Ji-Jing Pang

University of Florida, Gainesville, FL, United States

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Publications (24)108.91 Total impact

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    ABSTRACT: PURPOSE. The retinal degeneration 11 (rd11) mouse is a newly discovered, naturally occurring animal model with early photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration. Rd11 mice carry a spontaneous mutation in the lysophosphatidylcholine acyltransferase 1 (Lpcat1) gene. Here, we evaluate whether gene replacement therapy using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can arrest retinal degeneration and restore retinal function in this model. METHODS. AAV8 (Y733F)-smCBA-Lpcat1 was subretinally delivered to postnatal day 14 (P14) rd11 mice in one eye only. Ten weeks after injection, treated rd11 mice were examined by visually guided behavior, electroretinography (ERG) and spectral domain optical coherence tomography (SD-OCT) and then killed for morphologic and biochemical examination. RESULTS. Substantial scotopic and photopic ERG signals were maintained in treated rd11 eyes, whereas untreated eyes in the same animals showed extinguished signals. SD-OCT (in vivo) and light microscopy (in vitro) showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Almost wild-type Lpcat1 expression in photoreceptors with strong rod rhodopsin and M/S cone opsin staining, and normal visually guided water maze behavioral performances were observed in treated rd11 mice. CONCLUSIONS. The results demonstrate that the tyrosine-capsid mutant AAV8 (Y733F) vector is effective for treating rapidly degenerating models of retinal degeneration and moreover is more therapeutically effective than AAV2 (Y444, 500, 730F) vector with the same promoter-cDNA payload. This is the first demonstration of phenotypic rescue by gene therapy in an animal model of retinal degeneration caused by Lpcat1 mutation.
    Investigative ophthalmology & visual science 02/2014; · 3.43 Impact Factor
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    ABSTRACT: [This corrects the article on p. e35250 in vol. 7.].
    PLoS ONE 01/2014; 9(1). · 3.73 Impact Factor
  • Xu-Feng Dai, Ji-Jing Pang, Juan-Juan Han, Yan Qi
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    ABSTRACT: To investigate the distribution and biological roles of voltage-dependent calcium channel (VDCC) α1F subunit in murine retina. Experimental study.α1F(-/-) (homozygous mutant) mice (n = 35) and α1F(+/+) (wild type) mice (n = 35) were used in this study. Immunohistochemistry was performed to determine the expression of VDCC α1F subunit in the mouse retina. Retinae in α1F(-/-) mice and age-matched control mice at 3, 6, 9, 14-day and 3-month after birth were paraffin embedded, sectioned and HE stained, and full-field electroretinogram (ERG) were also recorded at these time points.Statistics were based on independent samples t-test. (1) α1F subunit was absent in α1F(-/-) mice retina. But in α1F(+/+) mice retina, α1F subunit was expressed most strongly in the outer plexiform layer (OPL), less in the inner plexiform layer (IPL) and ganglion cell layer (GCL). (2) OPL thickness in the subunit deficient mice gradually reduced after birth and lost at adult age. (3) In dark-adapted ERGs,standard response showed that the b-wave amplitude of α1F(-/-) mice [(163.8 ± 26.7) µV] significantly decreased compared with that of α1F(+/+) mice [(408.4 ± 54.5) µV] (t = -9.017, P = 0.000), whereas the a-wave amplitude of α1F(-/-) group [(208.2 ± 27.3) µV] was similar to that of control group [(196.0 ± 24.2) µV] (t = 0.748, P = 0.476). This study demonstrates that the lack of VDCC α1F subunit affect the structure and function in the OPL of the murine retina.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 06/2013; 49(6):521-525.
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    ABSTRACT: Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.
    Human gene therapy. Clinical development. 03/2013; 24(1):23-8.
  • Xu-Feng Dai, Ji-Jing Pang
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    ABSTRACT: Achromatopsia is an early onset retinal dystrophy that causes severe visual impairment. To date, four genes have been found to be implicated in achromatopsia-associated mutations: guanine nucleotide-binding protein (GNAT2), cyclic nucleotide-gated channel alpha-3 (CNGA3), cyclic nucleotide-gated channel beta-3 (CNGB3) and phosphodiesterase 6C (PDE6C). Even with early onset, the slow progress and the good responses to gene therapy in animal models render achromatopsia a very attractive candidate for human gene therapy after the successful of the Phase I clinical trials of Leber's congenital amaurosis. With the development of molecular genetics and the therapeutic gene replacement technology, the adeno-associated viral (AAV) vector-mediated gene therapy for achromatopsia in the preclinical animal experiments achieved encouraging progress in the past years. This article briefly reviews the recent research achievements of achromatopsia with gene therapy.
