Human genome map beginning to pay dividends in ophthalmology

The release of the map of the human genome was meant to usher in a new wave of medicine.

A working copy of the map was released in 2000, and the nearly complete code was released in 2003. Although the potential of genetic research and testing has been widely publicized, successes in applying this technology to improve diagnosis and to develop new treatments, such as gene therapy, have been hard to come by.

“There has been a lot of interest in the area of gene therapy because it is potentially very exciting, but also because it has faced a number of challenges,” James W.B. Bainbridge, PhD, FRCOphth, consultant ophthalmologist at Moorfields Eye Hospital, London, said. “The number of definitive successes in gene therapy have been relatively few.”

However, that may change soon. Recently, Dr. Bainbridge led a team investigating the therapeutic potential of genetic code delivered to damaged retinal cells of patients with Leber’s congenital amaurosis (LCA) type 2 with an adeno-associated viral vector.

The trial was a phase 1 safety study, so it warrants only cautious optimism. But the study showed that the eye may be an ideal medium for genetic research. The eye’s natural qualities — and the fact that there are a variety of ocular disorders with genetic components, ranging from classic Mendelian monogenic disorders to complex heterogeneous disorders — may make it a “proof of principle” concept for gene replacement therapy. The discoveries made in the eye may someday translate into other therapeutic areas.

James W.B. Bainbridge, PhD, FRCOphth, is consultant ophthalmologist at Moorfields Eye Hospital, where researchers are optimistic about the potential for ocular gene therapy. Image: Bainbridge JWB

Research in ocular genetics also has clinical utility because at the end of all the research is the patient and the promise that genetics may ultimately be a boon to clinical practice.

“What the LCA study does show is that in a particular condition that was previously considered untreatable, we have found that gene therapy can help,” Dr. Bainbridge said.

LCA studies

Before the publication of the studies on the use of adenovirus-associated vector manufactured with the genetic code for the human RPE65 gene in patients with LCA, gene transfer had been attempted only twice in human ophthalmology.

The three LCA reports – two in New England Journal of Medicine and one in Human Gene Therapy – were the initial findings in nine patients involved in separate phase 1 safety and dose-ranging studies.

“Our study, and those conducted in the United States, offers a strong proof of principle that gene replacement therapy can improve vision in this particular disorder,” Dr. Bainbridge said. “The implications are that similar approach may have further benefit over a wide range of disorders.”

LCA was specifically chosen as a target of the research. The hallmark retinal degeneration of LCA typically does not occur until the third generation of life; it is almost certain to progress into worse disease, but patients in their teens or 20s have relatively stable retinal structure. Early intervention offers an opportunity for demonstrable improvement in visual function.

“I think this is likely to be most effective when performed in a relatively early stage in the condition, so that is young people because there is often a progressive degeneration that will limit the degree to which the retina can benefit from gene replacement,” Dr. Bainbridge said.

According to Dr. Bainbridge, there is the potential to restore functionality to the well-described molecular chain of events that leads to the phenotypic pathology caused by a single gene defect in LCA. Specifically, a point mutation in the RPE65 gene, which occurs in roughly 6% of LCA patients, is known to stop downstream protein production in a chemical pathway that helps regenerate visual pigment after exposure to light.

Malfunction in this process results in degenerating rod cells. Cone cells have an alternate pathway for chromophores essential for light absorption, which explains cone-driven visual function in children with LCA. However, continued degeneration of cone cells eventually leads to late-stage blindness.

Although not specifically powered for determining visual function, each study suggested some biological activity in treated eyes. Of greater significance, Dr. Bainbridge said, none of the three trials reported any serious adverse events or systemic complications. While this was an early phase 1 study, proving the safety of the procedure was an important first step, as well as a potential harbinger of what can come.

“The results are limited, but to be able to demonstrate a positive result in gene research has been a challenge,” Dr. Bainbridge said. “The three studies all address a similar question in similar groups of patients using similar vectors, but with some slight differences in terms of promoter sequence and surgical approach used. The information, the data that is generated from these trials is in many ways complementary to each other.”

Why the eye?

According to Dr. Bainbridge, the anatomy and architecture of the eye are uniquely situated for gene-based research.

“[The eye] is relatively small, so we can use small volumes of vector, which is expensive to produce. It is highly compartmentalized, which means we can target specific parts of the eye using the microincision techniques we have available,” he said. “The cells in the eye are relatively stable, so if we can transfuse them or infect them with the vector, we can expect the effect to last for a long time.”

Additionally, vectors used in gene therapy in the eye theoretically have less chance of making it into the bloodstream, and the optic media is transparent, improving the ability to monitor both therapy effects and local reaction. All of this means that the eye is an ideal environment for genetic therapy.

