By David N. Leff

Editor¿s note: Science Scan is a roundup of recently published, biotechnology-relevant research.

Ages ago, when life on Earth was getting its act together, a proto-bacterium ¿ so the theory goes ¿ invaded a proto-cell, and stayed there. Like ¿The Man Who Came To Dinner,¿ the intrusive microorganism set up intracellular housekeeping as an organelle ¿ the mitochondrion.

Hundreds of these imported bodies swarm in the cytoplasm of every cell in every higher life form. Their main schtick is to supply the cell with its energy needs. As a memento of the mitochondrion¿s free-living bacterial origin, it boasts a genome of its own, harboring a unique, circular chromosome that codes for 13 proteins. But even more curious, 90 percent of a mitochondrion¿s proteins are donated by genes resident in the nucleus of its host cell.

Not all of that genetic endowment is benign. Mitochondria inflict a select group of hereditary diseases, notably blindness due to degeneration of the retina. The pioneer German ophthalmologist, Theodor Leber (1840-1917), described Leber hereditary optic atrophy, Leber plexus and Leber idiopathic stellate neuroretinitis, which have a mitochondrial etiology, as does retinitis pigmentosum. He also named the much rarer Leber congenital amaurosis (LCA).

LCA causes nearly total blindness in infancy. (See BioWorld Today, July 24, 2000, p. 1.)

¿Diagnosis of this disease,¿ observed molecular ophthalmologist Jean Bennett, ¿usually begins when the baby¿s parents notice soon after its birth that their child has roving eye movements, and apparently doesn¿t see. If LCA is confirmed by eye examination, the patient is resigned to a life of special schooling, canes, seeing-eye guide dogs and Braille. The disease results in significant hardship and morbidity for the child, the family and society.¿

Bennett is an associate professor of ophthalmology at the University of Pennsylvania¿s Scheie Eye Institute in Philadelphia. She is senior author of a paper in the May issue of Nature Genetics, titled: ¿Gene therapy restores vision in a canine model of childhood blindness.¿

¿This study,¿ Bennett told BioWorld Today, ¿takes a great stride forward in demonstrating that gene therapy does not just slow down a retinal degenerative disease, but can actually provide recovery of vision to an animal that was previously blind. There is currently no treatment for LCA,¿ she pointed out, ¿but the disease ¿ and retinal degeneration in general ¿ are good candidates for gene therapy, because the photoreceptor cells, which are typically affected, are relatively intact, and accessible to injected agents.¿

Her animal model consisted of three large Briard dogs afflicted with the same LCA defects that blind people. Briards are long-haired dogs, the size of a small pony, originally bred in France.

¿Under optimum conditions,¿ Bennett explained, ¿normal protein transports a vitamin A-like compound to the retina, which is necessary for sight in some cases of animal and human LCA. The evolutionarily conserved protein that delivers this compound, RPE65 ¿ retinal pigment epithelium ¿ is missing from its mutant gene, (RPE65), which results in blindness. The protein the normal gene encodes is abundantly expressed in RPE cells adjoining the neural retina. These cells provide nutrients to photoreceptor cells in the retina. They convert vitamin A into a chemical that combines with a molecule to form rhodopsin ¿ the visual purple¿ light-sensitive pigment of the eye.¿

Bennett and her co-authors used recombinant adeno-associated virus (AAV), a common gene-therapy vector, to inject a normal copy of the RPE65 cDNA gene into the unilateral (right-side) retinas of three Briards that suffered from early and severe visual impairment similar to that seen in human LCA. A fourth LCA animal received no treatment, as a control.

¿Following the therapy,¿ she said, ¿measurements of vision indicated that the treated eye responded almost as well as eyes of seeing dogs. Specifically, 90 days after the vector injection, the waveforms of the treated eyes were similar, but not identical, in scope to those of sighted dogs. They were reduced in amplitude, according to the dose of the viral vector.

¿Four months after treatment,¿ she went on, ¿the dogs were also tested for such behavioral tasks as avoiding obstacles in dim light. The treated canines all avoided collision with objects in front and to the right ¿ the side injected ¿ yet consistently bumped into objects to the left. In contrast, the untreated control canine did not display avoidance behavior in any direction.¿

Bennett made the point that ¿a logical question leading from our results is whether sub-retinal injection of AAV-RPE65 would also correct the functional defects found in humans with LCA due to RPE65 mutations.

¿There are a number of important steps that must be taken before we can initiate human clinical testing,¿ she said. ¿Primarily, we have to determine how long term the therapeutic effect is, and make sure that there are no toxic side effects. And along with this, determine the optimal dose for treatment. These steps are in progress.¿

Describing the co-authors¿ in vivo proof-of-concept study as ¿the first successful gene therapy that restores sight in blind dogs,¿ Bennett concluded, ¿The results are spectacular ¿ in fact they are the sort of findings that a scientist usually only hopes to ¿ but rarely does ¿ see in the course of a career.¿

Retinal Pigment Epithelium Protein, PEDF, Curbs Diabetic Retinopathy Macular Degeneration

Scientists at the Wilmer Eye Institute in Baltimore, and at GenVec Inc., of Gaithersburg, Md., reported Wednesday, May 2, 2001, that ¿Increased expression of pigment epithelium-derived factor [PEDF] inhibits ocular neovascularization.¿ They presented this finding to a session on ¿Transgenic approaches to study ocular angiogenesis,¿ at the annual meeting of ARVO ¿ the Association for Research in Vision and Ophthalmology, in Fort Lauderdale, Fla.

Their results demonstrated that PEDF administered in the eyes of transgenic mice, ¿using GenVec¿s proprietary PEDF gene delivery technology, achieved substantial reduction in new blood-vessel formation in these preclinical models of diabetic retinopathy and macular degeneration,¿ GenVec¿s vice president of clinical research, Henrik Rasmussen, told BioWorld Today.

¿PEDF is normally produced by cells in the retina¿s RPE cells,¿ he noted. ¿It¿s a potent inhibitor of vascularization ¿ 30 to 100 times more potent than endostatin and angiostatin. Patients with diabetic retinopathy and macular degeneration suppress PEDF levels. Giving it as a gene construct, we are able to get long-term expression of PEDF in the eye.¿

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