Anda di halaman 1dari 14

Recombinant AAV-mediated gene transfer to the retina Gene therapy perspectives

Name: Mohamed Ahmed Zaki No: 984 / 2006

Under supervision of:

DR. Rania Abdesalam Dr. Mahmoud Soliman

Abstract
Retinal degenerative diseases such as retinal macular degeneration and retinitis pigmentosa constitute a broad group of diseases that all share one critical feature, the progressive apoptotic loss of cells in the retina. There is currently no effective treatment available by which the course of these disorders can be modified, and visual dysfunction often progresses to total blindness. Gene therapy represents an attractive approach to treating retinal degeneration because the eye is easily accessible and allows local application of therapeutic vectors with reduced risk of systemic effects. Furthermore, transgene expression within the retina and effects of treatments may be monitored by a variety of noninvasive examinations. An increasing number of strategies for molecular treatment of retinal disease rely on recombinant adeno-associated virus (rAAV) as a therapeutic gene delivery vector. Before rAAV-mediated gene therapy for retinal degeneration becomes a reality, there are a number of important requirements that include: (1) evaluation of different rAAV serotypes, (2) screening of vectors in large animals in order to ensure that they mediate safe and long-term gene expression, (3) appropriate regulation of therapeutic gene expression, (4) evaluation of vectors carrying a therapeutic gene in relevant animal models, (5) identification of suitable patients, and finally (6) manufacture of clinical grade vector. All these steps towards gene therapy are still being explored. Outcomes of these studies will be discussed in the order in which they occur, from vector studies to preclinical assessment of the therapeutic potential of rAAV in animal models of retinal degeneration.

Introduction
Significant progress in understanding the molecular basis of a variety of retinal diseases has led to the development of gene therapy approaches for treatment of these diseases. Of all the vectors that have been evaluated in the eye, recombinant adeno-associated virus (rAAV) is potentially the most useful for treatment of retinal diseases as its tropism and transduction patterns lead to efficient and stable gene transfer in retinal pigmented epithelium (RPE), photoreceptor and ganglion cells. rAAV-mediated gene therapy studies to date have involved gene transfer to spontaneous and transgenic animal models of the diseases. The retinal diseases that have been studied include neovascular diseases (that are features of age-related macular degeneration, diabetic retinopathy and retinopathy of prematurity) and retinal degenerative diseases (such as retinitis pigmentosa and Leber congenital amaurosis).

rAAV serotypes and tropism


Adeno-associated virus type 2 (AAV-2) is a human parvovirus that has gained increasing interest because of its use as a successful gene transfer vector in a variety of rodent and large animal models. The AAV-2 genome consists of a 4.7 kb single-stranded DNA molecule, which is composed of two open reading frames, rep and cap, flanked by two 145 bp inverted terminal repeats (ITRs). Recombinant AAV-2 (rAAV-2) vectors used for gene therapy are derived from the wild-type virus by deleting the entire viral coding region (rep and cap) and replacing them by the reporter or therapeutic transgene. More recently, seven other rAAV serotypes (AAV-1, -3, -4, -5, -6, -7 and 8) have been isolated and cloned. Recombinant AAV-2 vectors are capable of mediating efficient and prolonged transgene expression in a number of tissues and have been used to deliver therapeutic genes to correct

defects in animal models of various human disorders. In the retina, following subretinal delivery, AAV-2 vectors transduce RPE and photoreceptor cells. Intravitreal injection of rAAV-2 leads to efficient ganglion cell transduction. We and others have studied rAAV chimeric serotypes in which the vector is flanked by AAV-2 ITRs, but encapsidated in an AAV-1, -2, -3, -4 or -5 shell generating rAAV-2/1,-2/2, -2/3, -2/4 and 2/5, respectively. In the mouse, subretinal injection of rAAV-2/1egfp shows early onset of EGFP expression (34 days) with transgene expression restricted to the RPE. In contrast, onset of transgene expression occurs at 24 weeks for AAV-2/2 and AAV-2/5 with both vectors transducing RPE and photoreceptors cells. No transduced cells are detected following intravitreal injection of AAV-2/1 and AAV-2/5. Subretinal injection of AAV-2/1, -2/2, -2/3, 2/4 and -2/5 in rats results in a hierarchy in the levels of transgene expression, with rAAV-2/4 and -2/5 capsids being the most efficient. Additional studies with these two vectors have confirmed that AAV-2/5 transduces both RPE and photoreceptor cells in rat and dog, with photoreceptors being more efficiently transduced than with AAV-2/2. More importantly, this study demonstrated that the type 4 capsid allows exclusive and stable transduction of RPE cells, and that this feature is conserved among rodent, canine and non-human primate models. Similar to the chimeric rAAV-2/5 vector, subretinal injection of a complete rAAV-5/5 vector in mouse shows faster expression kinetics and results in higher transduction efficiency compared with rAAV-2/2,27 with RPE cells and photoreceptors being transduced in both case. Tropisms of rAAV vectors in different species are reviewed in Table 1.