“If you want to inject certain genetic constructs where a gene is manipulated, the eye is a good place to start because it is totally isolated from the rest of the body, in terms of immune response. You can also do a good phenotypic assessment to evaluate the outcome of the genetic manipulation,” Hemin R. Chin, PhD, ocular genetics program director at the National Eye Institute (NEI) of the National Institute of Health, Division of Extramural Research, said.

Hemin R. Chin

Under the direction of Paul Sieving, MD, PhD, the NEI has taken a great interest in genetic research over the past decade across multiple ophthalmic disciplines. The NEI’s objective, according to Dr. Chin, is to understand how the molecular etiology of ocular pathologies can reveal new targets and new therapeutic options, which will ultimately foster the discovery of the next wave of therapeutics.

“Through gene research, we hope to come up with more targeted drug compounds that can be used for certain kinds of diseases, and we are going for very specialized, well-targeted diseases based on the knowledge we are gaining from genetics research. Hopefully, some day, we will be able to come up with a gene replacement or cell-based therapy for the complex diseases of the eye,” Dr. Chin said.

In addition to funding several research activities, the NEI established genotype-phenotype databases that allow researchers to mine for data rather than have to go through the arduous and expensive task of collecting data samples.

Understanding the variable factors that influence a patient’s susceptibility to a given drug may allow researchers to design smaller clinical studies with specific target populations. As well, phenotype mapping and genotype-phenotype correlation will help researchers identify the next targets in gene research.

“More often than not, the discovery of and identification of genes and genetic variation is not the end, but the first step in our understanding of the genetic basis of a disease,” Dr. Chin said.

The knowledge gained in monogenic disorders such as LCA may someday be useful in addressing complex pathologies.

“We are moving towards a greater understanding of complex eye diseases based upon our knowledge that we accumulated in studying monogenic eye disease,” Dr. Chin said.

Common diseases vs. uncommon mutations

In addition to the more rare diseases caused by classic Mendelian mutations, there are a host of more common ocular pathologies that may be attributed to several single nucleotide polymorphisms (SNPs). These SNPs are present in 100% of the population. Recognition of certain patterns of SNPs distributed among the human genome led to the success of the next phase of human genetics with the International HapMap Project. Understanding which particular SNPs correlate to function in health and disease may be the next obstacle in genetic research.

“Genes provide the architectural blue print of a cell, then an organ and ultimately the organism. But a fundamental question is, how do these genes manifest themselves in terms of cellular and organ function?” Sayoko E. Moroi, MD, PhD, an associate professor of ophthalmology and visual sciences and a genetic researcher at the Kellogg Eye Center of the University of Michigan, said.

Sayoko E. Moroi

At present, gene markers for common ocular pathologies are a moving target, with a conservative estimate of current gene markers for specific ocular diseases occurring only in about 5% to 20% of the population, according to Dr. Moroi. As researchers identify more gene markets for specific ocular diseases, there will be many more questions raised to determine whether or not these specific genes can be used to predict with certainty disease onset and disease severity, both of which are clinically relevant for assessing prognosis. Furthermore, other gene markers may potentially allow researchers to predict treatment outcomes in terms of both efficacy and adverse outcomes.

“Once you identify a strongly associated SNP with your disease of interest, then you start a new series of research questions. Some questions include, is the gene regulation changed? Is the protein expression changed? How do these change cell function? What is the impact of the environment on that change?” Dr. Moroi said.

Gene mutations do not occur in a vacuum. A change in an individual’s genetic structure may have consequences to the function of that gene and any byproduct of that code. However, once that structure-function correlation is determined, it can have profound clinical consequences. Dr. Moroi pointed to the use of genetic testing to determine a patient’s sensitivity to the anticoagulant warfarin as an example.

With the aid of the results from the human genome and the HapMap projects and through traditional genetic approaches with genotype-phenotype correlations, researchers have identified several gene mutations in the genetic code that are associated with ocular disorders. The next step, according to Dr. Moroi, will be to explain how those structural gene changes affect cell function so that future research can design agents that intervene in that process.

“We have the gene structure, but how does that impact the cell function, the organ function, the organism function?” she asked. “That is why we have to understand proteomics, or the proteins that come from those gene codes, and then how the proteins play a role in signal pathways, and that’s metabolomics.”

Molecular medicine

One example of the potential of molecular medicine and the integration of genetics, proteomics and metabolomics can be found in research on age-related macular degeneration. In a 2006 issue of Human Gene Therapy, Peter A. Campochiaro, MD, and colleagues described an adenovirus vector called AdPEDF (GenVec) that stimulated local protein production of human pigment epithelium-derived growth factor (PEDF) in retinal cells.

