to the 50 ng IL-12/10(6) cells/48 h obtained with Flexi-12. The IRES from PV was the most efficient in human melanoma cells.


CONCLUSIONS
Bicistronic retroviral vectors have been constructed that effectively transduce target cells and produce high levels of protein. Target cell specificity of IRES function was observed. The combination of Flexi-12 and the IRES from PV will be useful in the generation of vectors expressing IL-12 with a second protein such as IL-2 for transduction of melanoma cells.
Harries
M
M
Department of Immunology, University College London, The Windeyer Institute of Medical Sciences, UK.
Phillipps
N
N
Anderson
R
R
Prentice
G
G
Collins
M
M
D005537:Aphthovirus / Q000235:genetics; D002478:Cells, Cultured; D004686:Encephalomyocarditis virus / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D018014:Gene Transfer Techniques; D005796:Genes; D005822:Genetic Vectors; D006801:Humans; D018664:Interleukin-12 / Q000096:biosynthesis / Q000235:genetics*; D009052:Leukemia Virus, Murine / Q000235:genetics; D017955:Poliovirus / Q000235:genetics; D012190:Retroviridae / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*
D018664:Interleukin-12