Table 1. Tropism of rAAV serotypes following subretinal injection


rAAV-2/2 rAAV-2/1 rAAV-2/4 rAAV-2/5 rAAV-5/5 Mouse RPE+PR RPE RPE+PR RPE+PR Rat RPE+PR RPE RPE+PR Dog RPE+PR RPE RPE+PR Primate RPE+PR RPE __ RPE+PR

Gene transfer in large animals


Evaluation of rAAV-mediated gene transfer in the eye of large animals is highly relevant with respect to future clinical development as it is anatomically more similar to the human eye than either the mouse or rat counterpart. The non-human primate possesses ocular anatomic features virtually identical to those of the human, its components are of similar proportion and it possesses a macula. Also, surgical procedures for vector delivery and amount of injected vector in dog and primate will be similar to those that will be used in humans. In addition, primates have greater immunological and biological similarity to humans, and this is essential for determining the tolerance of vectors before clinical trials. As direct visualization of the green fluorescent protein (GFP) expression in the retina can easily be carried out by fluorescence fundus photography on anesthetized animals, vectors encoding for the GFP gene were used for the evaluation of rAAV-mediated gene transfer. Subretinal injection of rAAV-2/2 in dogs results in efficient transduction of RPE and rod and cone photoreceptors. In the primate, delivery of this same vector using an identical route leads to efficient transduction of rod photoreceptors, while cones are poorly transduced. A recent study has shown that in primates, rAAV-5/5 also has preferential tropism for rod photoreceptors.

For chimeric vectors rAAV-2/5 and rAAV-2/4, subretinal injection in dogs results in stable expression of GFP for more than 18 months (Rolling et al, data not shown), with the tropism being identical to the one observed in the rat: transduction of RPE and photoreceptor cells with type 5 and exclusive transduction of RPE with type 4.

Biodistribution and safety


There is now evidence for the potential therapeutic value of rAAV vectors for treatment of retinal diseases in different animal models. The primary requirement for safe and successful ocular gene therapy trials using rAAV is an accurate evaluation of vector biodistribution after subretinal or intravitreal administration. A number of studies have evaluated the biodistribution of rAAV following various routes of administration. Not only are multiple cell types in a target organ exposed to the vector but also some scatter of virus to the systemic circulation occurs with most routes of delivery. Non-human primate studies have demonstrated that rAAV-2/2 delivery to the muscle or lung results in detectable vector in the serum at 6 days (muscle) to 14 days (lung). Examination of tissue by PCR 18 months after rAAV intramuscular administration indicated the presence of rAAV genome in the liver, peripheral blood mononuclear cells (PBMC) and scattered lymph node sites. Primates that received intrabronchial rAAV showed very low level of rAAV DNA at these same extrapulmonary sites as well as in the heart, kidney, pancreas and brain. With regard to gene transfer in the retina, biodistribution of these vectors in distant organs following intraocular administration has never been documented. We have reported vector shedding after subretinal delivery of rAAV in the dog and non-human primates. Vector DNA could be detected in the serum as early as 15 min after rAAV administration and for up to 25 days in some instances. In lacrymal and nasal fluids, viral genome was also detected as early as 15 min after injection and up to 4 days postinjection (p.i.). Regarding intravitreal injection, it has been demonstrated that following delivery of rAAV-2/2gfp, high levels of GFP protein persist for at least 6 months in the optic nerves and brains of mice and dogs. In addition, injection of rAAV-2/2lacZ in the vitreous cavity of guinea pigs leads to -galactosidase expression in fibers, glial cells and blood vessels of the optic nerve. More recently, it was shown that GUSB activity can be detected in the brain after intravitreal injection of rAAV2/2gusb in mucopolysaccharidosis VII mice, suggesting that the protein, not the vector, spreads within the brain by both neuronal transport and diffusion. Although transgene product has been observed in the brain and optic nerve following intravitreal injection of rAAV-2/2, to date, biodistribution in distant organs has yet to be reported. We have now designed a study to evaluate biodistribution of rAAV vectors in distant organs following subretinal or intravitreal injection in the rat, dog and non-human primate. Following subretinal and intravitreal injections, rAAV sequences were not detected in the liver or in the gonads but were sometimes found in PBMC at early time points after vector delivery. The most striking result was the detection of rAAV sequences in different regions of the brain after intravitreal injection in dog (Rolling et al, data not shown). Although additional biodistribution studies in large animal are required, these preliminary results suggest the ability for rAAV-2/2 to achieve trans-neuronal transport.

