David N. LeffScience Editor

Most blindness in the world today is due to unneeded blood vesselsovergrowing the retina of the eye. This proliferative retinopathyafflicts its victims at every stage of life, from premature newborns toworking adults to the elderly.

When the blood delivers too little oxygen to the eyeball, for whateverreason, the ischemic (oxygen-starved) retina responds by blindlyassuming it needs more blood vessels, to deliver more O2. So itswitches on vascular endothelial growth factor (VEGF), a moleculethat calls in the endothelial cells that pave the walls of veins, arteriesand capillaries.

It's not that VEGF doesn't do a good job of its vascular networking;it just doesn't know when to stop. (See BioWorld Today, Aug. 11,1995, p. 1.)

Childhood retinopathic blindness begins with the birth of a "preemie"_ a neonate that jumped the gun on the normal human gestationalperiod of 40 weeks, and came into the world late in the secondtrimester or early in the third.

"The more premature such an infant is," observed pediatricophthalmologist Lois Smith, "the higher the likelihood that he or shewill suffer from the retinopathy of prematurity. So we examine allneonates weighing under two kilograms (4.4 lbs.) at birth. Thecondition usually shows up after the child has developed to what itwould have at 30 weeks of gestation. That's when we examine themvery closely for retinopathy."

Because of their immature lung development, preemies used to beplaced routinely in incubators that raised their oxygen intake.Nowadays, Smith, who teaches at Harvard Medical School, toldBioWorld Today, "We prefer to monitor oxygen levels, and keeptheir blood-oxygen at just under 100 percent saturation.

"Too much oxygen has been associated with the increased incidenceof retinopathy of prematurity, now running at about 12,000 cases ayear," Smith said. "Not all of these result in loss of sight, but it is theleading cause of blindness in the childhood population.

"Among working-age adults above the age of 40, she added, "themain cause of blindness is diabetic retinopathy. Despite the greatinroads on this disease that have been made by pan-retinalphotocoagulation and laser treatment, recent figures cite the numberof such blind at 4.8 million."

After the age of 65, macular degeneration of the retina, for which nosuch therapy exists, accounts for the large majority of the aged blind,worldwide. Some 13 million Caucasian Americans suffer from thedisease, of which 1.2 million have its late stage, marked by retinalneoproliferation of blood vessels.

Although oxygen deprivation instigates the angiogenesis, the hitmolecule that's on the most-wanted list is vascular endothelial growthfactor.

At Hybridon Inc., in Worcester, Mass., molecular biologist GregoryRobinson and his associates have devised and tested a smartantisense bullet that knocks out the rogue VEGF's expressionmachinery. Robinson, of Hybridon's cell targets division, is firstauthor of a paper in the latest Proceedings of the National Academyof Sciences (PNAS) dated May 14, 1996. Harvard's Lois Smith is itssenior author. The article's title: "Oligodeoxynucleotides inhibitretinal neovascularization in a murine model of proliferativeretinopathy."

"The mouse makes a good model," Robinson told BioWorld Today,because it has the blood-vessel growth on the retina that you see inhumans."

This overgrowth doesn't blind a patient by covering the retina with adense, opaque mat of blood vessels, Robinson explained, "butbecause these new vessels are leaky, the blood leaks out into thevitreous humor of the eyeball and causes scarring. Fluid gets underthe retina, causing it to detach from the sclera beneath.

"In diabetic retinopathy," he added, "some patients complain aboutdark blobs wandering across their field of vision. These spots areblood floating across the vitreous chamber."

The Hybridon scientists began by synthesizing and testing 33phosphorothioate oligodeoxynucleotides, aimed at specific regions ofthe VEGF gene. Two of these antisense sequences proved to be themost effective at inhibiting VEGF expression. One, Vm by name,targeted the nucleotide termination codon, which halts proteinsynthesis. The other, M3, hit a region of VEGF's messenger RNAsequence that doesn't translate amino acids.

Mice OD'd On Oxygen Contract Retinopathy

To generate retinopathy in their mice, Lois Smith, at her researchlaboratory in Childrens Hospital (a Harvard teaching facility),obliged newborn animals to breathe high levels of oxygen for fivedays, then returned them to normal concentrations. Five days later,they had full-blown retinal neovascularization.

Robinson explained: "What happens is that under hyperoxygenationyou get a reduction in retinal vasculature, because it already hasenough blood vessels; it already has the oxygen it needs. Then, whenyou bring it back down to normoxia, the retina _ by now feelingischemic after that hyper diet of O2 _ goes: `Hey, I need some moreoxygen.' So it starts up that VEGF to get back that blood flow, andthat leads to neovascular proliferation."

Smith injected Robinson's antisense sequences into the vitreoushumor of one eye of the now-retinopathic mice, and a dummy controlcompound into the other. The results, PNAS reported, "showed astatistically significant reduction in the level of retinalneovascularization," compared to the control eyes.

Since that proof-of-principle study last year, Robinson said, "We'vemainly moved on into the human system. That's Hybridon's realgoal.

"It's hard to look at humans as an animal model," he continued."Right now we have developed a human oligo, and it seems to beworking better than the murine one. So far, we're using it in humanglioblastoma cells, in vitro, at the RNA and protein levels of VEGF."n

(c) 1997 American Health Consultants. All rights reserved.