title: Co-expression of human adenosine deaminase and multidrug resistance using a bicistronic retroviral vector.
abstract: Current gene therapy protocols designed to treat adenosine deaminase (ADA) deficiency and other metabolic disorders suffer from low-efficiency delivery to target cells and a lack of long-term stability in expression of the therapeutic proteins. These problems may be resolved by use of an in vivo dominant selection. The multidrug transporter (MDR1) has been suggested as a useful selective marker for gene therapy. In this work, we co-expressed ADA and MDR1 cDNA in a retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. This system produced a bicistronic mRNA containing both ADA and MDR1, which enables co-expression of ADA and MDR1, and also allows the two proteins to be translated separately. After in vitro selection using a cytotoxic MDR1 substrate, vincristine, we demonstrated that functional ADA was co-expressed with MDR1 in proportion to the expression level of MDR1, whereas MDR1 expression was proportional to the stringency of the vincristine selection. Because the efficiency of IRES-dependent translation was much lower than that of cap-dependent translation in this system, we observed lower expression of the genes positioned after the IRES. This asymmetric expression caused a lower viral titer when MDR1 was placed downstream from the IRES, but it also provided a way of modulating the relative expression of ADA and MDR1. The retroviral system described in this work may serve as a useful tool to evaluate the strategies involving in vivo dominant selection for gene therapy of ADA-deficient patients.
authors.affiliation: National Cancer Institute, Laboratory of Molecular Biology, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Zhou
authors.forename: J
authors.initials: J
authors.lastname: Aran
authors.forename: M M
authors.initials: MM
authors.lastname: Gottesman
authors.forename: I
authors.initials: I
authors.lastname: Pastan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000096:biosynthesis; D000243:Adenosine Deaminase / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D004789:Enzyme Activation; D015870:Gene Expression; D005796:Genes; D019450:Genes, MDR; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D012190:Retroviridae / Q000235:genetics*
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000243:Adenosine Deaminase