Ординатура / Офтальмология / Английские материалы / Ocular Therapeutics Eye on New Discoveries_Yorio, Clark, Wax_2007

to “tunnel vision”. Within this category, we can put Leber congenital amaurosis (LCA) and the syndromic conditions such as Usher syndrome, Bardet–Beidl disease, etc., in which RP is present along with other sequelae such as hearing loss, neurological problems, etc. The second major grouping is the macular degenerations in which central vision, sharp vision and color vision are primarily affected. Within this category are the early-onset macular degenerations such as Stargardt’s disease and the cone-rod dystrophies. The largest grouping here, though, is with age-related macular degeneration (AMD), a late-onset macular degeneration as the name implies. Finally, there are a number of disease entities that are usually referred to as the rare retinal degenerations. These encompass RDs such as refsum disease, retinoschisis, etc.

Clinically, the disease process in these degenerative conditions results in photoreceptor dysfunction and possible death of the photoreceptor cell. As mentioned above, dim light vision is poor in RP patients and peripheral vision becomes restricted. The visual field generally continues to constrict to a few degrees of vision or eventual extinction. Phenotypically, there is great variation in onset and progression in the RPs. Visual dysfunction could be severe and be apparent from birth in cases such as LCA, or could be slower and later in onset as in some of the dominant types of RP. The same wide variation is seen in the macular degeneration group which can manifest very early in life (Stargardt’s disease) or much later (AMD). AMD itself has several stages or forms. Small discrete deposits in the retina, drusen, are often an early manifestation of the disease, even when vision may not yet be compromised. Larger drusen can then often be seen with progression to the end stage forms of dry AMD (geographic atrophy) or wet AMD (choroidal neovascularization).

A unifying feature of this family of diseases is that they all have genetic causes or at least have a significant genetic component in the etiology. Most of the RP group

of diseases are simple, inherited, retinal degenerations where a mutation in a single gene results in the retinal degeneration. Most often, inheritance patterns are classically dominant, recessive or X-linked although there are some exceptions as with digenic inheritance. With most of the earlyonset macular degenerations and other rare degenerations, the situation is similar with a single gene mutation precipitating the disease process. AMD is very different, however. Although there is a strong genetic component in AMD, environmental factors play a significant role in the disease process. Smoking, for example, is a significant risk factor in AMD. All this makes AMD best described as a complex disease with both genetic and environmental components.

III. CLINICAL OBJECTIVES

The simple clinical objectives in attempts at designing therapies for the inherited retinal degenerations are (a) if possible, maintain the viability and function of photoreceptor cells; and (b) if the cells are already dead, seek a mechanism, natural or artificial, that will take the place of the lost photoreceptors in signaling the secondary neurons of the retina with a visual image.

A number of strategies can be employed to maintain or at least lengthen photoreceptor life and function. For example, neurotrophic factors are being employed that increase photoreceptor neuron longevity and perhaps function as well. Nutritional therapy has also been found to be helpful in some instances. Replacement of the mutated gene to once again produce the critical normal gene product can certainly help to prolong photoreceptor cell life and function. When few or no photoreceptor cells remain, as in very rapid degenerations or when the degenerative process is not addressed early in its course, the use of natural or artificial substitutes for photoreceptor cells must be considered. Transplantation of photoreceptor cells from a normal donor

eye to the affected retina is an obvious theoretical solution that has been under study for a number of years. Likewise, the use of an electronic device implanted on the retina or even brain of the patient is possible to create “artificial vision”.

All in all, proof of principle has been established in several research directions now such that clinical trials are either in progress or can be planned for treating the retinal degenerations.

