Gene Gunmediated Gene Therapy In Other Systems

A. Gene Gun Applications for Wounds and Burns

The gene gun is most useful in applications on exposed tissues. Given this, a number of investigators are testing gene guns for their ability to increase repair of wounds or burns as well as to reduce pain signaling at sites of tissue injury [reviewed in (154)]. Examples of repair applications include delivery of keratinocyte growth factor (155), platelet-derived growth factor (156), and epidermal growth factor receptor (157) to speed wound healing. Examples of approaches to limit pain are delivery of semaphorin A (18) and delivery of the gene for pro-opiomelanocortin (158).

B. Gene Gun Applications in the Eye

Given that the eye is exposed on the surface of the body, this tissue is a natural target for gene gun applications. The first demonstration of transfection by the gene gun was in the delivery of plasmids encoding the green fluorescent protein to corneal epithelial cells (17). In this work in rabbits, it was demonstrated that gene gun particle penetration could be readily titratedby changing the helium pressure driving particle acceleration and that little corneal damage was observed after particle bombardment. This work was followed by a true gene therapy approach where the gene for semaphorin A was delivered into the eye to modulate neuronal growth and architecture

(18). This was a good example of a gene therapy approach that avoids immunogenicity problems because (1) the transgene product is already present in the host, but is being increased locally; and (2) the gene gun delivers simple plasmids encoding only the therapeutic transgene protein and no vector-specific antigens. This contrasts with viral methods of gene delivery to the eye that can provoke strong immune responses even in this ''immunopriviledged'' site (159).

Subsequent approaches using the gene gun in the eye have delivered fluorescent tracer chemicals and the genes for fluorescent proteins to the eye to characterize morphology (120,160,161) and to characterize eye-specific regulatory elements in enhancer/promoters of genes (162). Another interesting gene therapy application of the gene gun in the eye was to deliver genes encoding immunosuppressive proteins like IL-4 and CTLA4 into the cornea after an allotypic corneal transplant (163). In this case, gene gun delivery was able to prolong allogeneic graft survival in mice. This interesting application, like that for semaphorin A, delivers an endogenous gene product by simple plasmid to avoid immune responses against the transgene proteins. This work also provides proof of principle for the use of the gene gun to genetically modify a variety of tissues to increase transplant survival.

One limitation of the gene gun is that transfection generally occurs within 10 to 20 cells of the surface that is bombarded. As such, the gene gun will likely have utility in applications to genetically modify the surface of the eye, including the corneal epithelium and the conjuctiva. In contrast, applications in the retina or other buried structures of the eye will likely be out of reach of this technology.

C. Neuronal Gene Gun Applications

Most gene gun applications in neuronal systems have been performed for basic research studies to characterize the biology of these cells. The gun provides the unique ability to deliver substances directly into not only neuronal cells in culture (164,165), but also into whole brain slices (120,166). The gene gun has been used to deliver not only DNA, but also fluorescent tracer molecules to track neuronal phenotype and survival (165). Direct gene gun delivery into the brain in vivo has been reported for transfection of the lamprey brain neurons and glia in the exposed floor of the fourth ventricle (167), and for direct transfection of the exposed brain in live mice (168). An alternate in vivo approach of gene guns has been to manipulate neuron behavior, but not to directly transfect the neurons themselves. In this case, the gene for semaphorin A was delivered into corneal epidermal cells of rabbits (18). In this model, local production of semaphorin A after gene gun transfection effectively repelled established and growing A-delta and C-fiber trigeminal sensory afferents. This application provided proof of principle for gene therapy applications to repel pain-sensing neurons from locals to attenuate chronic pain or prevent problematic pain responses in surgical fields.

D. Gene Gun Applications in Other Organs

Although the gene gun is most easily applied to exposed tissues such as the skin or the eye, the original demonstrations of gene gun transfections in animals showed that internal tissues could be surgically exposed to allow gene gun delivery (13,14). Therefore, any tissue that can be surgically exposed can also be transfected by the gene gun. Applications to date on internal organs include the liver (13,14), the muscle (13,24), the spleen (25), the bladder (169), and the heart (170,171). In many cases, the gene gun has been used to probe the biology of cells or genes in these organs. In particular, the gene gun has been used to deliver genes to analyze the specificity and level of gene expression by different enhancer/ promoter constructs into the liver (172) and into cardiomyo-cytes (173). In other cases, preclinical approaches are being tested to deliver antigen genes directly into the liver to prevent the liver stage of malaria (174). Future applications of the gene gun in deep tissues may be focused not only on understanding the biology of genes and tissues, but also for direct gene therapy. One interesting future application in organs will be to test whether the gene gun can effectively protect transplanted tissue from immune rejection. The work in corneal transplantation protection in the eye (163) provides good proof of principle for the use of the gene gun to modify tissues after removal without the need for immunogenic viral vectors or the need for prolonged culture ex vivo.

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