By David N. Leff

The largest organ in the human body is that body’s skin.

Its tough outer layer, the epidermis, consists mainly of keratin, which protects the body’s interior organs from the slings and arrows of solar radiation, aggressive chemicals, mechanical trauma.

To renew the constantly shedding outer layer of epidermis, a seamless expanse of rapidly dividing epithelial cells, the keratinocytes, express keratin, virtually non-stop.

“A single epidermal stem cell,“ observed molecular epidermal biologist Elaine Fuchs, “has enough proliferative capacity to completely cover an adult human. It makes a very attractive candidate for gene therapy.“

Fuchs, now at the University of Chicago, cloned the first human keratinocyte gene in 1984, and others since.

“We felt that the promoters on those extremely actively transcribing genes,“ Fuchs told BioWorld Today, “would be useful in targeting various different genes of interest to the epidermis, for potential use in gene therapy. We showed that the human promoters were faithfully expressed in transgenic animals.“

Now Fuchs has a demonstrator model to back up her decade-old gene therapy surmise. Her paper in the current Proceedings of the National Academy of Sciences (PNAS), dated Jan. 7, 1997, bears the title: “Transgenic studies with a keratin promoter-driven growth hormone transgene: Prospects for gene therapy.“

It reports two innovations: “Here we show the first use of a human keratin promoter in prospective gene therapy,“ Fuchs said, “and the first successful demonstration that the promoters remain effective over time as well.“

In so doing, she explained, they resolve the two key issues that have hampered efforts by others to recruit keratinocytes as gene therapy vectors. Other experiments done thus far,“ she pointed out, “trying to get keratinocytes to express various genes, have suffered from the fact that their promoters, being foreign, were shut off. Our endogenous, natural promoters remain active.“

Dividing epidermal cells express two major genes — K-5 on human chromosome 12; K-14 on chromosome 17.

“What we’ve shown now,“ she explained, “is that these promoters will continue to be active when we attach them to a foreign gene, in this case, recombinant human growth hormone [hGH].

Making Mice Make Human Hormone

She and her co-authors linked the hGH gene, separated from its own promoter, to a 2,100-base-pair human K-14 sequence, microinjected that chimeric gene into single-cell fertilized mouse embryos, and implanted these in foster mother mice.

After the resulting transgenic animals were born, the team grafted patches of their skin onto biopsied areas of nude mice skin-surface. These immunity-lacking animals, by definition, were unable to reject the alien tissue.

“All 10 lines of K-14-hGH mice,“ the PNAS paper reported, “expressed sufficiently high levels of circulatory human growth hormone to elicit marked physiological and metabolic changes over time . . . characteristic of GH.“

At 2.5 weeks of age, the transgenic mice had 11 nanograms per milliliter of the human hormone in their blood; rising to 49 nanograms at 36 weeks. This level was 1,000-fold higher than that of growth hormone in endogenous mouse. Indeed, at 36 weeks, the transgenic mice, males and females, were 1.6 times larger than their control litter mates.

Assuming that the skin of an adult mouse contains 100 million k-14-expressing keratinocytes, Fuchs and her team estimated that “it could produce high enough serum concentrations of the growth hormone to meet the needs of a growing human child.“

Besides growth hormone, Fuchs said, “we’ve used these promoters in transgenic animal systems to drive the expression of other genes, including those expressing growth and transcription factors and cytokines. In all of those cases, the promoters faithfully targeted the expression of the gene to the keratinocyte.“

One of several potential clinical applications she sees is the treatment of chronic ulcers. Another is replacing non-functional or mis-functional genes, for example, insulin. In this instance, her experiments show that “additional parameters have first to be considered,“ before keratinocyte gene therapy can be tried in diabetes.

For eventual therapy in patients, Fuchs suggests that the size of the skin graft “in the ideal situation, would be on the order of a dime to a nickel to a quarter. The effectiveness of the patch size would depend upon the efficiency of the promoter, and of a particular factor to enter the blood stream.“

Besides transplanting transgenic skin, the system also envisages culturing the chimeric cells, and applying them, just as similar skin cultures are widely applied today for treating extensive burn injuries.

Despite its “singular advantage over a viral promoter,“ Fuchs does not presume that her epidermal gene therapy will usurp the field of DNA-delivering vectors. “The advantage of an adenoviral vector system,“ she grants, “is transfectability. That is an issue we’re still faced with; in fact it’s our next step. How do we efficiently put these genes into human keratinocytes?“

One answer that she entertains is “the possibility that we could combine the use of a viral delivery system with our natural promoters. In that sense, we would be able to use the virus only for delivering the gene into the cell, and then rely on our promoters for long-term expression. That’s an area we’re certainly going to be trying.“

A non-viral alternative in her sights as well “is to put genes into cells by slightly permeabilizing the cell membrane. The cell essentially ingests the gene up into itself by phagocytosis. Some of that DNA will enter the nucleus and integrate into the chromatin.“

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