Sight for the blind

First bioengineered cornea transplant

UNIVERSITY OF California Davis, School of Medicine and Medical Center scientists have restored or improved eyesight in 10 of 14 patients suffering from severe corneal damage, using a new technique in which replacement cornea tissue is grown in a laboratory dish. The cornea is the transparent outer coating of the eye that covers the iris and pupil. Before treatment all the patients had damage to their ocular surface, many with poor vision, and had failed standard treatments, including conventional cornea transplants. After the new treatment, 10 of the patients regained some or most of their sight.

Only two other bioengineered tissue replacements are currently commercially available: Bioengineered skin is widely used to treat burns and chronic skin wounds, and bioengineered cartilage is increasingly used to treat certain knee injuries. UC Davis researchers believe that bioengineered cornea could very well become the third type of bioengineered replacement tissue for humans that's available.

"We learned about skin, and then used that knowledge to create biological skin replacements for burn victims," says Dr. R. Rivkah Isseroff, professor of dermatology at the UC Davis School of Medicine and Medical Center. "Now we have transplanted that knowledge to the eye. In the future, we hope to translate it to the lung, gastrointestinal tract, bladder and other epithelial tissues throughout the body."Dr. Irvan Schwab says tissue engineers may be able to produce readily available, "off-the- shelf" cornea replacements that can be easily transplanted into patients with severe damage to the cornea, "Our research is a unique and important step toward that goal," he says.

In the technique pioneered by Drs. Isseroff and Schwab, a few corneal stem cells are first removed from a healthy cornea. If a patient has one good cornea, the stem cells are removed from that cornea. If both of a patient's corneas are damaged, stem cells are taken from the cornea of a related donor.

Corneal stem cells are the "mother" cells of the cornea; they lie deep within a protected area adjacent to the cornea, continually giving birth to new corneal cells to replace aging corneal cells, and to repair corneal injuries. (Corneal stem cells are distinct from fetal stem cells. Fetal stem cells possess the unique ability to develop into any tissue in the body. Corneal stem cells, in contrast, can produce only corneal cells).

Severe injuries to the cornea, such as a chemical, fire or radiation burn, can destroy corneal stem cells. So can certain diseases that affect the eye, including some tumors and a handful of rare disorders such as pemphigoid, an autoimmune condition, and Stevens-Johnson syndrome, which results from a severe allergic response. Without its stem cells, the cornea loses its ability to repair itself, and can become scarred and opaque, causing blindness.

Conventional corneal transplantation, in which a superficial layer of donor cornea is placed over a damaged cornea, does not transfer enough stem cells to be sufficient in these cases. In the UC Davis technique, the harvested corneal stem cells are divided among multiple laboratory dishes. In the dishes, the stem cells produce a fragile film of corneal cells just one cell thick. Scientists transfer the corneal cells, including the surviving stem cells, to the surface of a matrix of sterile amniotic membrane. Some cells from the other dishes are frozedn and banked for possible later use.

On the matrix, the corneal cells grow into a layer 5 to 10 cells thick, forming a sturdy composite tissue that combines the elasticity and resilience of amniotic membrane with the biological properties of corneal tissue. This bioengineered composite tissue is then stitched onto the patient's eye, after the abnormal corneal tissue has been removed. Amniotic membranes have been widely used in tissue engineering and corneal repair for many years. The membranes, which are acquired by donation from mothers after babies are born, make an ideal matrix because they do not trigger an immune reaction in the recipient.

The stem cells are harvested by corneal biopsy in a quick, painless procedure that poses minimal risk to the donor eye. Dr. Schwab emphasized that the transplants remain investigational, and for now are appropriate only for patients with corneal problems that have not responded to more conventional therapies. More needs to be known about the transplants' long-term success and risks before the procedure can be considered as a first-line treatment, he says.

The number of patients who might benefit from corneal stem cell transplant in this country is relatively small. Only a few thousand people in the United States suffer diseases or injuries each year that cause devastating cornea damage leading to vision loss. The numbers are much greater in developing nations, however, where infectious diseases of the eye remain common.

But the technique may have exciting applications outside ophthalmology. Corneal tissue, like skin, is an example of epithelial tissue, or epithelium. In fact, more than 60 percent of the cell types in the human body are epithelial cells.

We learned about skin, and used that knowledge to create biological skin replacements for burn victims," Dr. Isseroff says. "Now we have transplanted that knowledge to the eye. In the future, we hope to translate this knowledge to the lung, the gastrointestinal tract, the bladder and other epithelial tissues throughout the body." "The really exciting thing is where this can take us," Dr. Schwab agreed. "Replacing diseased tissues and organs with bioengineered tissue is rapidly moving from the realm of science fiction to reality."

- Cornea

Success with mice

SOME CHILDREN born blind may one day see. Mr. Krzysztof Palczewski of the University of Washington, and Mr.Samuel Jacobson of the University of Pennsylvania, and colleagues, have restored vision in sightless mice, genetically engineered to model a rare, incurable heritable condition called Leber congenital amaurosis (LCA).

LCA sufferers lose their sight at birth or very early in childhood. Normally, vitamin A travels to certain pigment cells in the eye's retina.These 'RPE' cells convert dietary vitamin A into a form that the retina can use.

One cause of LCA is a mutation in the gene that codes for the protein `RPE65'. Without this protein, a part of the biochemical pathway of sight, called the visual cycle, becomes blocked causing blindness. Mutations in this gene account for nearly ten percent of all LCA cases.

Mr. Palczewski and Mr. Jacobson fed mice lacking the RPE65 protein with a chemical derivative of vitamin A. The group report that the animals' visual functions improved to almost normal levels after just 48 hours; untreated mice remained blind.

Mr. Gerald Chader of The Foundation Fighting Blindness says, "that Palczewski and Jacobson were able to restore lost vision in an animal model with a severe retinal degenerative disease offers hope.

With advances in genetic research we are at last able to understand the causes of vision loss and develop treatments that overcome a genetic defect."

RPE65 is thought to make 'visual vitamin A'. In the visual cycle this form of vitamin A binds to the protein opsin, to make rhodopsin. When light falls on the cells, it bleaches rhodopsin back into opsin, sending an electrical message to the brain.

At this point a new visual vitamin A molecule binds to opsin and the cycle beings again, allowing cells to process light over and over again. Without rhodopsin, light falling on the eye goes unrecognised.

Rpe65-blind mice cannot make the visual form of vitamin A and therefore rhodopsin, so the researchers reasoned, that by providing a visual vitamin A substitute, they could by-pass the blockage in the visual cycle.

Their alternative vitamin A, which makes a different form of rhodopsin, created an artificial but fully functional visual cycle``Dramatic reversal of retinal dysfunction in such disorders is a rarity. Our experiments that reverse dysfunction in the Rpe65-deficient mouse prompt a multitude of questions," says Mr. Palczewski.

The researchers are now hoping to test the drug on dogs that lack RPE65. Before they can try it on humans they must ensure that the vitamin has no harmful side effects.

But as one LCA patient notes, "this particular treatment will only be useful for a small portion of patients with LCA who carry this specific mutation." "Nevertheless, the observation should lead to more work on the pathogenes is of inherited diseases at the cellular level," says Alan Bird of the Moorfields Eye Hospital, London, UK.

"There is clear justification to initiate a trial if it is shown that treatment of the rodent model has a sustained effect."

- Proceedings of the National Academy of Science