IV. BASIC MECHANISMS

A. Molecular Biology

1. RP and the rare degenerations

The starting point for all the retinal degenerative diseases is that they are inherited. Essentially, the mutation initiates a process which, after many steps, culminates in photoreceptor cell dysfunction and death. The number of genes known to be associated with the degenerations has grown steadily over the last two decades. It was in 1984 that the locus for the first gene associated with a specific type of retinal degeneration, X-linked RP, was linked to a specific chromosomal location (Bhattacharya et al., 1984). It was not until 1996 though that the actual gene (RPGR) was identified, underscoring the extreme difficulty in such gene identification. Quicker identification came with the first gene actually pinpointed as mutated in a retinal degeneration. After chromosomal localization by Humphries and coworkers, Dryja et al. (1990) soon reported a point mutation in the opsin gene as the cause of this type of autosomal dominant RP. Classically, the genetic forms fall into the dominant, recessive and X-linked categories. However, a digenic mutation has been reported in RP involving the Peripherin/ RDS and ROM1 genes (Dryja et al., 1977).

As of the writing of this chapter, RetNet, a website compendium of genes mutated in retinal degenerations, lists a total of 177 loci and identified genes causing RD, of

which 124 have been cloned and identified. Within this, there are specific disease entities in which several genes have been identified whose mutations cause the particular phenotype. For example, 11 different genes or loci have been pinpointed in Bardel–Beidl syndrome. RetNet now lists 10 different genes whose mutations cause Usher syndrome. Other conditions are simpler – Stargardt disease, the most common form of early-onset macular degeneration, has a rare dominant form and one major gene (ABCA4) whose mutations account for the recessive cases. Great progress has been made in identifying gene mutations in Leber’s congenital amaurosis (LCA). Until recently, 8 different genes had been found whose mutations cause LCA. Together, these gene mutations were thought to account for 40–50% of the LCA cases. However, den Hollander et al. (2006) have identified a ninth gene involved in LCA, the CEP290 gene, whose mutation is detected in about 21% of LCA cases. This makes it the most prevalent gene mutation known in LCA and brings the genotyped LCA patient number to about 70%.

It is thought that perhaps we now know about half of the genes involved in the “orphan” retinal degenerations. Finding the remaining genes may be a slow process as it is probable that the more obvious gene mutations have already been elucidated. However, with the advent of gene therapy clinical trials (see below), it may one day be possible to treat most of the patients with RP (including LCA, Usher, etc.) and all the other rare retinal degenerative diseases.

2. Age-related macular degeneration

A few short years ago, no genes were specifically associated with AMD although it has been clear from familiar aggregation studies, twin studies, etc., that AMD had a distinct genetic component in its etiology. Now, several genes have been linked to AMD. In a small number of patients, mutations in the ABCA4 (Allikmets et al.,

1999) and the Fibulin 5 (Stone et al., 2004) genes appear to lead to AMD. An increased frequency of AMD, for example, has been observed in older generational family members of Stargardt patients exhibiting ABCA4 mutations.

Somewhat more recently, several gene mutations have been uncovered that may account for a much larger percentage of AMD cases. At about the same time, several groups linked mutations in important immunological proteins to increased risk in AMD (Hageman et al., 2005; Edwards et al., 2005; Haines et al., 2005; Klein et al., 2005). In the CFH gene, a specific haplotype was found to predispose to AMD with an increased odds ratio of up to 5.57. It may be that a triggering event, coupled with the genetic variation in the CFH gene, could underlie up to 50% of AMD cases. Complement Factor H (CFH, HF1) is the major inhibitor of the alternative complement pathway in the immune system. A screening of similar genes in immunological regulatory pathways has led Gold et al. (2006) to two other genes whose variations lead to AMD. They found that variation in Factor B (BF) and complement component (C2) in the major histocompatibility complex class III region increases risk for AMD by 2–3-fold. Gold and coworkers calculate that the variations in the CFH, C2 and BF loci combined can “predict the clinical outcome in 74% of affected individuals”. Over and above this, Yang et al. (2006) have compelling evidence that mutations in the HTRAI gene play a key role in AMD susceptibility – with undoubtedly other such mutated genes yet to be identified.

