Tissue Specific Expression

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A. Skeletal Muscle

Since the initial report by Aihara and Miyazaki (51), in vivo electroporation of the skeletal muscle for delivery of therapeutic proteins has become widely used. Table 2 summarizes some of the recent literature reports in which electroporation was used to enhance plasmid delivery to skeletal muscle. Although the devices, conditions, methods, and animal models substantially differ, all studies conclude that plasmid injection followed by electroporation can be successfully used to deliver therapeutic genes. The electrokinetic enhancement of plasmid delivery allows the muscle to be used as a bioreactor for the persistent production and secretion of proteins into the bloodstream. The expression levels are increased by as much as 2 to 3 orders of magnitude over plasmid injection alone, to levels comparable to those of adenoviral-mediated gene delivery and may reach physiological ranges. The applications of intramuscular elec-troporation gene transfer are innovative and intriguing, and span a large array of pathologies: malignancies, renal disease, anemia, prevention of drug toxicity, etc. Thorough review articles address aspects of these proposed therapies (6,52-54). The duration of gene expression was reported to be at least 9 to 14.5 months after in vivo gene electrotransfer into skeletal muscle (55-57). Collectively, these studies provide the evidence that adequate levels of secreted proteins can be achieved using plasmids in a simple, safe, and efficient manner, with significant potential for gene transfer and vaccination for large animals and humans. Interestingly, more and more scientists are now addressing a new problem, the regulation of gene expression, raised by the adequate levels of protein production, to maintain levels of expression in concordance with therapeutic needs (15,18,58,59).

B. Liver

The liver represents one of the primary targets for gene therapeutic treatment of numerous metabolic diseases, cancers, hepatitis, and other pathologies. Although recombinant viral vectors have been widely used to introduce new genes into the liver, their usefulness may be mitigated by side effects and potential safety concerns (60-63). Plasmid delivered to hepatic vasculature (64), by hydrodynamic methods (65,66), or by electropora-tion constitutes an alternative method to deliver transgenes to the liver. Mechanistic studies to characterize electric field dis tribution for drug or plasmid administration under different conditions were performed in the liver (23). Electroporation is traditionally performed in conjunction with chemotherapy for different malignancies. This treatment, known as electro-chemotherapy, has been successful for liver malignancies in animal models (67). Tumor reduction has been recently achieved by locally injecting DNA to the site of interest in the liver followed by the electric field application (see also Table 4). Long-term expression of bcl-xs and tumor regression has been observed after plasmid delivery and electrotherapy using tweezers electrodes (68). In addition, nutritionally regulated foreign gene expression in vivo is attainable locally in the liver by this method (69). A method for efficient gene transfer to the liver by electroporation following tail vein administration of the naked DNA has been recently described (31). According to the authors, systemic injection has the advantage of delivering genes to more hepatocytes when compared with the local injection of plasmid to the liver. These advances in liver gene delivery may provide powerful tools for basic research or potential clinical application studies.

C. Skin

DNA delivery to skin may be useful for the treatment of skin disorders, DNA vaccinations, and other gene transfer applications requiring local or systemic distribution of a transgene product (33). This choice is facilitated by information that shows that electroporation of skin induces a mild and reversible impairment of the barrier function of the skin, a decrease in skin resistance, and a transient decrease in blood flow. Neither inflammation nor necroses is generally observed (70,71). Reporter genes are expressed in the immediate area surrounding the injection site. After direct plasmid injection into skin, transfected cells are typically restricted to the epidermis. However, in different animal species, when electroporation is applied after the injection, larger numbers of adipocytes and fibro-blasts and numerous dendriticlike cells within the dermal and subdermal tissues, as well as lymph nodes draining electroper-meabilized sites, are transfected (72-74). Compounds such as aurintricarboxylic acid have been found to enhance plasmid expression in rodent, primate, and pig muscles (73). Therapeutic molecules such as erythropoietin or HBV antigen can be efficiently produced by skin cells and remain functionally active up to 7 weeks (34,72).

D. Tumors

Electrochemotherapy, or enhanced delivery of chemotherapeu-tic drugs, especially bleomycin, to solid tumors has been used successfully for many years (76,77). Clinical trials using this method for the treatment of solid tumors have been conducted in humans and other species (9,78-80). Recently, investigators focused on plasmid delivery to tumors as a means to increase long-term antitumor immunity successfully (7,81,82), to inhibit angiogenesis (83,84), or to reduce tumor volume (85). Interestingly, in some cases, CpG islands within the plasmid backbone may contribute to the antitumor effect in the absence of therapeutic cDNA expression or eukaryotic sequences (86).

Figure 2 In vivo expression of plasmid fragments delivered by electroporation to the skeletal muscle. (A) The construct pSP-SEAP contains a SPc5-12 synthetic promoter, a human SEAP gene, the SV40 polyadenylation signal (expression cassette), and a plasmid backbone with bacterial replication origin, Uori, an antibiotic resistance gene (ampicillin), and a packaging origin for the SEAP gene, F1ori. Different regions of the plasmid were cut using restriction enzymes to yield the following:

Figure 2 In vivo expression of plasmid fragments delivered by electroporation to the skeletal muscle. (A) The construct pSP-SEAP contains a SPc5-12 synthetic promoter, a human SEAP gene, the SV40 polyadenylation signal (expression cassette), and a plasmid backbone with bacterial replication origin, Uori, an antibiotic resistance gene (ampicillin), and a packaging origin for the SEAP gene, F1ori. Different regions of the plasmid were cut using restriction enzymes to yield the following:

• KpnI/SalI, containing the expression cassette

• KpnI/AhdI, containing the expression cassette and Uori

• ApaLI/SalI, containing the expression cassette and F1 ori

• KpnI/SalI/AseI, containing the expression cassette and other 2 additional fragments of the plasmid backbone.

(B) SCID mice were injected with supercoiled DNA or plasmid fragments. Expression levels were not different among groups.

In a comparative study, 50 micrograms of plasmid was found to be as efficacious as 5 X 109 i.u. adenovirus. Furthermore, adenovirus leakage induced mild to moderate liver damage, while practically no leakage occurred after electroporation (87). Table 3 contains reports that show that improved vectors, delivered using highly effective electroporation methods, may form the basis for future human applications. Updated over views of the therapeutic perspectives of antitumor drug and DNA electrotransfer are also of significant interest (88,89).

Recently, electric pulse-mediated plasmid transfer has been used to deliver transgenes to cornea in an effort to ensure long-

Table 2 Electroporation for Plasmid Delivery in Muscle

Transgene

Species

Endpoint

Ref.

Restriction endonuclease Smal

Hamster

Cytochrome C oxidase activity

(108)

Erythropoietin

Mouse

SEAP, erythropoietin levels

(41)

Melanocyte antigen tyrosinase-related

Mouse

T cells, spleen CD8+T cells, outgrowth of s.c.

(109)

protein-2 H-2K(b)-restricted epitope

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