The gene Prph2 encodes a photoreceptor-specific membrane glycoprotein, peripherin-2 (also known as peripherin/rds), which is inserted into the rims of photoreceptor outer segment discs in a complex with rom-1 (ref. 2). The complex is necessary for the stabilization of the discs, which are renewed constantly throughout life, and which contain the visual pigments necessary for photon capture. Mutations in Prph2 have been shown to result in a variety of photoreceptor dystrophies, including autosomal dominant retinitis pigmentosa and macular dystrophy. A common feature of these diseases is the loss of photoreceptor function, also seen in the retinal degeneration slow (rds or Prph2 Rd2/Rd2) mouse, which is homozygous for a null mutation in Prph2. It is characterized by a complete failure to develop photoreceptor discs and outer segments, downregulation of rhodopsin and apoptotic loss of photoreceptor cells. The electroretinograms (ERGs) of Prph2Rd2/Rd2 mice have greatly diminished a-wave and b-wave amplitudes, which decline to virtually undetectable concentrations by two months. Subretinal injection of recombinant adeno-associated virus (AAV) encoding a Prph2 transgene results in stable generation of outer segment structures and formation of new stacks of discs containing both perpherin-2 and rhodopsin, which in many cases are morphologically similar to normal outer segments. Moreover, the re-establishment of the structural integrity of the photoreceptor layer also results in electrophysiological correction. These studies demonstrate for the first time that a complex ultrastructural cell defect can be corrected both morphologically and functionally by in vivo gene transfer.

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The retinal degeneration slow (rds or Prph2(Rd2/Rd2)) mouse, a model of recessive retinitis pigmentosa, lacks a functional gene encoding peripherin 2. This membrane glycoprotein is required for the formation of photoreceptor outer segment discs. The striking feature of the rds mouse is the complete failure to develop outer segments. We have previously examined the short-term effect of gene replacement therapy using an adeno-associated (AAV) vector and demonstrated induction of outer segments and improvement of photoreceptor function. Here we have extended our analysis and have demonstrated that the potential for ultrastructural improvement is dependent upon the age at which animals are treated, but the effect of a single injection on photoreceptor ultrastructure may be long-term. However, there was no significant effect on photoreceptor cell loss, irrespective of the date of administration, despite the improvements in morphology and function. Our investigation excluded procedure-related damage, vector toxicity and immune responses as major factors which might counteract the benefits of photoreceptor restoration, but suggested that transgene over-expression is of significance. These findings suggest that successful gene therapy in patients with photoreceptor defects may ultimately depend upon intervention in early stages of disease and upon accurate control of transgene expression.

In this study AAV-mediated gene replacement in Prph2(Rd2/Rd2) mice resulted in a significant functional improvement over a period of 14 weeks. These results support the utility of gene therapy approaches as treatment for photoreceptor dystrophies.

Using confocal microscopy we have examined in detail the temporal and spatial pattern of green fluorescent protein expression following sub-retinal injection of recombinant adeno-associated virus (rAAV) in the mouse and have determined the effect of viral titre on the number and type of cells transduced. Our results suggest that some transgene expression occurs as early as three days after injection, and that transgene expression occurs beyond the area of retinal detachment. Vector titre appears to have a substantial effect on both transduction efficiency and the speed of onset of photoreceptor cell transduction. Our data suggests that we have not yet reached the limits of photoreceptor transduction efficiency using AAV vectors. An increase in titre could still lead to an improved transduction efficiency and faster onset of photoreceptor transduction. We failed to detect transfected cones even in areas where nearly 100% of the rods were transduced, but we found efficient and sustained RPE transduction.