B. Cell Biology

1. RP and the rare degenerations

Most of the RP-like and other rare degenerative retinal diseases are simply genetic in that, after an initial triggering mutation, complications from other factors such as the environment do not seem to have a large influence on the disease process. That said, it is obvious that the many

mutations now known to cause the different RD forms lead to a bewildering array of phenotypes – fast/slow, severe, mild, etc. Also, genetic variation is well known within families with all affected members often not following the same course.

A unifying feature, though, at the cell level is that it appears that apoptosis is the final common pathway of photoreceptor death. In a seminal publication, Wong and his collaborators (Chang et al., 1993) examined three different murine models of retinal degeneration and found classical signs of apoptosis, including DNA fragmentation due to internucleosome cleavage. It is now generally felt that apoptosis, or programmed cell death, is the dominant form of cell death in the RDs due to the silent and relatively non-destructive manner of cell removal. The situation may be somewhat more complex in that Hao et al. (2002) have presented evidence for two separate apoptotic pathways in light damageinduced retinal degeneration, and also that additional genetic factors regulate the retinal sensitivity to the light-induced damage.

2. Age-related macular degeneration

Given that AMD is a complex disease with different phenotypic manifestations, many theories have been proposed to account for the cellular pathology(ies) that are seen during the course of the disease. It seems clear now, though, that inflammation plays a key role in the pathogenesis of AMD. Hageman et al. (2001) studied drusen as biomarkers of the disease process and concluded that AMD was an immunemediated disease. In particular, the variations cited above, in genes such as CFH, are strongly associated with AMD development. These associations have been well summarized by Donoso et al. (2006). Other evidence also implicates immune regulation. For example, Seddon et al. (2005) investigated the relationship between inflammatory biomarkers of cardiovascular disease and AMD. They found that higher levels of C-reactive protein (CRP) and interleukin 6 (IL-6) are associated with AMD

progression. Interestingly, CRP and IL-6 levels were found to be linked to higher body mass index and current smoking, the latter being one of the most significant environmental factors associated with AMD. Nozaki et al. (2006) have presented evidence that complement components C3 and C5 are present in early subRPE deposits and that ablation of C3a and C5a receptors reduces the expression of VEGF and CNV induction after laser injury. Also, an animal model of AMD further substantiates the important role of immune dysfunction in AMD. Ambati et al. (2003) have produced mice deficient either in monocyte chemoattractant protein-1 (Ccl-2) or C-C chemokine receptor (Ccr-2) and found that they exhibit ocular characteristics of AMD including drusen formation, photoreceptor cell death and, ultimately, choroidal neovascularization (CNV). These results strongly implicate immune (monocyte) involvement in AMD pathologies and indicate promising pathways for future interventions.

The role of inflammation and the immune system does not, however, rule out other processes being involved – either as precipitating or promoting factors. Crabb et al. (2002) have also analyzed drusen and Bruch’s membrane using sophisticated proteomic techniques involving liquid chroma- tography-mass spectrometry. They detected extensive oxidative protein modifications in the components of these structures. For example, advanced glycation products and carboxyethyl pyrrole (CEP) adducts, unique products of oxidation of docosahexaenoic acid (DHA), were found to be in higher concentration in Bruch’s membrane material from AMD patients than from normal subjects. DHA is a long-chain, polyunsaturated fatty acid found abundantly in photoreceptor outer segment membranes whose oxidation directly leads to the CEP adducts. Thus, oxidative protein damage could have a critical role in drusen formation and may otherwise contribute to the pathogenesis of AMD. Others such as Beatty et al. (2000) have also provided evidence that oxidative stress plays a role in AMD etiology.

What are the crucial factors in the development of CNV? Especially, why do only a small percentage of AMD patients (about 10%) go on to the neovascularization end stage? Unfortunately, the answer to this question is not yet known. However, it has been proposed that choroidal neovascularization might be thought of as a result of a wound healing or tissue repair process (Kent and Sheridan, 2003). In many body functions, wound healing involves a cascade of events initiated by growth factors and cytokines that affect the extracellular matrix and surrounding cells. Angiogenesis is often a component of this process and perhaps CNV in AMD can be thought of as “just another component of this wound healing process” along with inflammation, etc.