Regulation of transgene expression in vivo


As described above, the ability of rAAV to efficiently transduce retinal cells (RPE, photoreceptors and ganglion cells) in rodent and large animal models is well documented. The restoration of vision in RPE65 dogs, and the morphological and functional rescue of photoreceptors in the prph2 mouse model and in the RCS rat provides evidence to support rAAV-mediated gene therapy replacement strategies. An alternative to these gene-specific therapies, which target a particular gene defect, is a generic approach using factors promoting cell survival. Transgenes encoding survival factors might constitute an appropriate choice for the treatment of RP. rAAV-mediated gene transfer using neurotrophic factors, such as ciliary neurotrophic factor (CNTF), fibroblast growth factors (FGF) and glial cell line-derived neurotrophic factors (GDNF), have increased photoreceptors survival in different rodent models of retinal degeneration. However, a key issue for successful ocular gene therapy using such neurotrophic factors is the ability to regulate expression for pharmacological and safety reasons. Many proteins have more narrow therapeutic windows, and overproduction could result in toxicity. Several regulation systems, including rapamycin, mifepristone, ecdysone and tetracycline (tet) have been developed to control transgene expression in vivo. Rapamycin-mediated regulation of erythropoietin (Epo) expression has been demonstrated in rats and in one non-human primate following subretinal injection of two rAAV vectors, one encoding for the transactivator, the other for Epo. The tetracycline system has also proved to be efficient and reliable in controlling transgene expression in vivo following intramuscular injection of rAAV carrying the tetracycline transactivator and Epo cDNA in mice and in primates. In the retina, tetracycline-controlled expression of GFP in RPE and photoreceptor cells has been demonstrated in rats following subretinal injection of rAAV-2/2 vector. However, restricting transduction to the neuroretina, and more specifically to ganglion cells, following intravitreal (not subretinal) injection, would be a more attractive approach for neurotrophic factor gene transfer as it should allow transduction over the entire retina. We have demonstrated that a single rAAV-2/2 carrying the tTA and the GFP cDNAs can provide controlled transgene expression in the rat ganglion cells after intravitreal injection. In this last study, reiterated regulation of the transgene was carried out for 6 months. One alternative to these strategies, which are dependent on administration of exogenous pharmacological agents, is to couple transgene expression to changes in the local tissue environment. For example, it is possible to use a hypoxia-responsive element (HRE) that allows low basal expression in normoxic conditions with high-level activated expression when the oxygen concentration is low. Following subretinal delivery of rAAV-2/2HRE.gfp in murine models of retinal and choroidal neovascularization, transgene expression was targeted to sites of neovascularization and resulted in expression that is not sustained beyond the period of active angiogenesis.

Gene transfer in animal models of retinal degeneration


For several decades, research on acquired and inherited retinal degeneration was based on a variety of animal models, most of them originating from spontaneous mutations, others induced by damaging external agents. More recently, progress in genetic engineering has led to a rapidly growing number of transgenic animals, mostly mice, carrying constructs that lead to disruption or over expression of genes resulting in retinal degenerations. These animal

models were firstly used for the study of the molecular mechanism leading to retinal degeneration. These animal models often have a similar phenotype to humans and therefore constitute a powerful tool for investigation of treatment by gene therapy. Demonstrating 'proof of principle' and safety in a number of animal models and species allows us to approaching clinical trials with increased confidence. A summary of the rAAV-mediated gene therapy studies is listed in Table 2.

Table 2. Recombinant AAV-mediated gene therapy studies


Transgene AntiPEDF angiogenic K1K3 protein sFlt1 Sendostain TIMP-3 Survival factor FGF-2 FGF-5 FGF-18 GDNF CNTF Gene Peripherin-2 replacement Mertk therapy Rpe65 Animal model ROP mouse,CNV mouse ROP mouse ROP mouse, CNV rat ROP mouse ROP mouse S334 rat S334 rat S334 rat S334 rat Opsin-/- mouse Prph2Rd2/Rd2 mouse, RCS rat
Briard dog, RPE65-/- mouse

Disease ROP,CNV ROP ROP ROP ROP RP RP RP RP RP RP RP LCA

Reference 61 ,63 61 62 , 65 63 63 37 38 38 39 71 35,74,75 36 33,34,82

Nonspecific gene therapy Antiangiogenic proteins

Neovascular diseases of the retina include retinopathy of prematurity (ROP), diabetic retinopathy (PDR) and age-related macular degeneration (AMD). Diabetic retinopathy and AMD are the leading causes of blindness in developed countries. Angiogenesis is a complex multistep process that involves out-sprouting of vascular endothelial cells from existing vessels. Regulation of vascularization in the mature retina involves a balance between endogenous positive growth factors, such as vascular endothelial growth factor (VEGF) and inhibitors of angiogenesis, such as pigment epithelium-derived factor (PEDF). All current treatments (surgical, laser and photodynamic therapy) merely delay severe vision loss, because they are directed at destroying new vessels but do not address the underlying angiogenic stimuli that frequently cause recurrences. Currently, there are no antiangiogenic treatments available for patients with ocular neovascularization, but several new approaches such as intraocular injections of an aptamer that binds VEGF or an anti-VEGF antibody or proteins with antiangiogenic activity are being considered. Recombinant AAV-mediated gene transfer offers an alternative for local sustained delivery of antiangiogenic proteins to intraocular tissues. Regarding retinal neovascularization (NV), the efficacy of rAAV-2/2-mediated expression of PEDF, Kringle domains 13 of angiostatin (K1K3) and the soluble VEGF receptor sFlt-1, in reducing aberrant vessel formation in a mouse model of ischemia-induced retinal neovascularization, have been evaluated. rAAV-2/2pedf and rAAV-2/2k1k3 vectors were

