Ординатура / Офтальмология / Английские материалы / Retinal Degenerations biology, diagnostics, and therapeutics_Tombran-Tink, Barnstable_2007

of disease-associated variation of the ABCA4 gene still represents a difficult task for ophthalmologists and geneticists, the ABCR400 microarray offers a diagnostic tool that can advance our knowledge substantially, specifically in genotype/phenotype correlation studies of STGD. The latter will facilitate the counseling of patients on their visual prognosis. This information will also enhance future therapeutic trials in patients with STGD (see below in Emerging Therapeutic Options).

FUNCTIONAL STUDIES OF ABCA4

The ABCA4 protein was first described in mid 1970s as an abundant component of photoreceptor outer segment disk rims (44,45). Hence, it was called a Rim protein for the next 20 yr. After the encoding gene, ABCA4, was cloned in 1997 and characterized as a member of the ABC transporter superfamily, it suggested a transport function of some substrate in photoreceptor outer segments (1,16). All-trans retinal, the isoform of rhodopsin chromophore, was identified as a potential substrate of ABCA4 by its ability to stimulate ATP hydrolysis by reconstituted ABCA4 protein in vitro, suggesting that retinal could also be the in vivo substrate for ABCA4 (46). Studies of Abca4 knockout mice fully supported this hypothesis, proposing ABCA4 as a “flippase” of the complex of all-trans retinal and phosphatidylethanolamine (N-retinylidene-phos- phatidylethanolamine) (47). The most recent experimental data from Dr. Molday’s laboratory fully confirmed this hypothesis (48).

Mice lacking the functional Abca4 gene demonstrated delayed dark adaptation, increased all-trans retinal following light exposure, elevated phosphatidylethanolamine (PE) in rod outer segments, accumulation of the N-retinylidene-PE, and striking deposition of a major lipofuscin fluorophore (A2E) in RPE. Based on these findings, it was suggested that the ABCA4-mediated retinal degeneration may result from “poisoning” of the RPE owing to A2E accumulation, with secondary photoreceptor degeneration due to loss of the RPE support role (47). A2E, a pyridinium bis-retinoid, which is derived from two molecules of vitamin A aldehyde and one molecule of ethanolamine, has been characterized as one of the major components of retinal pigment epithelial lipofuscin (49). Accumulation of lipofuscin in the macular region of RPE is characteristic to aging eyes and is the hallmark of both STGD1 and age-related macular degeneration (AMD) (Fig. 1).

Together, these data define ABCA4 as the “rate keeper” of the retinal transport in the visual cycle. ABCA4 is apparently not absolutely essential for this process, as individuals completely lacking the functional protein (e.g., some patients with RP-like phenotype) maintain some eyesight for several years. Over time, however, even mild dysfunction of ABCA4 affects the vision irreparably.

EMERGING THERAPEUTIC OPTIONS

The scientific progress in determining the role of the ABCA4 gene in STGD and other retinal pathology has been remarkable. We have significantly expanded our knowledge of the extensive range of phenotypes caused by various combinations of ABCA4 mutations. ABCA4 research has lead to the screening of thousands of patients with STGD, encompassing large cohorts of ethnically diverse samples. We also know ABCA4 function as the transporter of N-retinylidene-PE, and have a mouse model that reproduces

some features of the human disease. Considering all the above, our efforts should now be directed towards finding therapeutic solutions to benefit patients with STGD. I will discuss the available options and most recent advances in this area below (Fig. 2).

Modifying Exposure to Environmental Factors

Avoiding Sunlight to Delay the Disease

Experimental and clinical data have documented the damaging effects of light exposure on photoreceptor cells and several lines of evidence point to retinoids or retinoid derivatives as chromophores that can mediate light damage. Studies from Jeremy Nathans’ laboratory have concluded that ABCA4 is unusually sensitive to photooxidation damage mediated by all-trans-retinal in vitro (50). Specifically, it was demonstrated that photodamage to ABCA4 in vitro causes it to aggregate in sodium dodecyl sulfate gels and results in the loss of retinal-stimulated ATPase activity. These observations suggested ABCA4 and several other photorecepter (PR) outer segment proteins as targets of photodamage. This is especially relevant to assessing the risk of light exposure in those individuals who already have diminished ABCA4 activity owing to mutation in the ABCA4 gene, such as patients with STGD.