As in RP, it seems that perhaps the final common denominator of photoreceptor cell death is apoptosis. Dunaief et al. (2002) have presented evidence that not only photoreceptor cells, but RPE cells and cells in the inner nuclear layer are removed by programmed cell death. Specifically, TUNEL-positive rod and RPE cell nuclei were found near the edges of RPE atrophy in AMD eyes as well as Fas labeling in photoreceptor cells. This and other evidence supports the supposition that apoptosis plays a prominent if not singular role in AMD-induced retinal cell death.

V.CURRENT THERAPY

A. Retinitis Pigmentosa and Allied Diseases

To date, the only therapeutic regimen available to RP patients and those with the rare, rod-based retinal degenerations has been vitamin A therapy. For cone-dominant degenerations such as Stargardt disease, no effective therapies have yet emerged.

In 1993, Berson and associates (1993) published that the disease course of common forms of RP could, on average, be significantly slowed with the use of vitamin A palmitate at a dose of 15,000 IU/day. Other

more recent work has studied the effects of vitamin A supplementation in RD animal models with opsin mutations. Mixed results were obtained with the treatment – saving ERG amplitude and photoreceptor nuclear layer integrity in one animal mutant (T-17-M), but not in another (P-347-S) model (Li et al., 1998). Importantly, safety of the treatment seems clear in healthy individuals since Sibulesky et al. (1999) examined patients supplemented with vitamin A, aged 18–54, as to long-term supplementation. Even though serum retinol levels were elevated 1.7-fold at 5 years of treatment, no clinical signs of liver toxicity were detected. Thus, for most otherwise healthy RP patients, vitamin A treatment appears to be safe, although its positive effects are limited to a smaller subset of patients.

Low blood levels of the omega-3 fatty acid docosahexaenoic acid (DHA) have long been reported in RP patients (reviewed by McColl and Converse, 1995). As pointed out above, DHA is an important and plentiful component of outer segment membranes, thus, any deficiency could be involved (primary or secondary) in RP pathology. To test if DHA supplementation were effective in RP, Birch and collaborators conducted a 4 year randomized clinical trial on X-linked RP patients who received either placebo or 400 mg/day DHA (Wheaton et al., 2003). Although biological safety was established, no positive effects of the treatment were reported in this study. Subsequently though, the group (Hoffman et al., 2004) reported on efficacy. Unfortunately, the overall rate of cone ERG functional loss (i.e. visual acuity and visual fields) was not significantly affected by the treatment. However, in subset analysis, DHA was found to be beneficial in reducing rod ERG functional loss and preserving cone ERG function in patients under 12 years of age. In a somewhat similar manner, Berson and collaborators conducted a clinical trial with DHA supplementation (12,000 mg/day) in patients already receiving vitamin A treatment (Berson et al., 2004a). Over a 4 year

period, they found no overall positive effect of the DHA supplementation. However, upon subset analysis (Berson et al., 2004b) found that, for patients just beginning therapy, use of DHA did slow the disease process in the first 2 years of treatment. Thus, some positive albeit weak effects of DHA have been noted in RP, indicating more work should be done to tease out possible patient subgroups in which DHA might be useful as a therapy.

B. Age-Related Macular Degeneration

1. Dry AMD

As with RP, a nutritional treatment is available for dry AMD. Moreover, since environmental factors are at play in AMD, as well as genetic factors, certain preventive measures such as oral supplementation with specific nutrients can be taken, as opposed to RP where “prevention” amounts to just slowing the course of the disease.