injected into one eye of postnatal day 0 (P0) newborn mouse pups. Retinal neovascularization was induced in P7 mice by exposure to elevated oxygen for 5 days followed by room air for another 5 days. Retinal NV was quantified by the number of vascularendothelial cells nuclei above the inner-limiting membrane in P17 eyes. The number of such vascular endothelial cell nuclei in eyes treated with rAAV-2/2pedf and rAAV-2/2k1k3 was significantly reduced compared with untreated eyes. For rAAV-2/2sFlt1 assessment, the vector was intravitreally injected at P2 and retinal neovascularization was quantified on retinal flatmounts following perfusion with dextran-conjugated FITC at p19. Eyes injected with AAV-2/2sFlt1 displayed fewer neovascular complexes than PBS-injected eyes. Similar promising results were obtained in the same mouse model of retinopathy of prematurity, using rAAV-2/1-carrying genes encoding for endostatin, tissue inhibitor of metalloproteinase-3 (TIMP-3) or PEDF. For choroidal neovascularization (CNV), an rAAV-2/2pedf was intravitreally or subretinally injected in mice where the Bruch's membrane was ruptured with laser photocoagulation, a mice model of induced neovascularization. At 46 weeks after intraocular injection of rAAV2pedf, the area of CNV at the Bruch's membrane rupture sites had significantly decreased compared with the CNV area at rupture sites in eyes injected with rAAV-2/2gfp. It was also demonstrated that, in a similar rat model of CNV, subretinal injection of an rAAV-2 encoding soluble VEGF receptor 1 (sFlt-1) was able to suppress choroidal NV at the laser lesions. These data provide evidence that intraocular expression of antiangiogenic proteins such as PEDF or sFlt-1, via rAAV vectors, may offer a new treatment for ocular neovascularization.

Survival factors

Inherited retinal degenerations are triggered by mutations in a variety of different genes. Although the mechanisms by which the genetic defects lead to photoreceptor death are still not clear, the final common pathway is apoptosis. Since specific gene therapies cannot readily be used to treat a significant fraction of patients with retinal dystrophy, there is a major interest in developing a more generally applicable survival factor therapy, which does not target the mutant gene product but rather alters the photoreceptor environment in a manner that promotes cell survival. Neurotrophic factors have the ability to modulate neuronal growth during development to maintain existing cells and to allow recovery of injured neuronal populations. In the retina, Lavail et al have had success in ameliorating photoreceptor cell death with direct protein injections of different survival factors in several mice model of photoreceptor degeneration. More recently, recombinant AAV-2/2 encoding fibroblast growth factor family members FGF-2, FGF-5 and FGF-18 have been evaluated using a transgenic rat model of retinitis pigmentosa, the S334ter-4 rat line that expresses a mutated rhodopsin gene. Despite the lack of significantly increased electroretinograms (ERGs), treated rats displayed a greater number of surviving photoreceptors following subretinal injection of these vectors, suggesting the ability of these factors to protect photoreceptors from apoptosis in this rat model of RP. The same RP rat model was used to evaluate the therapeutic potential of GDNF. In rAAV2/2gdnf-treated rats, photoreceptor cell death was slowed for at least 45 days and animals exhibited greater ERG a-wave mean amplitudes. Previous reports have indicated that intraocular delivery of encapsulated CNTF-producing cells is able to delay photoreceptor degeneration in rcd1 canine model of retinitis pigmentosa. These findings suggested that a continual supply of CNTF via rAAV-mediated gene transfer might be able to prolong

photoreceptor survival. One study of rAAV-2/2cntf-mediated gene transfer in an opsin -/mouse showed prolongation of photoreceptors survival following subretinal administration. Interestingly, the same vector was injected intravitreally in postnatal Prph2Rd2/Rd2 mice and in adult P23H and S334ter rhodopsin transgenic rats. In these two cases, retinas receiving rAAV-2/2cntf showed prominent morphological protection of photoreceptors, but with no improved ERG in the Prph2 Rd2/Rd2 mice and with lower ERG amplitudes in rAAV-2/2cntfinjected rat models compared with rAAV-2/2gfp-injected ones. This discordance of functional and morphological results suggested a deleterious effect of intraocular CNTF gene delivery on the retina. Two recent reports clearly demonstrated that intraocular CNTF expression using rAAV-2/2-mediated gene delivery, in wild-type mice, in Prph2Rd2/Rd2 mice and in mice with a P216L rds/peripherin mutation, resulted in a significant decrease of function, as assessed by ERG. The mechanisms for these side effects should be determined before clinical trials involving CNTF are considered.

