: 800 V/cm, 0.01-0.1 ms

degrade the plasmid (21). As animal models, pigs and dogs have muscle fibers that are quite large (37,38) and consequently are more suitable than rodent muscle for electrokinetic enhancement. Polymers such as polyvinylpyrrolidone (PVP) and poly-(L-glutamate) (PLG) at high or low concentrations or mild surfactants in low concentration such as poloxamer 188 have been used to enhance plasmid uptake, with some reduction in tissue damage. Poloxamer 188 may induce sealing of permeabilized lipid bilayers to rescue cells that were not extensively heat damaged; consequently, the expression levels may rise (21,39). Following electroporation of the skeletal muscle of mice, rats, dogs, or pigs (16,40-42), plasmid formulated with PLG or PVP has been observed to increase gene expression up to 10-fold compared with nonformulated plasmid. In mice, preinjection of the electroporated muscle with hyaluronidase, an enzyme that hydrolyzes hyaluronic acid, a ubiquitous component of the extracellular matrix, increases gene expression up to 5-fold with minimal tissue damage (43).

C. Delivery of Plasmid Fragments

Linear fragments of DNA derived from adeno-associated viral vectors have been delivered to the liver by an intra-arterial high-pressure hydrodynamic method, and have been shown to be efficacious and provide long-term expression of a secreted protein (44). Mice injected with a linear DNA expression cassette encoding human a-1-antitrypsin (hAAT) expressed approximately 10- to 100-fold more serum hAAT than mice injected with closed circular DNA over the length of the study.

We examined the effect of electroporation on expression of plasmid fragments containing expression cassettes, with or without residual plasmid backbone in skeletal muscle. There may be many advantages of delivering DNA fragments in vivo from which the antibiotic resistance gene and/or the bacterial origin of replication have been removed. First, the antibiotic resistance gene could render the host organism resistant to that particular antibiotic. In addition, some antibiotic resis tance genes, such as the ampicillin gene, contain multiple CpG motifs, which are known to enhance the immune response in muscle cells (45). A less immunogenic vector can reduce the possibility of immune or toxic responses and increase the therapeutic value of the vector (46). In addition, although undocumented for naked plasmid, the possibility of plasmid replication or recombination in vivo exists. It is possible to redesign plasmids with conditional origins of replication, as in the pCOR plasmids (47) or supercoiled minicircles (48) that lack the bacterial origin of replication. The creation of putatively safer plasmid constructs is difficult and will require sufficient resources for the design and testing of new plasmid systems that exceed the overall yield and performance of existing systems.

The pSEAP2 mammalian reporter vector (Clontech, CA) that contains the human placental secreted alkaline phospha-tase (SEAP) gene was used in these studies (Fig. 2A). The strong muscle-specific synthetic promoter SPc5-12 (49) was inserted into the pSEAP2 basic vector to create a pSP-SEAP vector. The SEAP coding sequence is followed by the SV40 late polyadenylation signal. The vector backbone also provides an f1 origin for single-stranded DNA production, a pUC19 (prokaryotic) bacterial origin of replication, and an ampicillin (prokaryotic)-resistant gene for propagation and selection in Escherichia coli. Several linear plasmid DNA fragments were generated by specific restriction enzyme digestions of the circular DNA. Electrophoresis was used to separate the linear fragments, which were then extracted from the gel. The SEAP gene is an immunogenic protein in most normal adult mammals (50). To avoid an immune reaction against the transgene, studies of the long-term expression of the different non-circular DNA fragments were conducted in severe combined immunodeficient (SCID) mice. On day 1, the mice (n = 10 per group) were weighed. Then, their left tibialis anterior muscles were injected with equivalent molari-ties of DNA fragments or supercoiled plasmid. Of the 6 groups, 1 received uncut, circular DNA; 4 received specific plasmid fragments; and 1 control group received an injection of phosphate buffered saline (PBS). The injection was followed within 2 min by electroporation, using external caliper electrodes and conditions of 6 pulses, 100 V/cm, 60 ms. A BTX T820 generator (Genetronics, San Diego, CA) was used to deliver square-wave pulses in all experiments. SEAP values were assayed to 54 days postinjection (Fig. 2B). The SEAP expression levels were not different among the treated groups. These results suggest that, when using the in vivo electropora-tion technique, gene expression, and probably intracellular bioavailability of the plasmid, is not dependent on the plasmid configuration.

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