What should a clinician know to be prepared for the advent of treatment of retinal dystrophies?

possible to identify the responsible gene in about 50% of families with autosomal dominant disease, and most if not all X-linked disease. For autosomal recessive disease the number is uncertain. In the commonest macular disease, Stargardt macular dystrophy, for which the gene is known, the proportion of mutations detected is low. This is in part because the gene is large and highly polymorphic such that there is uncertainty as to the signi¢cance of base changes to disease. It is clearly desirable for the mutation to be known in any patient entering a treatment trial, and essential for gene therapy.

Knowledge of the disease mechanisms

The following should be established:

. the cell expressing the mutant gene and the target cell of disease

.whether the gene causes gain or loss of function and the biochemical mechanisms of disease

. the temporal pro¢le of functional loss and cell death

The cell expressing the mutant gene and the target cell of disease

In most disorders the cell harbouring the mutation is recognizably the same as the cell that is most a¡ected by the disease. However this should not be assumed. In Stargardt disease the mutant gene is expressed in the photoreceptor cells, and yet the cell that initiates the process of visual loss is the retinal pigment epithelial cell that becomes overloaded with auto£uorescent material. This can be detected by auto£uorescence imaging, and is the ¢rst sign of disease.

Whether the gene causes gain or loss of function

Whether disease is due to haploinsu⁄ciency, or gain of function will determine a suitable approach to gene therapy. Knowledge of this will come from work by molecular geneticists, cell biologists and biochemists.

In haploinsu⁄ciency the objective is to insert a gene that will achieve long-term and appropriate expression of a protein that is correctly targeted. There is ample evidence that this can be achieved in animal models with subretinal injections and long-term rescue of function has been achieved.

Many instances of gain of function have been described. This has been nicely illustrated with mutant RetGC1 and GCAP1 (Newbold et al 2001, Wilkie et al 2000). In such cases the objective would be to block gene expression using ribozymes by which mRNA from the mutant gene would be cleaved with consequent reduction of the relevant mutant protein. Di¡erent ribozymes have variable speci¢city and e⁄ciency, but observations on rodent models indicate

that this is an approach with promise (LaVail et al 2000). In general it has been assumed that loss of function is a feature of recessive disease, and gain of function of dominant disease. However this is not always the case. In retinitis pigmentosa due to mutations in RDS (Goldberg et al 1998), and PRPF31, it appears that the degeneration is due to loss of function.

Recently it has been shown that some dogs with X-linked PRA, due to mutations in ORF15 of the RPGR gene, have an expanded rough endoplasmic reticulum (RER); a situation homologous to liver in a chymotrypsin de¢ciency (Zhang et al 2002). In this situation, it is possible that there is a combination of mechanisms, one of which may be more critical than the other.

Temporal pro¢le of cell death

It is evident that gene therapy will only succeed if the target cell of disease is physically present. It has been shown that in some disorders loss of function is due to cell dysfunction at least at some stage in the disease process, whereas in others it signals cell death (Massof & Finkelstein 1981, Lyness et al 1985). The two forms of disease were shown in dominant RP. In the ¢rst was termed as type 1 or di¡use, and the second regional or type 2. In the ¢rst there was widespread loss of scotopic function but relative preservation of photopic sensitivities. In the second there was patchy and equal loss of rod and cone mediated sensitivities. The rhodopsin levels were assessed by relfectometry (Kemp et al 1988). In type 2 loss of rod sensitivities, rhodopsin level was proportional to the loss of function. By contrast, in type 1 rhodopsin levels were much higher than would have been predicted from functional measurements; patients with more than three log units elevation of threshold had near normal concentrations of bleachable rhodopsin. In type 2 loss of function was determined by light catch, and all other functions of the visual system were normal. In type 1 loss of function might be due to loss of transduction gain or noisy photoreceptors due possible to constitutive activity of rhodopsin. The form of the disorder was consistent within a family implying that the di¡erences were intrinsic to the disease.

The implications for therapy are self-evident. In type 1, gene therapy might cause recovery of function, whereas in type 2 it would serve only to slow down the kinetics of loss. In type 1, cell transplantation would be inappropriate, but would be suitable for type 2.