Gene replacement therapy


Gene replacement therapy constitutes the most straight forward and therefore perhaps the most promising approach for treating autosomal recessive retinal disease. For this strategy, the therapeutic gene has to be directly transferred into the cells in which the gene is normally expressed, usually photoreceptors or the RPE. The first convincing gene replacement study using rAAV vectors was achieved in Prph2Rd2/Rd2 mice. The Prph2 Rd2/Rd2 mouse is homozygous for a null mutation in peripherin 2 gene, and completely fails to develop photoreceptor discs and outer segments. Consequently, the photoreceptor cell layer undergoes progressive apoptotic cell loss. Functionally, ERGs have greatly diminished a-wave and b-wave amplitudes, which decline to undetectable levels by 2 months. Subretinal injection of rAAV-2 encoding peripherin-2 transgene resulted in the re-establishment of the structural integrity of the photorecetor layer as well as in electrophysiological correctionExtended analysis of this gene therapy treatment in the same model demonstrated that the potential for ultrastructural improvement is dependent upon the age at which animals are treated and that overexpression of the prph 2 might be harmful to photoreceptor cells. The Royal College of Surgeons (RCS) rat is a widely studied animal model of retinal degeneration in which the inability of the RPE to phagocytose shed photoreceptor outer segments leads to a progressive loss of rod and cone photoreceptors. The defect, which has also been described in patients with retinitis pigmentosa, results from a mutation in the mertk gene, which is normally expressed in the RPE. The first demonstration of successful rescue of both a functional cellular defect (phagocytosis) and a photoreceptor degeneration was achieved by recombinant adenovirus-mediated gene transfer to the RPE. More recently, it was demonstrated that rAAV-mediated gene transfer slows photoreceptor loss in this model. Subretinal injection of rAAV-2/2mertk prolong photoreceptor cell survival in the RCS rat, as assessed by histology. Moreover, electroretinographic analysis of treated eyes showed that functional photoreceptors were still present at 9 weeks, when there is virtually no activity in untreated control eye. The last set of studies tested rpe65 gene replacement therapy in murine model of Leber Congenital Amaurosis (LCA) and in Briard dogs with the same genetic defect. The rpe65 gene encodes a 65-kDa-specific protein essential for synthesis of the rhodopsin ligand 11-cis-

retinal. Mutations in RPE65 are responsible for approximately 2% of autosomal recessive retinal dystrophy cases. Rpe65-null mutant transgenic mice and rpe65-/- affected Briard dogs do not generate adequate levels of visual pigment, they have very poor vision and show severely depressed light- and dark-adapted ERG responses. The fact that there are close similarities between the clinical characteristics of the diseases resulting from RPE65 gene mutations in humans and in the dog make the RPE65-/- Briard a valuable model for the evaluation of gene therapy. Two studies testing gene replacement therapy using AAV-2/2mediated delivery of a canine rpe65 gene have now been reported. They both demonstrated restoration of dark- and light-adapted ERG responses and improved psychophysical outcomes in the Briard dog. Although these results are promising, Narfstrom et al have reported that uveitis developed in 75% of the rAAV-2/2rpe65-treated eyes, which could be attributed either to the presence of a contaminant in the vector preparation or may result from an immune response to the RPE65 protein. The cause of this side effect should be determined before human clinical trials are considered. As the rAAV-2/2 vectors used in the two reported studies used a CMV promoter, high levels of rpe65 expression occurred in both the RPE and photoreceptors cells. Since the long-term effects of this are not known, it might be safer to use a RPE-specific promoter RPE65 promoter and/or an rAAV vector that target transgene expression specifically to the RPE, such as serotype 4 or 1. It has been recently reported that it is possible to restore visual function to the rpe65-/- mouse following in utero administration of an rAAV-2/1rpe65 vector. However, many more steps, ranging from safety studies to discussion of ethical implications, must be taken before in utero gene therapy for human retinal diseases can be envisaged.

Conclusion
The proof-of-principle of rAAV-mediated gene therapy in rodent models of RP and now in a species with a human-sized eye (ie the dog) suggests that ocular gene therapy might soon become a reality. The demonstration that rAAV-mediated intraocular expression of PEDF reduces CNV at sites of rupture of the Bruch's membrane in mice and the fact that PEDF has neuroprotective activity makes this gene therapy strategy a potential treatment for patients with AMD. The potential advantage of using PEDF as the antiangiogenic protein is that this protein is naturally produced by RPE cells and found at high concentrations within the retina as well as in the vitreous. The approach of delivering genes encoding neuroprotective factors is perhaps more complex as the expression of genes not naturally expressed or expressed at low levels in the retina might be deleterious. As observed with CNTF, although transgene expression slows photoreceptor cell loss in rodents, normal retinal function was not preserved. There is probably a requirement to express survival factors within an appropriate therapeutic window, therefore, rAAV vectors in which transgene expression can be tightly regulated will be required. Gene replacement therapy, which involves a gene that is naturally expressed within the targeted cells, constitutes the most straightforward and promising gene transfer approach. Current proof-of-principle in the Briard dog model suggests that gene therapy for childhood-onset retinal dystrophy, due to a defect in RPE65 gene, may become the first human ocular gene therapy trial. Although restoration of vision was demonstrated using AAV-2/2-mediated delivery of canine RPE65, rAAV serotypes that lead to exclusive transgene expression in RPE might provide better vehicles for long-term safe and stable gene transfer. While the therapeutic potential of this novel rAAV-mediated treatment appears promising for LCA, additional safety studies involving pharmacokinetics, biodistribution and toxicity will need to be accurately evaluated in large animal models before clinical trials might proceed.

