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The ultimate goal o f research into retinal degeneration in the rd mouse is to provide therapeutic and curative strategies that can be used to alleviate the symptoms o f human (and animal) eye disease. Morphological, biochemical and genetic

observations from the rd retina have led to two principal approaches in attempting to prevent/cure the disease: - to rescue/preserve photoreceptors or to reconstruct/replace lost photoreceptors. These methods, like those attempted in human RP, include drug treatment, growth factors, gene therapy, and retinal transplantation.

Drug treatment

As there is an abnormally high level o f cGMP in the rd mouse retina, D-cis-diltiazem, a calcium-channel blocker that also acts on light-sensitive cGMP-gated channels, has been shown to rescue cone photoreceptors and preserve their visual function in terms o f ERG amplitude at 25 and 36 days in the rd mouse (Frasson et a l, 1999). This work has yet to be confirmed by other investigators and meanwhile other studies using this drug on other animal models o f retinal degeneration were negative (Bush et a l, 2000; Pearce-Kelling et a l, 2001). Given the genetic heterogenieity o f RP in humans, this form o f treatment may only be beneficial in patients with mutations in

phosphodiesterase genes or probably other proteins o f the phototransduction cascade. Further studies will be required to assess whether this drug has a wider application.

Growth factors

In the retina, bFGF has been shown to slow the progression o f photoreceptor degeneration in RCS rats (Faktorovich et a/., 1990). This was the first time an

inherited retinal degeneration had been slowed significantly by a pharmacological agent. As a result there has been a surge in studies examining the possible therapeutic role o f growth factors in animal models o f retinal degeneration. Lambiase and Aloe (1996) have shown that intraocular and retro-ocular injections o f nerve growth factor (NGF), one o f the most highly characterised neurotrophins, given to young rd mice resulted in the preservation o f the ONL, when compared with untreated subjects. LaVail et a l (1998) later showed that intra-vitreal injections o f CNTF, but not brain- derived neurotrophic factor, leukaemia inhibitory factor, bFGF or insulin-like growth factor II, could slow the retinal degeneration in the rd mouse. The results with

Axokine were equivocal. New trophic factors are continuing to be identified that have an effect on photoreceptor rescue in rd mice, the most recent ones include glial cell line-derived neurotrophic factor (Frasson et al, 1999) and pigment epithelium- derived factor (Cayouette et a l, 1999). Recently, it was shown that growth factors in combination, rather than individually, rescued rd mouse photoreceptor in organ culture. These results demonstrate that trophic factors promote photoreceptor survival through a synergistic interaction (Ogilvie et a l, 2000). The protective action of growth factors seen in the above studies may be due to their inhibition o f apoptotic cell death which has been reported in the rd mouse retina (Porter-Calliau et a l, 1994). NGF has been shown to block apoptosis in rat embryo sympathetic neurones and chicken immune cells in vitro and it is possible that the effect o f growth factors in the rd mouse occurs through a similar mechanism (Lambiase and Aloe, 1996). Questions remain over the long term ability o f growth factors to provide photoreceptor rescue, these include possible toxic side effects, finding a safe mode o f delivering the growth factors, and establishing the correct choice o f growth factor for each particular

degeneration. Until these are addressed the therapeutic use o f growth factors in human eye disease is likely to be limited.

Gene therapy

Firstly gene therapy can be applied by gene augmentation. As retinal degeneration in the rd mouse is caused by a mutation in the gene encoding the pPDE, Lem et al.

(1992) expressed the normal bovine homologue o f PPDE in transgenic mice homozygous for the endogenous rd allele and showed that photoreceptors could be rescued. This indicated that the expression o f normal pPDE was sufficient to restore cGMP PDE activity and prevent photoreceptor degeneration. Furthermore, the photoreceptors showed normal morphological features up to the age o f three months, even ultrastructurally. Unfortunately, examination o f mice aged between four and seven months from one transgenic line (RP-33), revealed that a progressive degeneration was taking place in these retinae (Farber et al., 1994). In terms o f somatic retinal gene therapy, Bennett et al. (1996) showed that subretinal injections o f a recombinant replication-defective adenovirus that contained the murine cDNA for wild-type pPDE resulted in pPDE transcripts and increased PDE activity, delaying photoreceptor cell death by 6 weeks. This finding was the first to

demonstrate cell rescue by in vivo gene transfer, thus supporting the feasibility o f treating an inherited retinal degeneration by somatic gene therapy even though the effect was not lasting.

