Long-term expression of glial cell line-derived neurotrophic factor slows, but does not stop retinal degeneration in a model of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a group of diseases caused by mutations in hundreds of different genes that cause death of rod photoreceptors. After rods die, there is a marked reduction in oxygen utilization resulting in high levels of oxygen in the outer retina causing progressive oxidative damage to cones and gradual cone cell death (Yu et al. 2000; Shen et al. 2005; Komeima et al. 2006). One treatment strategy is to use gene therapy to replace mutant genes in patients with recessive RP, but this would not apply to patients with dominant RP, and as causative mutations for recessive RP can occur in hundreds of genes, each gene replacement therapy will apply to only a small percentage of RP patients. Therefore, there is strong incentive to develop treatments that provide benefit irrespective of the underlying mutation. Neurotrophic factors are agents that promote survival of neurons that are under duress from a wide variety of insults and thus may provide a treatment that is applicable to all patients with RP. Increased expression of glial cell line-derived neurotrophic factor (GDNF) in the retina slows the death of rod photoreceptors in models of retinal degeneration and reduces retinal degeneration from severe oxidative stress (McGee et al. 2001; Dong et al. 2007; Dalkara et al. 2011; Del Rio et al. 2011). In addition, high levels of GDNF reduced levels of reactive oxygen species in the retina after intraocular injection of paraquat (Dong et al. 2007). The combined activities of slowing mutation-induced death of rods and providing protection against oxidative damage caused us to hypothesize that sustained expression of GDNF would prevent death of cones and thereby provide stable cone function over time. One way to test this is by gene transfer using AAV vectors. Although viral vectors, non-viral gene transfer, or another mechanism to provide sustained delivery of GDNF would be needed to apply this approach in patients, the transgenic approach provides advantages to test proof-of-concept in animal models. This is because after subretinal injection of an AAV vector, transduction of photoreceptors and retinal pigmented epithelium (RPE) cells is limited to the area of the retina detached by the injection and thereby exposed to the vector. As the area of detachment created by subretinal injection can vary considerably from mouse to mouse, this adds to variability and generally causes an underestimation of the optimal achievable effects. Prior to embarking on gene transfer, it is useful to utilize transgenic mice with tissue-specific promoters to drive expression of the transgene of interest in a uniform, predictable manner to try and determine the maximum achievable effects. We have utilized the tet/on inducible expression system (Tet), which utilizes the reverse tetracycline transactivator (rtTA) coupled to a tissue-specific promoter and the tetracycline response element (TRE) coupled to a gene of interest to test the effects of over-expressing various proteins in photoreceptors or retinal pigmented epithelium (Ohno-Matsui et al. 2002; Nambu et al. 2004; Oshima et al. 2005; Nambu et al. 2005; Usui et al. 2009). The interphotoreceptor retinoid binding protein (IRBP) promoter drives expression in both rod and cone photoreceptors (Yokoyama et al. 1992). We previously generated IRBP/rtTA-TRE/GDNF (Tet/IRBP/GDNF) double transgenic mice and demonstrated that they are resistant to oxidative damage-induced retinal degeneration (Dong et al. 2007). In RP, rods die from the underlying mutation and even with expression of GDNF in rods one would expect that the number of rods would decrease over time. Thus, one source of GDNF production would decrease, which could lead to a substantial reduction in GDNF production although it should still be produced in surviving cones which could be ideal for cone survival. We reasoned that production of GDNF in RPE cells which are adjacent to photoreceptors, but not subject to the degeneration, would allow us to test the effect of sustained high-level expression of GDNF and would be useful for comparison. The VMD2 (vitelliform macular dystrophy two) promoter drives expression specifically in RPE cells (Esumi et al. 2004) and therefore we used Tet/VMD2/GDNF double transgenic mice to test the effects of expression of GDNF in RPE cells.