Method for introducing pharmaceutical drugs and nucleic acids into skeletal muscle

Example 1

Stimulated Versus Unsimulated Muscles

Transfection efficiencies were determined by injecting skeletal muscles with the pSV40-luc reporter construct into the soleus muscle. Three days after injection, the muscles were removed and luciferase activity was measured using the Promega Luciferase Assay System (Madison, Wis.) according to manufacturer's protocols. Unstimulated EDL muscles from the same rats were used as control. The data are shown below in Table 1.

Example 2

Transfection Efficiency Versus Frequency

Rats were injected with 50 .mu.l of 1 mg / .mu.l of a plasmid carrying lac Z gene. Immediately following injection, electrodes were placed between 2-3 mm apart and the muscle was stimulated with the following stimulation parameters: voltage=30 volts; pulse duration=0.2 ms (total 0.4 ms, bipolar); trains=30, 1 second on 1 second off for 1 minute. Transfected fibers were counted from a 1 mm slice from middle of muscle. The number of transfected fibers is shown below in Table 2 and illustrated in FIG. 7. These data also illustrate that the method of the present invention transfects more than just surface muscle fibers; muscle fibers several cell layers deep are also transfected.

Example 3

Transfection Efficiency Versus Pulses

Soleus muscles of Wistar rats (200-270 grams) were injected with 50 .mu.g of RSV luciferase DNA plasmid in 50 .mu.l 0.9% NaCl. Shortly after injection, the muscles were electrically stimulated using the following parameters: 1000 Hz, between 0-1000 bipolar pulses of 200 .mu.l duration in each train were applied to the muscle 30 times over a period of 1 minute. Muscles were removed 3 days after transfection and frozen in liquid nitrogen. Cryostat sections were taken from the of the muscles and stained with Hematoxolin, Eosin and Safran (see Example 9). The remaining pieces were homogenized as described in Example 4 below. As illustrated in FIGS. 10-12, transfection efficiency increased with the number of pulses delivered to the muscle.