References
1. Snyder RO. Adeno-associated virus-mediated gene delivery. J Gene Med 1999; 1: 166175. 2. Monahan PE, Samulski RJ. AAV vectors: is clinical success on the horizon? Gene Therapy 2000; 7: 2430. 3. Xiao W et al. Gene therapy vectors based on adeno-associated virus type 1. J Virol 1999; 73: 39944003. 4. Muramatsu S et al. Nucleotide sequencing and generation of an infectious clone of adeno-associated virus 3. Virology 1996; 221: 208217. 5. Chiorini JA et al. Cloning of adeno-associated virus type 4 (AAV4) and generation of recombinant AAV4 particles. J Virol 1997; 71: 68236833 6. Chiorini JA et al. Cloning and characterization of adeno-associated virus type 5. J Virol 1999; 73: 13091319.
7. Rutledge EA, Halbert CL, Russell DW. Infectious clones and vectors derived from adenoassociated virus (AAV) serotypes other than AAV type 2. J Virol 1998; 72: 309319.

8. Gao GP et al. Rep/Cap gene amplification and high-yield production of AAV in an A549 cell line expressing Rep/Cap. Mol Ther 2002; 5: 644649 9. Bohl D et al. Improvement of erythropoiesis in beta-thalassemic mice by continuous erythropoietin delivery from muscle. Blood 2000; 95: 27932798 10. Bosch A et al. Long-term and significant correction of brain lesions in adult mucopolysaccharidosis type VII mice using recombinant AAV vectors. Mol Ther 2000; 1: 6370 11. Daly TM et al. Neonatal gene transfer leads to widespread correction of pathology in a murine model of lysosomal storage disease. Proc Natl Acad Sci USA 1999; 96: 22962300 12. Elliger SS et al. Elimination of lysosomal storage in brains of MPS VII mice treated by intrathecal administration of an adeno-associated virus vector. Gene Therapy 1999; 6: 11751178. 13. Jung SC et al. Adeno-associated viral vector-mediated gene transfer results in longterm enzymatic and functional correction in multiple organs of Fabry mice. Proc Natl Acad Sci USA 2001; 98: 26762681. 14. Snyder RO et al. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med 1999; 5: 6470. 15. Wang B, Li J, Xiao X. Adeno-associated virus vector carrying human minidystrophin genes effectively ameliorates muscular dystrophy in mdx mouse model. Proc Natl Acad Sci USA 2000; 97: 1371413719. 16. Xiao X et al. Full functional rescue of a complete muscle (TA) in dystrophic hamsters by adeno-associated virus vector-directed gene therapy. J Virol 2000; 74: 14361442. 17. Ali RR et al. Gene transfer into the mouse retina mediated by an adeno-associated viral vector. Hum Mol Genet 1996; 5: 591594. 18. Ali RR et al. Adeno-associated virus gene transfer to mouse retina. Hum Gene Ther 1998; 9: 8186. 19. Bennett J et al. Stable transgene expression in rod photoreceptors after recombinant adeno-associated virus-mediated gene transfer to monkey retina. Proc Natl Acad Sci USA 1999; 96: 99209925. 20. Dudus L et al. Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV. Vis Res 1999; 39: 25452553 21. Liang FQ et al. Long-term protection of retinal structure but not function using RAAV. CNTF in animal models of retinitis pigmentosa. Mol Ther 2001; 4: 461472