For progression of AMD at moderate stages of advancement, the Age-Related Eye Disease Study (AREDS) has been the primary guide available for slowing the course of the disease (Age-Related Eye Disease Study Research Group, 2001). In this double masked clinical trial, the effectiveness of a group of antioxidative supplements, i.e. vitamins C and E, beta carotene and zinc, was assessed in AMD patients. Visual acuity, as well as photographic evidence, was assessed as a measure of AMD progression. It was found that persons with intermediate or moderately advanced AMD, i.e. (1) extensive intermediate sized drusen; (2) those with at least one large druse; (3) those with geographic atrophy in one or both eyes; and (4) those with advanced AMD or vision loss in one eye alone could benefit from taking the supplement. Safety was found to be good in that no significant serious side effects were noted. A contraindication is that smokers should probably avoid taking beta-carotene due to an independent risk of cancer.

The antioxidant theme has been extended to the potential of using carotenoids such

as lutein as a treatment in AMD. Reicher et al. (2004), for example, conducted a double masked, randomized clinical trial of lutein and antioxidant supplements as a possible intervention in AMD. They found that both lutein alone and lutein with the supplements improved visual function. As the study was only for one year, further studies are needed to assess the long-term effects of the nutrients. Similar studies are in progress and will be discussed below. Theoretically, the use of lutein and zeaxanthin could be beneficial since they have been shown to be efficient scavengers of superoxide and hydroxyl radicals as well as quenching singlet oxygen, thus inhibiting biological oxidation (Trevithick-Sutton et al., 2006).

2. Wet AMD

Specifically for wet AMD, a number of treatments are now available. A purely surgical approach was suggested several years ago by Machemer and Steinhorst (1993) involving macular translocation, i.e. retinal separation, retinotomy and macular relocation to move the macular region of AMD patients to a more favorable location at the back of the eye. De Juan and collaborators then developed a technique for limited macular translocation and subsequent laser photocoagulation of the neovascular complex. Improvement in a substantial number (about 40%) of eyes was reported at 1 year follow-up (Fujii et al., 2002). Persistence and reoccurrence of neovascular lesions, however, are common after such surgery as well as the difficulty of the initial surgery.

Photodynamic therapy using Visudyne (Verteporfin) was the first drug intervention specifically approved and available for wet AMD. Although it has been mainly used for only certain forms of wet AMD, it is now approved worldwide in most major countries. The Verteporphin Study Group found, for example, that visual acuity benefits for this treatment in patients with predominantly classic CNV were maintained for at least 2 years (Treatment of AMD with

Photodynamic Therapy (TAP) Study Group, 2001). Other studies have found some benefits in minimally classic CNV and in occult CNV (Verteporfin in Photodynamic Therapy Study Group, 2001), etc., but, on the whole, the benefits are mainly in stabilizing vision and slowing visual loss, and not in actual improvement. With the advent of other therapies (see below), it is probable that photodynamic therapy will remain as a niche market, used in combination with other treatment options.

Also approved as a treatment is Macugen, an anti-VEGF aptamer (Pegaptanib), which is applicable for use in wet AMD. An aptamer is an oligonucleotide designed to bind to a specific protein such as VEGF with high specificity and affinity, and thus inhibit its action. The 2 year efficacy of Pegaptanib treatment was found to be good in that it reduced the progression of vision to legal blindness. Some patients gained vision compared to controls (VEGF Inhibition Study in Ocular Neovascularization Clinical Trial Group, 2006). A positive aspect of the action of Macugen is that it is effective in all CNV subtypes as compared with Visudyne which is not equally effective in all neovascular lesion subtypes and sizes (Vavvas and D’Amico, 2006).

Lucentis (Ranibizumab) now seems to be the first treatment for neovascular AMD that actually improves vision in many patients (Rosenfeld et al., 2006). Ranibizumab is a recombinant, humanized, monoclonal antibody fragment that is effective on all forms of VEGF. Data from two large Phase III clinical trials (ANCHOR and MARINA) demonstrated the safety of Lucentis treatment, but also showed an improvement of many patients in vision with 40% of treated patients achieving vision of 20/40 or better. In the ANCHOR study, Ranibizumab was specifically found to be superior to Verteporfin in predominantly classic CNV with a low rate of serious side effects. Due to the relatively high cost of treatment with Lucentis though, the possibility of alternative use of Avastin, a somewhat similar but

much less expensive antiVEGF molecule, has been raised (Rosenfeld, 2006).