The protein PEDF has anti-angiogenic properties, including promoting cell death in endothelial cells undergoing neovascularization as well as possibly acting as an inhibitor of several downstream angiogenic factors, including VEGF. Although it is not fully proven in humans, the protein may also have neurotrophic activity.

In the study, 28 patients with advanced wet AMD received a single intravitreous injection of AdPEDF in several different doses. There were no serious adverse events reported and no dose-limiting toxicities.

In addition, there was some suggestion of biological activity in terms of visual function, according Dr. Campochiaro, a professor of ophthalmology and neuroscience at the Wilmer Institute of Johns Hopkins University School of Medicine, Baltimore. Fluorescein angiograms and optical coherence tomography findings suggested that increased expression of PEDF may potentially have some beneficial effects in the setting of advanced neovascular AMD, he said. Fundus images showed resorption of subretinal fluid and blood in patients receiving higher doses compared with patients receiving one of the lower doses.

As well, “in the high doses compared to the low doses, patients had either a decrease in size or stabilization of their choroidal neovascularization measured in a reading center with completely masked grading, and there was a tendency toward better maintenance of visual acuity,” Dr. Campochiaro said.

One of the inherent theoretical shortcomings of the vector used in the study was that it was an adenovirus, which is known to have a short duration of expression. However, in the study, patients treated with higher doses appeared to demonstrate more prolonged stabilization of lesion size, raising the possibility that the duration of adenoviral vector-mediated expression might be longer in human compared with rodent eyes. Nonetheless, studies with adeno-associated virus or lentiviral vectors would be useful and informative, he said.

The implications of the biological activity and the extent of duration would have to be elucidated in larger and longer studies, according to Dr. Campochiaro, but the early results point to a preliminary demonstration of proof-of-concept for ocular gene therapy for ocular neovascular diseases.

“The importance of that study is that it suggests it is feasible to express anti-angiogenic proteins in the eye, they may have biological effects, and, therefore, using different viral vector platforms, such as AAV or lentiviral vectors, that a long-term effect may be achieved,” Dr. Campochiaro said.

Commercial potential

According to statements on GenVec’s Web site, an additional 22 patients with less severe AMD have been administered AdPEDF, and while data mining is still ongoing in these patients, “very much like the first 28 patients, no dose-limiting toxicities or drug-related severe adverse events were observed.”

As a commercial product, PEDF could have potential because of the protein’s possible neuroprotective properties, which could be useful in other ocular pathologies such as diabetic retinopathy, diabetic macular edema, dry AMD, glaucoma and retinitis pigmentosa.

But translating the therapeutic possibility of AdPEDF to reality may be an uphill battle because the AdPEDF study was done before the widespread adoption of Lucentis (ranibizumab, Genentech) and Avastin (bevacizumab, Genentech). According to J. Timothy Stout, MD, PhD, MBA, vice president at the Oregon Health and Science University and one of the co-investigators in the AdPEDF trial, any new therapeutic option for AMD, even one based in genetics, must prove equivalence or superiority to current treatments.

J. Timothy Stout

“The bar is a little different now than it was pre-Avastin or Lucentis,” Dr. Stout said.

Timing of dosing is another consideration. The AdPEDF trial employed an adenoviral vector and is, therefore, expressed only transiently.

“The transgene is only expressed for about a month, maybe 2 months. If you have blood vessels growing and you are able to inhibit that growth by the genetic expression of PEDF, but it’s only going to work for a month, are you going to have to do monthly reinjections? We do not know,” Dr. Stout said.

A possible solution is to use lentivirus, a known integrating vector. It may be possible to package the genetic code for PEDF, or any other anti-angiogenic protein, into a lentivirus and extend the duration of effect.

“Perhaps we could inject a virus so that the therapeutic gene could become a stable integrant into the host cell’s DNA, and that recipient cell would make the anti-angiogenic protein forever,” Dr. Stout said.

In a study in primates receiving a single injection of an anti-angiogenic gene packaged in lentivirus, or lentivirus without the gene or saline, there was less neovascularization induced by laser in animals receiving the anti-angiogenic gene. More importantly, Dr. Stout said, that effect persisted in animals re-exposed to laser 6 to 8 months after the initial injection.

“It was clear that there was an inhibitory effect in the animals that received the lentivirus that contained the anti-angiogenic gene. There was less neovascularization, and it subsided relatively quickly,” he said.

