Co-Expression of Human Adenosine Deaminase and Multidrug Resistance Using a Bicistronic Retroviral Vector

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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. The human multidrug transporter (MDR1) and adenosine deaminase (ADA) were co-expressed in a bicistronic retroviral vector using an internal ribosome entry site (IRES) from encephalomyocarditis virus. Two retroviral vector configurations were examined with ADA and MDR1 placed either before or after the IRES. Using this system, we were able to produce a desirable amount of functional ADA through in vitro selection using cytotoxic MDR1 substrate at various concentrations. We also found that in this system the efficiency of IRES-dependent translation was much lower than that of cap-dependent translation. 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.