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

Anecortave Acetate (Retaane, Alcon)

Anecortave acetate is an angiostatic steroid. Owing to structural modifications, it has no glucocorticoid activity and does not elevate intraocular pressure nor increase the risk of cataract formation. The drug inhibits both urokinase-like plasminogen activator and matrix metallopeptidase 3, two enzymes necessary for vascular endothelial cell migration during blood vessel growth. Anecortave acetate seems to inhibit neovascularization independent of the angiogenic stimulus and it, therefore, has the potential of nonspecific angiogenesis inhibition. The drug is administered by means of a posterior juxtascleral injection with a specially designed cannula. Preclinical and clinical trials have shown that Retaane is far superior to placebo in maintaining positive visual outcomes, preventing severe visual loss, and inhibiting lesion growth (21).

Fig. 5. VEGF is blocked from binding with its natural receptor after Macugen® (pegaptanib sodium injection) binds with VEGF. Courtesy of Eyetech Pharmaceuticals and Pfizer, Inc.

Squalamine (Genaera)

Squalamine is a naturally occurring, pharmacologically active, small molecule that belongs to the aminosterols family. It is the first compound to be tested as a clinical drug candidate in this group of agents. Squalamine is a potent molecule with a unique multifaceted mechanism of action that blocks the formation of the cytoskeleton, integrin expression, and the action of a number of angiogenic growth factors, including VEGF (26,28). The drug is administered intravenously (25–50 mg/m2). In a phase I/II study, patients were treated weekly with an iv dose of Squalamine. After 4 mo of follow-up, 10 out of 40 subjects had three or more lines of visual improvement, and 29 (72.5%) maintained their initial visual acuity or did not lose less than three lines of vision.

Other Agents Under Investigation

1.Combretastatin A4 Prodrug (CA4P From Oxigene Inc.)

Combretastatin represents a new class of therapeutic compounds known as vascular targeting agents. It was originally derived from the root bark of the Combretum caffrum tree, also known as the Cape Bushwillow. Zulu warriors utilized a substance made from this tree to poison the tips of their arrows and spears as a charm to ward off their enemies. Combretastatin works by microtubule inhibition present in the cytoskeleton of endothelial cells lining the abnormal blood vessels. When this tubulin structure is disrupted, endothelial cell morphology changes from flat to round, stopping blood flow through the capillary. The drug was tested in two different models of ocular neovascularization. Combretastatin suppresses the development of VEGF-induced retinal neovascularization and also blocks and promotes regression of choroidal neovascular membranes (29).

2.Small interfering RNA (siRNA) targeting VEGF (Cand5 from Acuity Pharmaceuticals) RNA interference (RNAi) mediated by siRNAs is a technology that allows the silencing of mammalian genes with great specificity and potency. The highly potent RNAi mechanism of action of Cand5 stops production of VEGF at the source, whereas other compounds inhibit VEGF after it is produced, with VEGF remaining present at significant and potentially pathogenic levels. Cand5’s potency reflects the ability of a single RNAi drug molecule to stop the

production of VEGF protein molecules. RNAi mechanism also has the potential to translate into a longer duration of action resulting in a lower required dosing frequency compare to other compounds, which bind directly to VEGF (30).

CONCLUSION

Several different classes of agents that target angiogenesis have been developed recently for the treatment of wet AMD, in which angiogenesis is thought to be the primary mechamism. It is well known that VEGF plays a critical role in the genesis of the abnormal vessels. Antiangiogenic therapy targeting the VEGF signaling pathway is a promising new strategy for the management of wet AMD. The development of dosing strategies and combination therapies will become an important clinical challenge for the vitreo-retinal specialist.

ACKNOWLEDGMENTS

The authors have no proprietary interest in any aspect of this study.

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Fig. 1. Fundus photo of a patient diagnosed with STGD. Note characteristic yellowish flecks around the macula and a defined area of central macular degeneration.

is characterized by a highly variable age of onset and clinical course. Most cases present with juvenile to young-adult onset, evanescent to rapid central visual impairment, progressive bilateral atrophy of the foveal retinal pigment epithelium (RPE) and photoreceptors, and the frequent appearance of yellow-orange flecks distributed around the macula and/or the midretinal periphery (3,4) (Fig.1). In a large fraction of patients with STGD, a “dark” or “silent” choroid is seen on fluorescein angiography, which reflects the accumulation of lipofuscin. Electroretinographic (ERG) findings vary and are not usually considered diagnostic for the disease. A clinically similar retinal disorder, fundus flavimaculatus (FFM), often manifests later onset and slower progression (5–7). Despite historical separation (8,9), results of linkage and mutational analysis confirmed that STGD and FFM are allelic autosomal recessive disorders with slightly different clinical manifestations, caused by mutations of a single gene located within an approx 2- cM interval between markers D1S406 and D1S236, at chromosome 1p13-p21 (3,10–13).