22. Guy J et al. Reporter expression persists 1 year after adeno-associated virus-mediated gene transfer to the optic nerve. Arch Ophthalmol 1999; 117: 929937 23. Folliot S et al. Sustained tetracycline-regulated transgene expression in vivo in rat ganglion cells using a single type 2 adeno-associated viral vector. J Gene Med 2003; 5: 493501 24. Auricchio A et al. Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model. Hum Mol Genet 2001; 10: 30753081. 25. Rabinowitz JE et al. Cross-packaging of a single adeno-associated virus (AAV) type 2 vector genome into multiple AAV serotypes enables transduction with broad specificity. J Virol 2002; 76: 791801. 26. Weber M et al. Recombinant adeno-associated virus serotype 4 mediates unique and exclusive long-term transduction of retinal pigmented epithelium in rat, dog, and nonhuman primate after subretinal delivery. Mol Ther 2003; 7: 774781 27. Yang GS et al. Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size. J Virol 2002; 76: 76517660. 28. Bainbridge JW et al. Stable rAAV-mediated transduction of rod and cone photoreceptors in the canine retina. Gene Therapy 2003; 10: 13361344 29. Lotery AJ et al. Adeno-associated virus type 5: transduction efficiency and cell-type specificity in the primate retina. Hum Gene Ther 2003; 14: 16631671 30. Favre D et al. Immediate and long-term safety of recombinant adeno-associated virus injection into the nonhuman primate muscle. Mol Ther 2001; 4: 559566. 31. Conrad CK et al. Safety of single-dose administration of an adeno-associated virus (AAV)-CFTR vector in the primate lung. Gene Therapy 1996; 3: 658668. 32. Hennig AK et al. Intravitreal gene therapy reduces lysosomal storage in specific areas of the CNS in mucopolysaccharidosis VII mice. J Neurosci 2003; 23: 33023307 33. Acland GM et al. Gene therapy restores vision in a canine model of childhood blindness. Nat Genet 2001; 28: 92 95. 34. Narfstrom K et al. Functional and structural recovery of the retina after gene therapy in the RPE65 null mutation dog. Invest Ophthalmol Vis Sci 2003; 44: 1663 1672. 35. Ali RR et al. Restoration of photoreceptor ultrastructure and function in retinal degeneration slow mice by gene therapy. Nat Genet 2000; 25: 306 310. 36. Smith AJ et al. AAV-mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa. Mol Ther 2003; 8: 188195 37. Lau D et al. Retinal degeneration is slowed in transgenic rats by AAV-mediated delivery of FGF-2. Invest Ophthalmol Vis Sci 2000; 41: 36223633. 38. Green ES et al. Two animal models of retinal degeneration are rescued by recombinant adeno-associated virus-mediated production of fgf-5 and fgf-18. Mol Ther 2001; 3: 507515. 39. McGee Sanftner LH et al. Glial cell line derived neurotrophic factor delays photoreceptor degeneration in a transgenic rat model of retinitis pigmentosa. Mol Ther 2001; 4: 622629 . 40. Rivera VM et al. A humanized system for pharmacologic control of gene expression. Nat Med 1996; 2: 10281032. 41. Ye X et al. Regulated delivery of therapeutic proteins after in vivo somatic cell gene transfer. Science 1999; 283: 8891. 42. Serguera C et al. Control of erythropoietin secretion by doxycycline or mifepristone in mice bearing polymer-encapsulated engineered cells. Hum Gene Ther 1999; 10: 375383

43. No D, Yao TP, Evans RM. Ecdysone-inducible gene expression in mammalian cells and transgenic mice. Proc Natl Acad Sci USA 1996; 93: 33463351. 44. Gossen M, Bujard H. Tight control of gene expression in mammalian cells by tetracyclin-responsive promoters. Proc Natl Acad Sci USA 1992; 89: 55475551. 45. Gossen M et al. Transcriptional activation by tetracyclines in mammalian cells. Science 1995; 268: 17661769 46. Auricchio A et al. Pharmacological regulation of protein expression from adenoassociated viral vectors in the eye. Mol Ther 2002; 6: 238 47. Bohl D, Naffakh N, Heard J-M. Long-term control of erythropoietin secretion by doxycycline in mice transplanted with engineered primary myoblasts. Nat Med 1997; 3: 299305. 48. Bohl D et al. Control of erythropoietin delivery by doxycyclin in mice after intramuscular injection of adeno-associated vector. Blood 1998; 92: 15121517. 49. Favre D et al. Lack of immune response against the tetracycline-dependent transactivator correlates with long-term doxycycline-regulated transgene expression in nonhuman primates after intramuscular injection of recombinant adeno-associated virus. J Virol 2002; 76: 1160511611. 50. McGee Sanftner LH et al. Recombinant AAV-mediated delivery of a tet-inducible reporter gene to the rat retina. Mol Ther 2001; 3: 688 696. 51. Bainbridge JW et al. Hypoxia-regulated transgene expression in experimental retinal and choroidal neovascularization. Gene Therapy 2003; 10: 10491054 52. Hafezi F et al. Molecular ophthalmology: an update on animal models for retinal degenerations and dystrophies. Br J Ophthalmol 2000; 84: 922927. 53. Witmer AN et al. Altered expression patterns of VEGF receptors in human diabetic retina and in experimental VEGF-induced retinopathy in monkey. Invest Ophthalmol Vis Sci 2002; 43: 849857. 54. Behling KC, Surace EM, Bennett J. Pigment epithelium-derived factor expression in the developing mouse eye. Mol Vis 2002; 8: 449454. | 55. Chader GJ. PEDF: raising both hopes and questions in controlling angiogenesis. Proc Natl Acad Sci USA 2001; 98: 21222124 56. Ciulla TA, Danis RP, Harris A. Age-related macular degeneration: a review of experimental treatments. Surv Ophthalmol 1998; 43: 134146 57. Krzystolik MG et al. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch Ophthalmol 2002; 120: 338346 58. O'Reilly MS et al. Angiostatin: a novel angiogenesis inhibitor that mediates the suppression of metastases by a Lewis lung carcinoma. Cell 1994; 79: 315328. 59. O'Reilly MS et al. Endostatin: an endogenous inhibitor of angiogenesis and tumor growth. Cell 1997; 88: 277285. 60. Stellmach V et al. Prevention of ischemia-induced retinopathy by the natural ocular antiangiogenic agent pigment epithelium-derived factor. Proc Natl Acad Sci USA 2001; 98: 25932597. 61. Raisler BJ et al. Adeno-associated virus type-2 expression of pigmented epitheliumderived factor or Kringles 1-3 of angiostatin reduce retinal neovascularization. Proc Natl Acad Sci USA 2002; 99: 89098914. 62. Bainbridge JW et al. Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1. Gene Therapy 2002; 9: 320326 63. Auricchio A et al. Inhibition of retinal neovascularization by intraocular viralmediated delivery of antiangiogenic agents. Mol Ther 2002; 6: 490.