In summary, antioxidant (AREDS) supplement therapy is available for patients with intermediate (dry) AMD as well as surgical and drug modalities of treatment for those whose disease has progressed to the neovascularization stage.

VI. FUTURE THERAPY

A. Retinitis Pigmentosa, Dry AMD and

Allied Degenerative Diseases

In the RP family of diseases, as well as for dry AMD, future treatments can roughly be divided into two categories. The first category would be those treatments that would be applicable to patients who have very few or no photoreceptors remaining. This situation could simply be due to a too rapidly progressing disease process or perhaps the inability to apply an appropriate treatment at an early enough age. Relevant for this category of patients would be photoreceptor transplantation or use of the retinal electronic prosthetic device. The second category of treatments would be those that sustain the life and/ or improve the function of photoreceptors when there are still some viable photoreceptor cells remaining in the retina. Interestingly, the number of remaining photoreceptors needed for functional vision may be fewer than one might think. In modeling degenerate photoreceptor arrays, Geller and Sieving (1993) have calculated that, in diseases such as Stargardt disease, “good visual performance can be achieved at cell counts far lower than those indicated by the acuity-eccentricity function”.

It is important to point out that, even though photoreceptor cells may have died in fairly advanced RD cases, most of the other layers of the retina remain fairly intact for a relatively long period of time. Milam and coworkers have performed morphometric analyses of macular photoreceptors and ganglion cells in a number of human

retinas with RP. Although degeneration was certainly noted in the secondary neuronal layers (i.e. bipolar, ganglion cells, etc.), a substantial number of these neurons remained alive (Stone et al., 1992; Santos et al., 1997). This “good news” must be balanced with the “bad news” that there can be progressive and extensive remodeling in the remaining cellular layers in RP that could complicate prospective RP treatment strategies. Fariss and Milam (2000) found that many remaining cells of the inner retina of an RD eye undergo neurite sprouting, rearrangement and inappropriate contact with Müller cells. Marc et al. (2003) have systematically examined the stages of degeneration and cataloged the array of abnormal changes that progressively develop. These features include end stage migration of neuronal somas on glial surfaces and “anomalous self-signaling via rewired circuits”. All in all, these changes must be taken into consideration when any treatment strategy is considered, although Marc and collaborators do state that “remaining neurons appear to be stable, active, healthy cells and, given evidence of their reactivity to deafferenation, it may be possible to influence their emergent rewiring and migration habits”.

In considering treatments for dry AMD, many of the same overarching considerations apply as discussed above for RP. A main consideration being, of course, whether enough photoreceptors remain alive to warrant a particular treatment. As with RP, preservation of inner retinal neurons has been observed in human AMD eyes, in fact, better preservation than in the RP eyes (Kim et al., 2002a,b). On average, the examined eyes showed up to 90% preservation of the inner retinal neurons. An interesting comparison (although not truly parallel) is that the cochlear implant requires as few as 10% of remaining neurons to function in hearing. If this holds true in most AMD cases, therapies such as the electronic prosthetic device could ultimately allow for reading vision and face recognition, as well as basic mobility. Other than prevention, wet AMD should be

considered as a separate entity in considering treatment due to the acute complications of neovascularization which overlay any problem of generalized cell loss – photoreceptor or inner retinal neuron.

The following are examples of future therapies for RP, dry AMD, and allied diseases for which proof of principle has been established as a minimum and, in some cases, the potential therapy has progressed to the clinical trial stage of development.

1. Transplantation

a. Photoreceptor and RPE cell transplantation–

Attempts at retinal transplantation date back many years. In 1959, Royo and Quay implanted rat fetal retinal tissue into the anterior chamber of the murine mother’s eye. Survival of the tissue and some development was noted. In 1985, del Cerro and colleagues transplanted embryonic rat retinal cells into the anterior chamber to test immune sensitivity. The first retinal replacement experiments were conducted by Turner and Blair in 1986 in transplanting fetal retinal tissue near the surface of rat and rabbit eyes.