The second approach to gene therapy is by anti-apoptosis therapy. As apoptosis appears to be the common death pathway for RP animal models, its manipulation by genetic means appears logical. The bcl-2 gene (an anti-apoptotic gene) significantly enhanced photoreceptor survival in the rd mice if a wild-type copy o f the pPDE gene was also delivered (Bennett et a l, 1998).

The third approach is through growth factor gene delivery. As described earlier, particular growth factors have the capability to delay photoreceptor degeneration. Cayouette and Gravel (1997) had shown that following intravitreal injection o f an adenoviral vector encoding a NGF/CNTF fusion gene into one eye o f rd mice, many strong CNTF-immunoreactive profiles were detected in various cell types o f the injected eyes. Semiquantitative analysis o f the corresponding retinae reveals that the ONL retains significantly more rows o f photoreceptor nuclei than that o f eyes treated with a control (LacZ) vector, or untreated, for at least 18 days after vector

administration.

The last approach is probably the most exciting and involves ribozyme therapy. The problem with dominantly inherited mutations is the production o f inappropriate gene products disturbing metabolism and causing cell death. Ribozymes, small RNA molecules, act by cleaving mutant transcripts in an allele-specific manner while leaving the wild-type transcripts intact. LaVail et a l (2000) recently showed that in a rat model o f autosomal dominant RP, the P23H transgenic rat, ribozyme rescue appears to be a potentially effective long-term therapy with effects lasting up to eight months post-injection. It is also effective in late-stage therapy.

Intraocular transplantation

In the literature the earliest attempt to transplant into the eye was in 1873, when Van Dooremaal placed a variety o f cells, including human labial mucosa into the anterior chambers o f rabbit eyes (cited in del Cerro et aL, 1997), the purpose was to assess the survival o f various tissues. Over the years, the anterior chamber served as a site for transplantation studies because the clear cornea allowed observation o f grafted tissue

and the unusually low immune response in the anterior chamber was beneficial. However it was not until 1959, that the first neural retinal transplants into eyes were performed (Royo and Quay, 1959), where foetal rat retinae were implanted into the anterior chambers o f the eyes o f the mothers. The aim was to study the survival and development o f retinal tissue. Photoreceptors differentiated and many o f them formed rosettes, where cells group in a circular fashion with their inner segments and

rudimentary outer segments directed towards the luminal centre. The next advance was the discovery that immunological closeness o f donor and host tissue was not apparently necessary for successful transplantation in the eye (del Cerro et al., 1987). This experiment reconfirmed the concept o f the immune privilege o f the anterior chamber, however it is now known that it is not entirely correct and further studies are required.

Turner and Blair (1986) performed the first transplantation o f retinal neurones to the posterior segment when they transplanted neonatal rat retina into a specially lesioned site in the adult rat retina. Physical continuity between the graft and host was seen and the graft developed a laminar structure. Since then a number o f laboratories around the world have attempted neural retinal transplantation and reported positive findings with respect to photoreceptor transplant survival in the rd mouse. Initially Gouras et al. (1991) transplanted reporter gene labelled normal mouse photoreceptors into rd mouse retina with up to 4 weeks survival and showed that rudimentary outer segments were formed. Silverman et al. (1992) transplanted sheets o f mature mouse photoreceptors harvested by vibratome into rd mouse retina and showed that they survived for 3 months. Using photoreceptors labelled by a transgenic transporter gene and two different cell preparations o f neonatal cells, Du et a/. (1992) found surviving grafts at 2 months post-operation and EM revealed that some surviving

photoreceptors showed relatively mature outer segments. Silverman et al. (1994) has reported maintenance and regeneration o f cone outer segments in 4-month-old rd mice following transplantation o f photoreceptors at 4 weeks o f age. This was later supported by further works from Sahel’s group (Mohand-Said et al., 1997; Mohand- Said et al., 2000) who showed that rod photoreceptor transplants increased the survival o f host cones. These findings suggest that photoreceptor transplants have a certain rescue, as well as reconstruction, potential. Long-term survival o f grafted photoreceptor varies tremendously (del Cerro et al. 1997). Gouras et al. (1994) have shown that allogeneic photoreceptor transplants survived for at least 7 months in the rd retina without immunosuppression. However, Jiang and del Cerro (1992a), using the reciprocal transplantation paradigm, found that grafts o f normal foetal retinae into rd eyes faired less well than normal grafts to normal retina and they had a lower percentage o f survival with time. It was also suggested that donor age influenced the success o f retinal grafts (Aramant et al., 1988) and also the immune privilege o f allogeneic retinal grafts is not indefinite (Jiang et a l, 1995).