A picornaviral 2A-like sequence-based tricistronic vector allowing for high-level therapeutic gene expression coupled to a dual-reporter system.
The 2A-like sequences from members of the picornavirus family were utilized to construct a tricistronic vector bearing the human iduronidase (IDUA) gene along with the firefly luciferase and DsRed2 reporter genes. The 2A-like sequences mediate a cotranslational cleavage event resulting in the release of each individual protein product. Efficient cleavage was observed and all three proteins were functional in vitro and in vivo, allowing for supratherapeutic IDUA enzyme levels and the coexpression of luciferase and DsRed2 expression, which enabled us to track gene expression.
Osborn
Mark J
MJ
Pediatrics, Hematology-Oncology, Blood and Marrow Transplant Program, Institute of Human Genetics, Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN 55455, USA.
Panoskaltsis-Mortari
Angela
A
McElmurry
Ron T
RT
Bell
Scott K
SK
Vignali
Dario A A
DA
Ryan
Martin D
MD
Wilber
Andrew C
AC
McIvor
R Scott
RS
Tolar
Jakub
J
Blazar
Bruce R
BR
D000818:Animals; D003546:Cysteine Endopeptidases / Q000235:genetics*; D015870:Gene Expression; D018014:Gene Transfer Techniques; D005796:Genes; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D007068:Iduronidase / Q000378:metabolism; D066298:In Vitro Techniques; D008156:Luciferases / Q000378:metabolism; D049409:Luciferases, Firefly; D008164:Luminescent Proteins / Q000235:genetics; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D008957:Models, Genetic; D009083:Mucopolysaccharidoses / Q000378:metabolism; D041681:NIH 3T3 Cells; D010849:Picornaviridae / Q000235:genetics*; D010957:Plasmids / Q000378:metabolism; D014176:Protein Biosynthesis; D013997:Time Factors; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics*
D008164:Luminescent Proteins; D014764:Viral Proteins; C413663:fluorescent protein 583; D008156:Luciferases; D049409:Luciferases, Firefly; D007068:Iduronidase; D003546:Cysteine Endopeptidases; C057400:picornain 2A, Picornavirus