64. Mori K et al. AAV-mediated gene transfer of pigment epithelium-derived factor inhibits choroidal neovascularization. Invest Ophthalmol Vis Sci 2002; 43: 19942000. 65. Lai YK et al. Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy. Gene Therapy 2002; 9: 804813. 66. Phelan JK, Bok D. A brief review of retinitis pigmentosa and the identified retinitis pigmentosa genes. Mol Vis 2000; 6: 116124. 67. Chang GQ, Hao Y, Wong F. Apoptosis: final common pathway of photoreceptor death in rd, rds, and rhodopsin mutant mice. Neuron 1993; 11: 595605 68. Chaum E. Retinal neuroprotection by growth factors: a mechanistic perspective. J Cell Biochem 2003; 88: 5775 69. LaVail MM et al. Protection of mouse photoreceptors by survival factors in retinal degenerations. Invest Ophthalmol Vis Sci 1998; 39: 592 602 70. Tao W et al. Encapsulated cell-based delivery of CNTF reduces photoreceptor degeneration in animal models of retinitis pigmentosa. Invest Ophthalmol Vis Sci 2002; 43: 32923298. 71. Liang FQ et al. Aav-mediated delivery of ciliary neurotrophic factor prolongs photoreceptor survival in the rhodopsin knockout mouse. Mol Ther 2001; 3: 241248 72. Bok D et al. Effects of adeno-associated virus-vectored ciliary neurotrophic factor on retinal structure and function in mice with a P216L rds/peripherin mutation. Exp Eye Res 2002; 74: 719735. 73. Schlichtenbrede FC et al. Intraocular gene delivery of ciliary neurotrophic factor results in significant loss of retinal function in normal mice and in the Prph2(Rd2/Rd2) model of retinal degeneration. Gene Therapy 2003; 10: 523527. 74. Schlichtenbrede FC et al. Long-term evaluation of retinal function in Prph2Rd2/Rd2 mice following AAV-mediated gene replacement therapy. J Gene Med 2003; 5: 757 764 75. Schlichtenbrede FC et al. Improvement of neuronal visual responses in the superior colliculus in Prph2(Rd2/Rd2) mice following gene therapy. Mol Cell Neurosci 2004; 25: 103110. 76. Sarra GM et al. Gene replacement therapy in the retinal degeneration slow (rds) mouse: the effect on retinal degeneration following partial transduction of the retina. Hum Mol Genet 2001; 10: 23532361. 77. Gal A et al. Mutations in MERTK, the human orthologue of the RCS rat retinal dystrophy gene, cause retinitis pigmentosa. Nat Genet 2000; 26: 270271. 78. Vollrath D et al. Correction of the retinal dystrophy phenotype of the RCS rat by viral gene transfer of Mertk. Proc Natl Acad Sci USA 2001; 98: 1258412589 79. Thompson DA, Gal A. Vitamin A metabolism in the retinal pigment epithelium: genes, mutations, and diseases. Prog Retin Eye Res 2003; 22: 683703. 80. Thompson DA et al. Genetics and phenotypes of RPE65 mutations in inherited retinal degeneration. Invest Ophthalmol Vis Sci 2000; 41: 4293 4299. 81. Lorenz B et al. Early-onset severe rodcone dystrophy in young children with RPE65 mutations. Invest Ophthalmol Vis Sci 2000; 41: 27352742. 82. Dejneka NS et al. In utero gene therapy rescues vision in a murine model of congenital blindness. Mol Ther 2004; 9: 182 188 83. Cao W et al. In vivo protection of photoreceptors from light damage by pigment epithelium-derived factor. Invest Ophthalmol Vis Sci 2001; 42: 16461652. 84. Dawson DW et al. Pigment epithelium-derived factor: a potent inhibitor of angiogenesis. Science 1999; 285: 245248.

Anda mungkin juga menyukai