Moreover, studies on Abca4-/- mice (47) suggested that A2E did not accumulate in RPE of animals kept in the dark. Therefore, patients with STGD can be advised to avoid light exposure by wearing (ultraviolet-blocking) sunglasses and limiting the exposure of eyes to direct light. However, the effectiveness of this approach has not been demonstrated in epidemiological studies, nor is it likely to be conductive to a productive and satisfying lifestyle.

Limiting Dietary Intake of Vitamin A

Based on our knowledge of the ABCA4 function in the visual cycle, a sound recommendation to all patients with ABCA4-associated pathology would be to limit the dietary intake of vitamin A. Vitamin A has been recommended to patients with RP51 and also as a dietary supplement for patients with AMD (52). However, in a substantial fraction of patients with both disorders, the disease phenotype is caused, at least in part, by ABCA4 mutations. Elevated intake of vitamin A by these patients would only worsen the disease prognosis by stimulating the visual cycle and, consequently, increasing the accumulation of A2E in RPE with predicted grave consequences. This observation further justifies thorough diagnostic screening of patients for underlying genetic variation to pinpoint the molecular cause and/or mechanism of the disease before recommending therapeutic intervention.

Drugs Modifying Functional (ATP-Binding and Hydrolysis) Activity

of ABCA4

The in vitro assay developed by Hui Sun and coworkers (46) suggested several compounds that either enhanced or diminished the (ATPase) activity of the ABCA4 protein. Small-molecule drugs, synergistically enhancing the ATPase activity of ABCA4, may represent potentially beneficial compounds for patients with STGD and/or for a subset of individuals at risk for AMD. Although clear predictions regarding the in vivo effect on ABCA4 function of compounds like amiodarone and digitonin are currently impossible,

the existence of such compounds suggests that environmental and/or drug effects may be relevant to ABCA4-associated degenerative retinal diseases. In this context, Sun et al. suggested to study visual function in patients receiving amiodarone, an Food and Drug Administration-approved drug that has been widely used in the treatment of cardiac arrhythmias at tissue concentrations at 40 M or greater (46).

Drugs Slowing Down the Visual Cycle

The vertebrate visual cycle consists of enzymatic reactions that contribute to the generation of 11-cis retinal, the visual chromophore. One consequence of the constant recycling of vitamin A, as described here previously, is the formation of the fluorophores, such as the bi-retinal conjugate A2E, its photoisomer iso-A2E, that constitute the lipofuscin of RPE cells (53–58). Because of its unusual pyridinium bis-retinoid structure (59), A2E cannot be enzymatically degraded and, thus, accumulates in RPE cells of patients with STGD and Abca–/– mice (47). At sufficient concentrations, A2E perturbs cell membranes (49,60), confers a susceptibility to blue light-induced apoptosis (61–63), and alters lysosomal function (64). In light of this adverse behavior, there is considerable interest in retarding A2E formation as a means to prevent vision loss in STGD and perhaps in AMD. Therefore, it is significant that studies have shown that RPE lipofuscin is substantially reduced when the 11-cis and all-trans-retinal chromophores are absent either because of dietary deficiency or gene knockout (65,66). Light exposure, an obvious determinant of the rate of flux of all-trans-retinal through the visual cycle, can also moderate the rate of A2E synthesis (57,58,67). In addition, Radu et al. (68) reported that isotretinoin (13-cis-retinoic acid), an acne medication Accutane known previously to delay dark adaptation (69), dampens the deposition of A2E in RPE cells. Although heralded as a potential therapeutic agent for STGD, Accutane (i.e., retinoic acids) cannot be used for chronic treatment of patients as a result of the numerous systemic side effects associated with the use of these drugs, including teratogenic effects, depression, birth defects, dryness of mucosal membranes, and skin flaking (70). Therefore, identification of other, better-targeted compounds, which will slow down the visual cycle with minimal and/or acceptable side effects (such as mild night blindness), could lead to the availability of treatment options for patients with juvenile-onset macular dystrophies and possibly to preventive patient care.

