After 20-odd years of optimism, hype and pessimism, gene therapy as we know it seems due for a top-to-bottom makeover. Clinical cardiologist and molecular biologist Frank Giordano, at Yale University School of Medicine, is trying out a novel technology in vivo.

"It's actually a new way to do gene therapy," he told BioWorld Today. "Every other approach up to this point has been predicated either on putting one piece of genetic material into a patient to express a single gene, or antisense of a gene to try to turn it off. But neither of those efforts is based on modulating expression of a natural, endogenous gene, already in the patient's body."

Giordano is senior author of a forthcoming report in Nature Medicine, released online Nov. 4, 2002, and scheduled for publication in December. Its title: "Induction of angiogenesis in a mouse model using engineered transcription factors."

"The overall message," Giordano observed, "is that it's feasible to design protein-based transcription factors de novo that can regulate genes in vivo in an intact animal. This allows modulation of a complex biological process, such as wound healing or blood vessel growth. Instead of putting a foreign gene in, it regulates a natural gene that's already there. Based on the technology we are testing, there's no restriction as to what gene you can target - up or down.

"Each gene encodes many genes," he went on. "Many genes encode not just one gene product, not just one protein, but multiple proteins. As it gets expressed, a process called splicing occurs such that multiple proteins are produced from a single gene. It's very different from conventional gene therapy per se," Giordano continued.

"Consider for instance, this particular gene, VEGF - the vascular endothelial growth factor. It's one of many different genes that control angiogenesis - blood vessel growth. Our initial approach has been to augment blood vessel growth in tissues that had poor blood supplies, such as peripheral ischemic limbs, or hearts with diminished coronary artery blood flow. The experiment described in this Nature Medicine paper shows for the first time that one can modulate gene expression by designing a molecule specifically targeted to a desired gene - in this case, VEGF."

A Step Beyond Conventional Therapeutics

"What happens in conventional gene therapy's classic approach," Giordano noted, "is to pick a single protein product and make a specific complementary DNA that expresses only it - not all of the natural proteins that the normal gene would express. To do that with standard gene therapy," he continued, "one would have to put complementary DNAs for all those genes into the same tissue in order to get the benefit of all those genes' protein products. This new technology is very manageable in regulating multiple genes at once." The technology, developed by Sangamo BioSciences Inc., of Richmond, Calif., "is a step above and a little beyond potential therapeutics."

"Vascular endothelial growth factor," Giordano pointed out, "is a very important protein that regulates blood vessel growth. Artificial molecular switches called zinc-finger proteins," he explained, "were designed by Sangamo to bind the specific areas of the VEGF gene that regulates its expression. Our in vivo experiment showed this would be reachable because of the structure of the protein that bound the DNA. We put a DNA blueprint that encoded the expression of these molecular switches into an adenovirus vector, and injected that gene delivery virus into the skeletal muscle of mice. This induced expression of the VEGF gene in all of its natural protein products. By injecting these viruses into the ears of mice, we induced blood vessel growth. Those blood vessels appeared to be different from vessels that resulted from a single protein. Also, we accelerated wound healing in the mice, using these gene-transferred viruses.

"The wounds we administered," Giordano recounted, "were 5-millimeter punch biopsies placed subcutaneously on the backs of the mice. They were the kind of lesions you would get if you had a small biopsy taken in a dermatologist's office.

"VEGF promotes angiogenesis - blood-vessel growth," he went on. "We were able to induce the formation of blood vessels - significant numbers of capillaries - after six days. In those trials we used a single splice variant of VEGF A - either VEGF 121 or 165 - the human forms of the mouse gene. The advantage of the zinc-finger approach is that by turning on the VEGF A gene, all the splice variants are expressed, which is quite different than the single splice variant expression in present clinical use. The murine VEGF-A 164 gene that we then targeted causes the formation of blood vessels that are very permeable - leaky. We compared those against the ones that came from turning on the natural gene, and found that all their different proteins were not leaky."

Giordano said, "This is the first time ever in an intact mammalian system that it's been shown one can design a transcription factor to a regulatory region based on the protein structures of the gene, and that this actually had a biologic effect."

Aiming At Drug-Like Pharmaceutical Approach

"Complex processes like angiogenesis, wound healing or arteriogenesis involve multiple genes," Giordano observed. "I find it doubtful that a single factor is going to be the magic-bullet panacea that will lead to mature blood vessels for treating vascular disease. We were able to find regulatory sequences near multiple genes of interest that we targeted with a single molecule.

"We're now proceeding to do more clinical work in preparation for bringing this technology to clinical trial. There are still issues to resolve, establishing safety and the optimal route of delivery. So we could turn on VEGF fibroblast growth factor in other genes that are part of the blood vessel growth cascade. To do that with a standard gene therapy approach, you'd have to put cDNAs for all those genes into the same tissue to get the benefit of all those gene products. Our new method is very manageable in regulating multiple genes at once.

"We are working right now on ways," Giordano concluded, "to regulate genes that can have a huge impact on bypassing blocked arteries. And we're anticipating," he concluded, "clinical trials within a year or a year and a half."