The wide variation in clinical expression of the disease in patients with STGD has been classified into three major clinical phenotypes according to Fishman (14). Phenotype I is characterized by an atrophic-appearing macular lesion, localized perifoveal yellowish-white flecks, the absence of a dark choroid, and normal ERG amplitudes. Patients in the phenotype II group present with a dark choroid, more diffuse yellowish-white flecks in the fundus, and inconsistent ERG amplitudes. Phenotype III

group includes patients with extensive atrophic-appearing changes of the RPE and reduced ERG amplitudes of both cones and rods.

GENETIC PREDISPOSITION: THE ABCA4 (ABCR) GENE

Several laboratories independently described ABCA4 in 1997 as the causal gene for arSTGD (1,15,16). There is no definitive evidence of genetic heterogeneity of arSTGD because, as described here previously, all families segregating the disorder have been linked to the ABCA4 locus on human chromosome 1p13-p22. Hence, the role of the ABCA4 gene as the only causal gene for arSTGD has not been disputed. Subsequently, several cases were reported where ABCA4 mutations segregated with retinal dystrophies of substantially different phenotype, such as autosomal recessive cone-rod dystrophy (arCRD) (17,18) and autosomal recessive retinitis pigmentosa (arRP) (17,19,20).

Disease-associated ABCA4 alleles have shown an extraordinary heterogeneity (1,14,21–24). Currently, about 500 disease-associated ABCA4 variants have been identified, allowing comparison of this gene to CFTR, one of the best-known members of the ABC superfamily, encoding the cystic fibrosis transmembrane conductance regulator (CFTR) (25). What makes ABCA4 a more difficult diagnostic target than CFTR is that the most frequent disease-associated ABCA4 alleles, e.g., G1961E, G863A/delG863, and A1038V, have each been described in only about 10% of patients with STGD in a distinct population, whereas the delF508 allele of CFTR accounts for close to 70% of all cystic fibrosis alleles (26).

Several studies have identified frequent “ethnic group-specific” ABCA4 alleles, such as the 2588G > C variant resulting in a dual effect, G863A/delG863, as a founder mutation in Northern European patients with STGD (22), and a complex allele (two mutations on the same chromosome), L541P/A1038V, in both STGD and CRD patients of German origin (24,27). Complex ABCA4 alleles are not uncommon in STGD (21), in fact, they are detected in about 10% of all patients with STGD (28).

Because the ABCA4 gene has been screened mainly in European patients with STGD, the estimates of allele frequencies and pathogenicity have been made based on these data. However, several studies have suggested substantial differences in frequencies of specific ABCA4 alleles between the Caucasian general population and those of other ethnic/racial groups. For example, an ABCA4 allele T1428M, which is very rare in populations of European descent, is apparently frequent (~8%) in the Japanese general population (29).

An even more intriguing case involves one of the most frequent ABCA4 mutations, G1961E. Although its frequency in the Caucasian general population is approx 0.2%, and in patients with STGD of the same ethnic origin it reaches greater than 10% (30), this allele has been detected at substantially higher rate in East African countries (tribes from Somalia, Kenya, Ethiopia, etc.). It is likely that approx 10% of the general population from Somalia carries the allele in a heterozygous form (31). At the same time, it is almost absent in individuals of West African descent, including African Americans (Rando Allikmets, unpublished data). Although the exact cause and consequences of this phenomenon are still to be determined, it is likely that the prevalence of STGD in East Africa is much higher than expected and/or observed.

The summarized data presented here establish allelic variation in ABCA4 as the most prominent cause of retinal dystrophies with Mendelian inheritance patterns. The latest estimates suggest that the carrier frequency of ABCA4 alleles in general population is in the range of 5 to 10% (22,32,33). This finding, that at least 1 out of 20 people carry a diseaseassociated ABCA4 allele, has enormous implications for the amount of retinal pathology attributable to ABCA4 variation and suggests re-evaluation of current prevalence estimates.

