Modulation of Erythropoietin Delivery from Engineered Muscles in Mice

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Abstract
In most relevant diseases, the permanent systemic delivery of a therapeutic protein from engineered cells might be proposed only if secretion levels can be regulated. The tetracycline resistance operon of Escherichia coli provides a transcriptional regulatory system effective in mammalian cells, which could be used for that purpose. A chimeric transactivator (tTA) consisting of the tetracycline repressor fused to the transactivation domain of the herpes simplex virus VP16 protein stimulates transcription by binding a minimal cytomegalovirus (CMV) promoter containing repeats of the tetracycline operator (tetO-CMV). Binding is abolished by tetracycline, thus impairing promoter activation. We have transduced C2.7 myoblasts with two U3-deleted retroviral vectors containing these regulatory elements. The tetP-Epo vector expressed the murine erythropoietin (Epo) cDNA under the control of the tetO-CMV promoter. The D-De-tTA vector expressed tTA under the control of the muscle-specific human desmin enhancer-promoter. Northern blot analysis showed background Epo mRNA expression in myoblasts. Myotubes differentiation induced tTA expression, leading to a 28-fold increase of Epo mRNAs, which was suppressed by tetracycline. Basal Epo secretion in myoblasts increased 23- to 41-fold during the formation of multinucleated myotubes, and turned back close to myoblast level when tetracycline was added. Myoblasts transduced with both vectors and treated with mitomycin with the aim to prevent tumor formation were engrafted in skeletal muscles of syngeneic C3H mice. Hematocrit levels were significantly higher in animals bearing cells transduced with both vectors than in control animals grafted with cells transduced with the Epo vector only. This difference was abolished when tetracycline was given to mice. These data indicate that the tetracycline regulatory elements can modulate transcription in the context of retroviral vector genomes, and that this system can be used to control the in vivo delivery of a therapeutic protein from genetically modified muscles. We examined whether the in vivo delivery of erythropoietin (Epo) from genetically modified muscles can be modulated over time. We used the tetracycline repressible system, described by Gossen and Bujard, in which a chimeric trans-activator stimulates transcription by binding to a minimal cytomegalovirus (CMV) promoter containing repeats of the tetracycline operator. The two components of this regulatory system were introduced into two retroviral vectors. We showed that Epo secretion can be regulated at the transcriptional level in a tissue-specific and regulatable manner in vitro when both vectors were introduced in C2.7 myoblasts. We also observed that the modulation of Epo secretion by tetracycline was maintained following transplantation of these cells in recipient mice.