Thus the relationship between cell death and functional loss must be established for each disorder for which treatment is contemplated. With respect to photoreceptor cells this can be achieved with auto£uorescence imaging, or optical devices that allow visualisation of the outer retina (von Rˇckmann et al 1995, Fitzke 2000). The latter might be achieved using confocal optical coherence tomography or corrective optics. It is believed that acquisition of

auto£uorescence in the retinal pigment epithelium (RPE) is determined by its metabolic activity, which is dependent largely upon photoreceptor outer segment renewal. Thus loss of auto£uorescence implies photoreceptor cell death or at least outer segment loss. The presence of normal auto£uorescence implies that the photoreceptor cell layer is intact and that there are outer segments that are being constantly renewed. Abnormally high levels of auto£uorescence indicate inability of the RPE to process phagosomal material. That function can be poor in patients with an apparently intact outer retina is illustrated by normal auto£uorescence in a 15 year old with Leber amaurosis whose vision has not been recorded at better than light perception from early life.

In most outer retinal dystrophies the RPE remains physically present throughout disease, despite appearing very abnormal following loss of photoreceptor cells. Exceptions to this would include choroideremia.

Detection of the therapeutic e¡ect

Under certain circumstances gene therapy may cause gain of function. In other disorders there may be reversal of a speci¢c clinical attribute of disease such as the photophobia so characteristic of some with mutations in RetGC1. Both of these should be readily evident clinically. By contrast the e¡ect may be slowing or cessation of progression. This would be much more di⁄cult to detect. It will require very accurate and reproducible tests of function and recording of the physical state of the retina. It would also be required that the tests be well tolerated by patients. Many psychophysical tests of function require a great deal from the patient, whereas electrophysiology, being objective is somewhat easier. Some laboratories have good longitudinal recording of function, and patients could be selected for therapy in whom the time-course of the disease is known, and reliability of recording veri¢ed.

Internationally agreed protocols for tests of electrophysiological responses have been established (Marmor & Zrenner 1993) but for psychophysical attributes have not; this clearly needs to be addressed. Methods by which alteration of pro¢les of cell loss have only recently been addressed.

Clinical setting for treatment

It is evident that a large well-documented patient pool is necessary to realise the full bene¢ts of treatment, and should thus be introduced into clinical practice. It will be required that mutations be known and that the patients have welldocumented disease. Techniques for recording the treatment e¡ect such as electroretinography, psychophysics and specialized imaging must be available. This requires extensive registers of disease with all the available data recorded

and easily accessible by the clinicians involved. The testing equipment exists although not all major centres have access to all equipment needed. The techniques are practiced by many centres; reproducibility and progression have been well documented. Finally the centres should be easily accessed by patients.

To achieve these objectives it will take a great deal of time and e¡ort on the part of clinicians, scientists and patients. This activity should take place in parallel with the establishment of clinical services for inherited ocular diseases.

Conclusions

It is evident that biological approaches to treatment have achieved success in experimental studies. There is also reason to hope that a minor change in the metabolic environment of the retina can have a profound and long lasting e¡ect on the course of disease. The extension of work from rodents to larger animal models is in its early stages, in part because identi¢cation and generation of such models is very recent. This extension is another important precedent to human trials since larger eyes provide the opportunity to develop delivery systems and assess safety.

Each therapeutic approach has its potential advantages and disadvantages. As the work evolves it is possible that all three techniques will be used in combination. For example, transplantation of transgenic cells that express a growth factor over long periods may prove viable.

Although the biological treatment may be a future dream, it must be encouraging to the clinician and their patients that progress is being made in providing therapy for these intractable diseases. It is important that the clinical community is in a position to take advantage of these advances when clinical application can be justi¢ed. It will require a large number of patients with wellcharacterized disease, protocols by which the bene¢ts of treatment can be tested and centres that are capable of carrying out such tasks. The model is illustrated by the therapeutic trial of vitamin A, which was a monumental task that required immense discipline (Berson et al 1993).

References

Acland GM, Aguirre GD, Ray J et al 2001 Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 28:92^95

Ali RR, Sarra GM, Stephens C et al 2000 Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. Nat Genet 25:306^310

Berson EL, Rosner B, Sandberg MA et al 1993 A randomized trial of vitamin A and vitamin E supplementation for retinitis pigmentosa. Arch Ophthalmol 111:761^772

Faktorovich EG, Steinberg RH, Yasumura D, Matthes MT, LaVail MM 1990 Photoreceptor degeneration in inherited retinal dystrophy delayed by basic ¢broblast growth factor. Nature 347:83^86

Fitzke FW 2000 Imaging the optic nerve and ganglion cell layer. Eye 14:450^453