Soon after the discovery of ABCA4, a disease model was proposed that suggested a direct correlation between the continuum of disease phenotypes and residual ABCA4 activity/function (34,35,36). According to the predicted effect on the ABCA4 transport function, Maugeri et al. classified ABCA4 mutant alleles as “mild,” “moderate,” and “severe” (22). Different combinations of these were predicted to result in distinct phenotypes in a continuum of disease manifestations, the severity of disease manifestation being inversely proportional to the residual ABCA4 activity. This model, although widely accepted, was recently disputed by Cideciyan and co-workers (37), who did not find the suggested inverse relationship between residual ABCA4 activity and clinical disease severity. The latter was defined by intensity of autofluorescence, rod and cone sensitivity, and rod adaptation delay. However, their study cohort included only 15 patients with known ABCA4 variants on both alleles and only a few of these were severe mutations, compared to mild mutations (3 vs 28). Furthermore, classification of mutations by severity through circumstantial evidence, e.g., by predicting the effect of an amino acid change on the protein function, would often lead to erroneous conclusions, as clearly demonstrated by the available in vitro assay, which currently provides the best estimate of the functional effect of ABCA4 variants (28,38). In summary, all experimental, clinical, and genetic variables have to be carefully assessed when making conclusions either on the proposed model of ABCA4, or on genotype/phenotype correlations in patients with STGD.

MOLECULAR DIAGNOSIS

Allelic heterogeneity has substantially complicated genetic analyses of ABCA4- associated retinal disease. In the case of arSTGD, the mutation detection rate has ranged from approx 25% (14,39) to approx 55–60% (21,22,24,40). In each of these studies, conventional mutation detection techniques, such as single-stranded conformational polymorphism (SSCP), heteroduplex analysis, and denaturing gradient gel electrophoresis (DGGE), were applied. Direct sequencing, which is still considered the “gold standard” of all mutation detection techniques, enabled a somewhat higher percentage of disease-associated alleles to be identified, from 66 to 80% (28,32).

Other important parameters influencing mutation analysis are the effort and the cost of screening the entire gene. ABCA4 presents a special challenge because, similar to most full-sized ABC transporters, it is comprised of 50 exons and has an open reading frame exceeding 6800 bp. Therefore, all mutation detection techniques that remain exclusively polymerase chain reaction-based, are relatively inefficient, expensive, and labor intensive. Efforts related to mutation detection and genotyping become especially crucial in allelic association or genotype/phenotype correlation studies, in which screening of thousands of samples is needed to achieve enough statistical power, as is the case with ABCA4, in which multiple rare variants must be studied (41).

Fig. 2. The microarray for mutational screening of the ABCA4 gene (the ABCR400 chip). Each allele (mutation) is queried by duplicate oligonucleotides from both strands, i.e., represented on the array by four dots. Four colors discriminate four dideoxynucleotide triphosphates labeled with four different fluorescent dyes.

To overcome these challenges and to generate a high-throughput and a cost-effective screening tool, the ABCA4 genotyping microarray was developed (33). The ABCR400 microarray contains all currently known disease-associated genetic variants (>450) and many common polymorphisms of the ABCA4 gene (Fig. 2). The chip is more than 99% effective in screening for mutations and it has been used for highly efficient, systematic screening of patients with ABCA4-associated pathologies, especially those affected by STGD. When a cohort of patients with STGD is analyzed by both the array and one of the conventional mutation detection methods, e.g., SSCP, the mutation detection rate reaches 70–75% of all disease-associated alleles, thus being comparable to direct sequencing (33). The chip alone will detect approx 55–65% of these alleles, an efficiency similar to that in the best studies with conventional (SSCP, DGGE, etc.) mutation detection methods (33,42,43). However, the effort and the expense of the array-based screening are each at least one order of magnitude lower than any other method, whereas the speed and reliability are much higher. A complete screening of one DNA sample takes only a few hours and the current all-inclusive cost is estimated at $0.20/genotype. The reliability of the chip, i.e., the efficiency of detection of any mutation on the chip, is more than 99% in any given experiment. Another feature of the ABCR400 array allows for detection of any sequence variation at each and all positions included on the chip, as it directly sequences all queried positions. Several new, previously not described, mutations have been found by the ABCR400 array in several studies (33,43). In conclusion, although the assessment