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Survival Factors as Potential Therapeutic Agents for Retinal Degenerations: Status and Prospects
Digital Journal of Ophthalmology 1999
Volume 5, Number 4
July 1, 1999
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Matthew M. LaVail | Beckman Vision Center and Neuroscience Program, University of California, San Francisco
Roy H. Steinberg | Beckman Vision Center and Neuroscience Program, University of California, San Francisco

Abstract
Objective
A number of years ago it was found that the subretinal or intravitreal injection of basic fibroblast growth factor (bFGF) could slow the rate of degeneration in an inherited retinal dystrophy in rats [1]. Soon thereafter, we found that bFGF also protected photoreceptors FROM the damaging effects of constant light [2]. Using this model, we then found that a number of growth factors, neurotrophins and cytokines also protected the retina FROM light damage. These agents included acidic FGF, neurotrophin-3, brain derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CNTF) and several others [3]. Functional protection as measured by electroretinography using various survival agents (also referred to as "rescue") has been seen in the light damage model using bFGF, [4] as well as in preliminary observations with transgenic rats with rhodopsin defects [5]. These findings raise the possibility that survival factors might be used to protect photoreceptors in age-related and inherited retinal degenerations in humans.

What general issues need to be addressed to determine if survival factors cam be used clinically, and if so, what is needed to make them effective therapeutic agents? If the factors should prove to be ineffective, then we may ask if they are useful when combined with other agents, or as adjuncts to other therapeutic approaches. These topics form the basis of this report.
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Discussion
Efficacy
The first issue is efficacy. Do the agents slow retinal degenerations in models other than the RCS rat and light damage, both for which there are no known human counterparts? In fact, we have been able to slow the progression of photoreceptor degeneration with various survival factors in two species, which comprise four different mutants, that have the same or similar gene defects found in human patients with retinitis pigmentosa. These include the retinal degeneration (rd/rd ) and Q344ter mutant rhodopsin transgenic mice [6], and P23H and S334ter mutant rhodopsin transgenic rats [5]. These findings, combined with the BDNF-enhanced regrowth of rod outer segments seen in the cat retinal detachment model by Fisher and colleagues [7] indicate that some of these agents may be efficatious in human retinal degenerations.

Toxicity
Another problem is toxicity. In our initial study on RCS rats using bFGF as a protective agent [1], we found an occasional eye with a significant proliferative response, including epiretinal membrane formation. This was due presumably to the combination of an injection-induced injury and the presence of bFGF, a known mitogen and angiogenic factor. For this reason we sought agents that had more specific neuronal survival-promoting activity, such as the neurotrophins and certain cytokines and other growth factors [3]. Thus far, we have not seen any consistently harmful side effects with single intravitreal injections of most of the survival factors, and this includes injection of some of the agents at very high concentrations [8]. In preliminary studies, where agents have been injected intravitreally multiple times over a number of weeks in rat eyes, cataracts have been seen in some of the eyes, but not in all. Moreover, some of the rats’ eyes that received PBS buffer in multiple injections also developed cataracts. Thus, the issue of toxicity of survival factors has not been resolved and remains to be critically studied.

What if survival factors were shown to be effective for the treatment of human retinal degenerations, yet produced a significant side effect with high incidence, such as cataract formation? Would this preclude their use? There obviously would be a debate over the use of the factors. They would potentially save vision in heretofore untreatable diseases, but they might require surgery to REPLACE a cataract with an intraocular lens implant. Hopefully, this complication or dilemma will not appear when formal toxicology studies are completed on prospective survival factors.

Delivery
Delivery of drugs is one of the most significant problems to be solved when contemplating survival factor therapy. The survival-promoting factors identified thus far are peptides that do not cross the blood-retinal barrier. This barrier must be bypassed, as we have accomplished with intravitreal injection. Unfortunately, the biological and pharmacological half-lives of the agents in the eye are short relative to the time course of the diseases in which they might be used. We have found, in preliminary studies, that BDNF and CNTF can protect the retina FROM constant light for perhaps up to 15 days after a single injection, and a single injection of bFGF protects the RCS rat retina for up to two months [1]. However, macular degeneration and retinitis pigmentosa progress over the course of years to decades, so if intraocular injection were the only possible method of delivery, many injections at relatively short intervals would be required. It is likely that this therapeutic approach would not be feasible. For those reasons, some form of sustained release of the factor within the eye will be needed.