Possible protein targets of these compounds, similar to Accutane, could include 11- cis-retinol dehydrogenase (71), or RPE65 (72). Recent studies have demonstrated that slowing the kinetics of the visual cycle curtails the accumulation of A2E (73). These results also point to RPE65 as a rate-limiting step in A2E formation that can be therapeutically targeted. Therefore, a class of compounds that could provide a specific, controlled inhibition of the visual cycle includes antagonists of the physiological substrate of RPE65, all-trans-retinyl esters. (74) Furthermore, the fact that RPE65 is essentially unique to the visual system strongly suggests that its inhibitors are unlikely to be generally toxic.

Gene Therapy

Although the therapeutic applications discussed here previously would only delay or modify the disease progress, the gene therapy approach should provide “the cure” for

STGD and other ABCA4-associated diseases. Because all ABCA4-associated diseases, including STGD, are recessive, introducing a normal, functioning copy of the gene to photoreceptors would restore visual function. Another advantage of the gene therapy approach in the specific case of STGD relies on the fact that degeneration of the retinal cells in this disease is relatively delayed, allowing a reasonable time window for therapeutic intervention.

The Abca4 knockout mice described earlier allows testing various gene therapy approaches in vivo. The extent of the restoration of the retinal function in the mouse model can be judged by a combination of biochemical and functional tests, such as noninvasive electroretinogram and quantitation of A2E accumulation either by highperformance liquid chromatography or autofluorescence. The functioning human ABCA4 gene can be introduced into mouse photoreceptors via several techniques, the most proven and robust of which are those utilizing viral vectors, which efficiently transduce photoreceptors. The two main groups of viral vectors used in gene therapy to date are adeno-associated viruses (AAV) and lentiviruses. Lentiviral vectors offer several key advantages in the specific case of ABCA4 (75,76). First and foremost, lentiviruses are capable of delivering genes stably and permanently into the genome of infected cells in vivo. Second, they can transduce nondividing cells, a crucial requirement for terminally differentiated cells such as photoreceptors. Third, they can carry large inserts, a distinct advantage over AAV-based vectors because the coding region of the human ABCA4 gene is extraordinarily large (>6800 bp), exceeding the capacity of AAV.

Although the efficiency of transducing photoreceptors by lentiviruses has been a certain concern, several studies, e.g., those from the laboratory of Inder Verma (77) utilizing HIV-based vectors, allow substantial optimism. Moreover, the photoreceptor transduction efficiency could be further increased by using vectors on the equine infectious anemia virus backbone (78), especially those pseudotyped with the rabies virus envelope protein, which has showed greatly improved neurotropism (79).

Alternatively, AAV-based vectors, which have demonstrated high PR-tropism, could be utilized. In this case, however, the ABCA4 gene has to be delivered to the same photoreceptor cell in two clones because of the capacity constraints. Subsequently, the functional ABCA4 gene can be reassembled by either trans-splicing technique (80) or the functional ABCA4 protein can be reconstituted from the two, structurally almost identical, ABCA4 domains (81). The well-publicized success in restoring vision in the dog model of Leber congenital amaurosis by introducing the functional RPE65 gene via AAV vectors (82) allows for optimism in other recessive retinal diseases, such as STGD.

OUTLOOK

The scientific progress in determining the role of the ABCA4 gene in STGD and in overall retinal pathology has been remarkable. We have significantly expanded our knowledge of the extensive range of phenotypes caused by various combinations of ABCA4 mutations, and have efficient diagnostic tools, such as genotyping arrays. ABCA4 research has lead to the formation of multicenter studies, encompassing large cohorts of ethnically diverse samples. Knowledge of the ABCA4 function as the transporter of N-retinylidene-PE, and availability of the mouse model that reproduces several features of the human disorders have allowed rapid advancement to the next

stage of research directed towards finding therapeutic solutions for ABCA4-mediated retinal disease. One, or a combination, of the therapeutic options discussed in this chapter should be available relatively soon for application toward the therapy of the entire spectrum of ABCA4-associated retinal disorders, which were most recently considered incurable.

ACKNOWLEDGMENTS

The author sincerely appreciates helpful discussions and useful suggestions from many colleagues and collaborators over the years of STGD and ABCA4 research. Support by National Eye Institute/National Institutes of Health, Foundation Fighting Blindness, and Research to Prevent Blindness is gratefully acknowledged.

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