Although the transplants survived, they often detached and many formed rosettelike structures. Subsequently, many other reports on animal transplantation studies have appeared both for retinal RPE cells (Li and Turner, 1988) and for photoreceptor cells (Silverman and Hughes, 1989).

Over the ensuing years, transplantation technologies have been developed for both RPE cells and photoreceptor cells. The area of photoreceptor transplantation in RP animal models has been particularly active in attempts to establish proof of principle in not only allowing photoreceptor graft survival, but in demonstrating actual visual function. Restoration of visual responses, for example, has been reported in rd mice following transplantation of intact retinal sheets (Arai et al., 2004). This was adjudged by restoration of superior colliculus responses subsequent to light flash stimuli. In other work in S-344-ter transgenic rats, recovery of retinotectal visual function has been confirmed after transplantation of fetal retinal sheets (Sagdullaev et al., 2003). Figure 20.1 shows representative recording

Normal rat

(n 12)

Rostral

(a)

light stimulus

Recovery of function after transplantation

RD rat, no surgery

50 ms

Transplanted age 3.4–6.3 months

(n 7)

2.6–5.4 mo. after surgery

(d)

Sagdullaev et al., IOVS. 2003; 44:1686–95.

from the superior colliculus (SC) of the S-344-ter rats at times after surgery. Untreated rats developed large scotomas while many (64%) of the treated rats regained retinadriven responses in appropriate SC areas representative of the retinal sector in which the transplant was placed. Transsynaptic viral tracings from host brain back to the subretinal transplant confirmed the presence of a viable pathway between retina and brain after transplantation (Seiler et al., 2005). Thus, theoretical proof of principle has been established in these, and many other, similar studies in animal models. The level of adequate integration of the transplant with the host retina (i.e. synaptic connections) remains a primary problem although factors such as the role of reactive astroglial cells and injury-provoked scar tissue are being investigated (Kinouchi et al., 2003). Also, the degree of signal transmission to relevant brain areas needs to be further elucidated.

In spite of these persisting questions, human transplantation studies have begun, mainly on RP patients. In 1997, Kaplan et al. (1997) published a safety study on human retinal transplantation indicating the relative safety of the procedure in RP patients without systemic immunosuppression. In 1999, Radtke and coworkers (Radtke et al., 1999) reported improved visual function in two blind patients with retinitis pigmentosa following retinal transplantation. In these cases, sheets of intact fetal retina were instilled in the subretinal space. By 6 months postoperatively, at least some subjective and objective improvement was reported. Importantly, no signs of transplant rejection were observed. Safety was also established in a separate study by Radtke et al. (2002) with no immunosuppressive medication. Along with transplantation of retinal sheets, transplantation of dispersed retinal cells has been studied. Humayun et al. (2000) used microaggregate suspensions of human fetal retinas, as well as sheet transplantation to examine effects in RP and AMD patients. No positive effects of the transplants were demonstrated

but they did find high tolerance for the grafted cells with no post-operative immunosuppression. Most recently, Radtke and coworkers have received FDA approval for a human clinical trial. Their latest transplant results on RP and AMD patients report improved vision in these patients in the first few months after surgery.

b. RPE cell transplantation – RPE cell transplantation has also been studied subsequent to the initial transplant studies by Li and Turner. In this, the rationale is that RPE cells could enhance the general survival and function of photoreceptor cells in RP or, as in cases where the primary lesion is actually in the RPE cell layer (e.g. RPE65 mutation), could supply competent cells to correct the defect. Also, in AMD, RPE cells are actively involved in the pathology, and replacement with fresh cells could allow for improved function. For example, transplantation of allogenic RPE sheets has been reported by Del Priore et al. (2004) in the normal pig. Although pathological changes were observed, “choroidal vessels and the choripcapillaris remained patent in the transplant bed”. The immunological consequences of transplantation have also been extensively examined. Jiang et al. (1994), for example, found that after allograft RPE cell transplantation, the RPE cells enjoyed immune privilege for a time, but ultimately were rejected in a cell-mediated manner. Aspecial problem encountered inAMD work is that Bruch’s membrane is altered in aging and also with the pathological changes engendered by the disease process (Ho and Del Priore, 1997). Cai and Del Priore (2006) have also reported that aging of Bruch’s membrane induces changes in gene expression in human ARPE-19 RPE cells seeded on the acellular membranes.