Other research by Dr. Stout relates to the VEGF expression pathway. Responding to an ischemic state, the body produces VEGF to indiscriminately generate new blood vessels. If those factors that trigger or inhibit VEGF expression can be identified, they potentially could be used to inhibit the synthesis of new VEGF protein and thus inhibit new blood vessel growth altogether.

According to Dr. Stout, regulators of the hypoxia/VEGF pathway have applications beyond AMD, as other retinal pathologies share a similar VEGF activation, including retinopathy of prematurity, diabetic retinopathy as well as vaso-occlusive diseases.

“These are clearly non-overlapping patient sets, but they have overlapping molecular mechanisms which drive their diseases,” he said. “Even though these are different patients, treating these patients with anti-VEGF drugs is potentially useful.”

Other research

Research currently being conducted in the field of ocular genetics is expanding what is possible in treating ocular disorders.

New hybrid vectors allow delivery of larger genetic sequences, expanding the catalog of genetic mediators available to researchers. With potential applications in disorders such as Stargardt’s disease, which may involve up to 19 mutations in the ABCA4 gene, and Usher’s syndrome, caused by mutations in any one of 10 genes, these new vectors are pushing the boundaries of what might be possible with genetic medicine.

Researchers at Trinity College of Dublin are investigating the potential of gene suppression technology that would stop disease code and allow healthy code to continue to function. Elsewhere, researchers are working on genotype-phenotype correlation in both rare Mendelian disorders and more common ocular pathologies.

Dr. Bainbridge said that further research in ocular genetics will reveal new therapeutic possibilities. Gene therapy is one potential application, but understanding genetics is expected to lead to the development of a huge field of medicine, especially in gene-directed therapy. – by Bryan Bechtel

What are the biggest impediments in translating genetic research to clinical applications?

References:

• Bainbridge JW. Prospects for gene therapy of inherited retinal disease [published online ahead of print Jan. 16, 2009]. Eye. doi:10.1038/eye.2008.412.
• Bainbridge JW, Ali RR. Keeping an eye on clinical trials in 2008. Gene Ther. 2008;15(9):633-634.
• Bainbridge JW, Ali RR. The eyes have it! Ocular gene therapy trials for LCA look promising. Gene Ther. 2005;15(17):1191-1192.
• Bainbridge JW, Smith AJ, Barker SS, et al. Effect of gene therapy on visual function in Leber’ congenital amaurosis. New Eng J Med. 2008;358(21):2231-2239.
• Campochiaro PA, Nguyen QD, Shah SM, et al. Adenoviral vector-delivered pigment epithelium-derived factor for neovascular age-related macular degeneration: Results of a phase I clinical trial. Hum Gene Ther. 2006;17(2):167-176.
• Hauswirth WW, Aleman TS, Kaushai S, et al. Phase I trial of Leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: Short-term results [published online ahead of print Sept, 7, 2008]. Hum Gene Ther.
• Maguire AM, Simonelli F, Pierce EA, et al. Safety and efficacy of gene transfer for Leber’s congenital amaurosis. New Eng J Med. 2008;358(21):2240-2248.
• Stout JT. Gene transfer for the treatment of neovascular ocular disease (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc. 2006;104:530-560.
• Wei L. Adenovector pigment epithelium-derived factor (AdPEDF) delivery for wet age-related macular degeneration. Retina. 2005;25(8 Suppl):S48-S49.

• James W.B. Bainbridge, PhD, FRCOphth, can be reached at Division of Molecular Therapy, Institute of Ophthalmology, University College London, 11-43 Bath St., London EC1V 9EL, United Kingdom; 44-020-7608-6889; fax: 44-020-7608-6991; email: j.bainbridge@ucl.ac.uk
• Peter A. Campochiaro, MD, can be reached at Johns Hopkins Hospital School of Medicine, Wilmer Ophthalmological Institute, Department of Ophthalmology, 719 Maumenee, 600 N. Wolfe St., Baltimore, MD, 21287-9277; 410-955-5106; fax: 410-614-7083; e-mail: pcampo@jhmi.edu.
• Hemin R. Chin, PhD, can be reached at National Eye Institute, 31 Center Drive, Building 31, Bethesda, MD 20892-2510; 301-496-2234; fax: 301-496-9970; email: hemin@nei.nih.gov.
• Sayoko E. Moroi, MD, PhD, can be reached at Department of Ophthalmology and Visual Sciences, University of Michigan, 1000 Wall St., Ann Arbor, MI 48105; 734-763-7974; fax: 734-615-0542; email: smoroi@umich.edu.
• J. Timothy Stout, MD, PhD, MBA, can be reached at Casey Eye Institute, 3375 SW Terwilliger Blvd., Portland, OR 97239-4197; 503 494-7891; email: stoutt@ohsu.edu.