Efficient and regulated erythropoietin production by naked DNA injection and muscle electroporation

Abstract

We show that an electric treatment in the form of high-frequency, low-voltage electric pulses can increase more than 100-fold the production and secretion of a recombinant protein from mouse skeletal muscle. Therapeutical erythopoietin (EPO) levels were achieved in mice with a single injection of as little as 1 microgram of plasmid DNA, and the increase in hematocrit after EPO production was stable and long-lasting. Pharmacological regulation through a tetracycline-inducible promoter allowed regulation of serum EPO and hematocrit levels. Tissue damage after stimulation was transient. The method described thus provides a potentially safe and low-cost treatment for serum protein deficiencies.

Figures

Figure 1

Hematocrit levels in C57BL/6 mice injected with 50 μg of pCMV/mEPO or with saline with (+ES) or without (−ES) electrical stimulation. Data are the mean ± SD of hematocrit and serum EPO measured in five animals. Serum EPO levels at different time points are indicated.

Figure 2

(A) Serum EPO levels in BALB/c mice injected with different amounts of pCMV/EPO. The horizontal line indicates the limit of detection of the assay. (B) Hematocrit levels. (C) Comparison of hematocrit levels in mice injected with different DNA doses with (+ES) or without (−ES) electrostimulation. Data are the mean ± SD of hematocrit and serum EPO measured in five animals. #, Significantly different from the saline control; ∗, significantly different from 50 μg.

Figure 3

Hematocrit levels in DBA/2J (A) and CD1 (B) mice injected with 3 or 50 μg of pCMV/EPO with (+ES) or without (−ES) electrical stimulation. Data are the mean ± SD of hematocrit measured in four animals. (C) Serum EPO levels. ∗, Significantly different from 50 μg −ES; #, significantly different from 50 μg +ES.

Figure 4

Readministration of pCMV/EPO. The right quadriceps of a group of eight mice was injected at day 0 (arrow) with 1 μg of plasmid (○). At day 56 (arrow), in half of the mice, the left quadriceps was injected with 50 μg of the same plasmid (■) or with saline (●). A group of four mice (▵) was injected with saline at day 0 and with 50 μg of pCMV/EPO at day 56. Each injection was followed by electrical stimulation. Data are mean ± SD.

Figure 5

(A) Hematocrit levels 10 days after injection in mice treated with 10 μg of pOr/EPO and different amounts of pMCK/rtTAnls as indicated. Serum EPO levels are indicated at the top of each column. (B) Hematocrit levels over time in mice injected with 10 μg of pOr/EPO and 0.5 μg of pMCK/rtTAnls. Solid lines indicate the presence of doxyxycline in the drinking water, dashed lines the absence. Group 1 mice were treated with DOX from day 0 to day 30; Group 2, from day 30 to day 70. Data are the mean ± SD of hematocrit as measured in four animals. ∗, Significantly different from mice not treated with DOX.

Figure 6

Histological analysis of electrostimulated muscles 1 week after injection; black arrowheads indicates the fibers with central nuclei. White arrowheads indicate the necrotic region. [Bar = 105 μm (A and C) and 45 μm (B and D).]