A study in the human involving transplantation of autologous RPE into eyes with AMD and foveal CNV has been reported (Binder et al., 2002). This also involved conventional surgical removal of the choroidal neovascular membranes. Visual acuity

improvement of “2 or more lines in 57% of the eyes” was reported with no recurrence of CNV formation during the study period (12–24 months). In spite of the preliminary nature of these results, the data indicated that such treatment might be generally effective in wet AMD. This technique could also be considered in conjunction with other (e.g. drug) treatments.

Variations on the general transplant theme have been tested. Human RPE cell lines have been studied in transplantation in the well-known RP model, the RCS rat (Lund et al., 2001). The authors report that subretinal transplantation of these cell lines results in “significant preservation of visual function as assessed by either behavioral or physiological techniques”. Transplantation of Schwann cells has also been investigated. The rationale behind these studies lies in the fact that Schwann cells produce neurotrophic factors such as CNTF, BDNF and GDNF which can slow retinal degeneration. As such, the cells act as a trophic factor delivery system. Keegan et al. (2003) report that such transplantation into the subretinal space of an RP mouse model does indeed prolong photoreceptor survival for a period of time but that the effect is subsequently lost. Moreover, a reactive Müller glial cell response was provoked.

Finally, a primary question has yet to be answered: Are the positive responses seen in all the aforementioned transplant studies due to the action of the transplanted cells themselves or are the responses due (at least in part) to a neurotrophic effect evoked by the surgery and/or implant?

In early RPE transplant studies, a surprisingly wide zone of rescue was seen in the treated eyes in regard to the relatively small number of transplanted RPE cells. This suggested that other factors were involved in the rescue besides the RPE cells themselves. Silverman and Hughes (1990) studied the effect of the surgery itself on rescue using the injection of saline solution as a control, as well as temporary retinal detachment. They concluded that substantial rescue does

not necessitate the presence of RPE cells, but that the surgical procedure as well as temporary retinal detachment “can induce photoreceptor rescue in the RCS rat”. This is reminiscent of the results with data on the implantation of an electronic prosthetic device that will be discussed below. In sum, the differential effects of the photoreceptor/ RPE cells vs secondary (positive) effects elicited by the transplantation surgery need to be fully evaluated. This question may be a moot point, though, if the current human clinical trial of Dr Radtke demonstrates long-term efficacy as well as safety.

c. Stem cell transplantation – There is no doubt that the use of stem cell therapy has enormous potential in treating diseases, including those of the eye. Repopulation of the retinal photoreceptor or RPE layers, for example, denuded by disease processes such as RP and AMD, is a compelling idea. Current work not only indicates that the instillation of foreign stem cells into the human eye might one day be feasible, but that there would even be the possibility for reinitiation of stem cell growth and differentiation at the peripheral margin of the adult retina. Also, cells derived from RPE, iris, Müller glial or other cell types, might also be induced to develop photoreceptorlike characteristics and function.

Classically, stem cells are of course obtained from embryonic tissue. Retinal progenitor cells obtained from embryonic retina can proliferate, survive when transplanted into the subretinal space, and express specific neuronal markers (Chacko et al., 2000). Much work, such as that of Qiu et al. (2005), has further evaluated stem cells transplanted into the mature retina. In this case, the retinal progenitor cells were found to express rhodopsin, organize into layers and at least partially integrate into the host retina in an RP animal model. A role for intrinsic signals is postulated as contributing to the development of the progenitor cells. Importantly, retinal stem cells are also found in the adult eye. Van der Kooy and