Use and comparison of different internal ribosomal entry sites (IRES) in tricistronic retroviral vectors.
BACKGROUND
Polycistronic retroviral vectors that contain several therapeutic genes linked via internal ribosome entry sites (IRES), provide new and effective tools for the co-expression of exogenous cDNAs in clinical gene therapy protocols. For example, tricistronic retroviral vectors could be used to genetically modify antigen presenting cells, enabling them to express different co-stimulatory molecules known to enhance tumor cell immunogenicity.


RESULTS
We have constructed and compared different retroviral vectors containing two co-stimulatory molecules (CD70, CD80) and selectable marker genes linked to different IRES sequences (IRES from EMCV, c-myc, FGF-2 and HTLV-1). The tricistronic recombinant amphotropic viruses containing the IRES from EMCV, FGF-2 or HTLV-1 were equally efficient in inducing the expression of an exogenous gene in the transduced murine or human cells, without displaying any cell type specificity. The simultaneous presence of several IRESes on the same mRNA, however, can induce the differential expression of the various cistrons. Here we show that the IRESes of HTLV-1 and EMCV interfere with the translation induced by other IRESes in mouse melanoma cells. The IRES from FGF-2 did however induce the expression of exogenous cDNA in human melanoma cells without any positive or negative regulation from the other IRESs present within the vectors. Tumor cells that were genetically modified with the tricistronic retroviral vectors, were able to induce an in vivo anti-tumor immune response in murine models.


CONCLUSIONS
Translation of the exogenous gene is directed by the IRES and its high level of expression not only depends on the type of cell that is transduced but also on the presence of other genetic elements within the vector.
Department of "Innovations thérapeutiques en Oncologie", INSERM U563, Institut Claudius Regaud, 20-24 rue du pont St Pierre, 31052 Toulouse, France. victorine.douin@toulouse.inserm.fr
Victorine
V
Douin
Stephanie
S
Bornes
Laurent
L
Creancier
Philippe
P
Rochaix
Gilles
G
Favre
Anne-Catherine
AC
Prats
Bettina
B
Couderc
D000230:Adenocarcinoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology; D000818:Animals; D015703:Antigens, CD / Q000235:genetics; D018122:B7-1 Antigen / Q000235:genetics; D001761:Bleomycin / Q000378:metabolism; D053282:CD27 Ligand; D045744:Cell Line, Tumor; D004351:Drug Resistance / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D018014:Gene Transfer Techniques; D005814:Genes, Viral / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D005839:Gentamicins / Q000378:metabolism; D006801:Humans; D007668:Kidney / Q000196:embryology / Q000821:virology; D015674:Mammary Neoplasms, Animal / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology; D008545:Melanoma / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology; D008546:Melanoma, Experimental / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D041681:NIH 3T3 Cells / Q000737:chemistry / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D012190:Retroviridae / Q000235:genetics*; D012270:Ribosomes / Q000235:genetics*; D012878:Skin Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology / Q000821:virology; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes / Q000235:genetics; D015678:Viral Structural Proteins / Q000235:genetics*
D015703:Antigens, CD; D018122:B7-1 Antigen; D053282:CD27 Ligand; C505771:CD70 protein, human; C505772:Cd70 protein, mouse; D005839:Gentamicins; D008565:Membrane Proteins; D012333:RNA, Messenger; D015678:Viral Structural Proteins; D001761:Bleomycin; C105427:Zeocin; C010680:antibiotic G 418