Is sustained release of a survival factor feasible? With continuous release, the issue of toxicity becomes even more significant, since the potential for negative effects of any agent may possibly be greater with continuous application compared with a single injection. We recently carried out an experiment that provides a proof-of-principle for these issues — that it is possible to bypass the blood-retinal barrier with a sustained release system, with at least one survival factor that protects the retina FROM the damaging effects of constant light, and that continuous application is apparently not toxic to the retina. To address these issues, we induced lens fiber cells in transgenic mice to become a sustained release system for neurotrophin-3. We found that the retinas of adult animals were protected FROM the damaging effects of constant light [9]. Furthermore, the continuous release of the factor had no apparent negative cytological or functional effect on the retina, despite the expression of the peptide throughout retinal cytogenesis, FROM about embryonic days 11-12 through adult life. It should be noted, however, that some other agents, such as leukemia inhibitory factor, when expressed by lens fiber cells in the same transgenic mouse system do have significant effects on the developing retina [10]. Thus, each agent considered for sustained release in the eye must be tested for toxicity.

What methods might be used for sustained release of survival factors in the human eye? There are many different methods of sustained release that are currently being tested in various organ systems, including other areas of the nervous system such as the brain, spinal cord and peripheral nerves, and which might be adaptable to the eye. These include biodegradable polymers; refillable or replaceable capsules or other release devices; and mini-pumps of various sorts (descriptions of these can be found throughout the medical literature). The devices or mechanisms for release may use the peptide survival factors, themselves, or they may use encapsulated cells that have been immortalized and genetically engineered to produce and release the survival factors.

Still another approach to sustained delivery is the genetic transduction of cells within the eye or retina that would cause the production and release of survival factors. This has recently been done with the rd/rd mouse, in which an adenoviral vector was used with a CMV promoter that caused the transduced cells to produce the survival factor, CNTF. In this case, injection of the vector complex at day 12 (even after significant photoreceptor cell loss) protected many of the photoreceptors up to 18 days after the injection [11]. This demonstration reveals that the transduction of cells within the eye may prove to be a useful approach for sustained intraocular delivery of survival factors.

Combining Survival Factors with Other Agents
Survival factors, alone, may not be able to delay indefinitely the loss of photoreceptor cells in hereditary or age-related degenerations. Perhaps survival factors could combined with other agents that exhibit protective effects on photoreceptor cell survival in retinal degeneration, the combination being more powerful and/or longer lasting than either agent alone. These include antioxidants [12,13], calcium channel blockers [14], anti-inflammatory agents [15,16], as well as other sorts of agents such as vitamins, fatty acids and plant products that have received some attention in the medical and lay literature.

Adjunct to Cell or Tissue Transplantation
Transplanation is another newly developing potential therapeutic approach for retinal degenerations, such as those with photoreceptor-specific gene mutations, In all transplantation procedures, many donor and host cells die. For this reason, the use of survival factors at the time of transplantation should allow more transplanted cells to survive the procedure. This is one application where the biological and pharmacological half-lives of the known survival factors should not be limiting.

Adjunct to Gene Therapy
Yet another possible use for survival factors is as an adjunct to gene therapy. It may be that when genetic rescue is effected, the rescue may not be permanent, as in the case of transgenic photoreceptor rescue in rd/rd and rds/rds mice, as well as in studies using adenoviral vectors. In addition, the degree of gene expression in any gene therapy experiment may be less than optimal for complete rescue. In both of these instances, survival factors may provide the extra boost that would ensure a better or complete protection. A related recent experiment is the preliminary report by Bennett and co-workers [17]. They used adenovirus-mediated delivery of the protoonocogene, bcl-2, which delays the onset of the photoreceptor degeneration in rd/rd mice [18], in conjunction with bPDE, finding that co-delivery protects photoreceptors to a greater extent than bPDE delivery alone [17]. Thus, the effect of specific mutant gene-targeted therapy can be enhanced by a cell-death inhibiting gene product. We would hope that a survival factor, administered either directly or genetically, along with specific gene therapy would be similarly additive.

In conclusion, just 7-10 years ago, firm evidence did not exist for effective therapy in human degenerative diseases. Now, there are several possibilities. We speculate that any single approach may not be optimal for rescuing photoreceptors completely FROM degeneration, but a mixed approach might give greater protection. Either way, whether alone or in combination, we can now be hopeful that human retinal degenerations can be treated by these potential new therapies.
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Acknowledgements
Supported by NIH Research Grants EY01919, EY06842 and EY01429, Core Grant EY02162 and funds FROM the Foundation Fighting Blindness, Research to Prevent Blindness and That Man May See, Inc.
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References
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