    [Zhonghua yan ke za zhi] Chinese journal of ophthalmology 08/2012; 48(8):755-758.
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    ABSTRACT: Achromatopsia is a rare autosomal recessive disorder which shows color blindness, severely impaired visual acuity, and extreme sensitivity to bright light. Mutations in the alpha subunits of the cone cyclic nucleotide-gated channels (CNGA3) are responsible for about 1/4 of achromatopsia in the U.S. and Europe. Here, we test whether gene replacement therapy using an AAV5 vector could restore cone-mediated function and arrest cone degeneration in the cpfl5 mouse, a naturally occurring mouse model of achromatopsia with a CNGA3 mutation. We show that gene therapy leads to significant rescue of cone-mediated ERGs, normal visual acuities and contrast sensitivities. Normal expression and outer segment localization of both M- and S-opsins were maintained in treated retinas. The therapeutic effect of treatment lasted for at least 5 months post-injection. This study is the first demonstration of substantial, relatively long-term restoration of cone-mediated light responsiveness and visual behavior in a naturally occurring mouse model of CNGA3 achromatopsia. The results provide the foundation for development of an AAV5-based gene therapy trial for human CNGA3 achromatopsia.
    PLoS ONE 01/2012; 7(4):e35250. · 3.73 Impact Factor
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    ABSTRACT: To determine the safety and efficacy of subretinal gene therapy in the RPE65 form of Leber congenital amaurosis using recombinant adeno-associated virus 2 (rAAV2) carrying the RPE65 gene. Open-label, dose-escalation phase I study of 15 patients (range, 11-30 years of age) evaluated after subretinal injection of the rAAV2- RPE65 vector into the worse-functioning eye. Five cohorts represented 4 dose levels and 2 different injection strategies. Primary outcomes were systemic and ocular safety. Secondary outcomes assayed visual function with dark-adapted full-field sensitivity testing and visual acuity with Early Treatment Diabetic Retinopathy Study charts. Further assays included immune responses to the vector, static visual fields, pupillometry, mobility performance, and optical coherence tomography. No systemic toxicity was detected; ocular adverse events were related to surgery. Visual function improved in all patients to different degrees; improvements were localized to treated areas. Cone and rod sensitivities increased significantly in the study eyes but not in the control eyes. Minor acuity improvements were recorded in many study and control eyes. Major acuity improvements occurred in study eyes with the lowest entry acuities and parafoveal fixation loci treated with subretinal injections. Other patients with better foveal structure lost retinal thickness and acuity after subfoveal injections. Gene therapy for Leber congenital amaurosis caused by RPE65 mutations is sufficiently safe and substantially efficacious in the extrafoveal retina. There is no benefit and some risk in treating the fovea. No evidence of age-dependent effects was found. Our results point to specific treatment strategies for subsequent phases. Gene therapy for inherited retinal disease has the potential to become a future part of clinical practice. clinicaltrials.gov Identifier: NCT00481546.
    Archives of ophthalmology 09/2011; 130(1):9-24. · 3.86 Impact Factor
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    ABSTRACT: To test the effects of adeno-associated virus encoding sFLT01 (AAV5.sFLT01) on the retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice, a model for age-related macular degeneration (AMD), AAV5.sFLT01 was injected into the subretinal space of the right eyes and the left eyes served as controls. Histology found no retinal toxicity due to the treatment after 3 months. The treated eyes showed lesion arrest compared with lesion progression in the left eyes by fundus monitoring monthly and histological evaluation 3 months after treatment. Retinal ultrastructure showed fewer lipofuscin and better preserved photoreceptors after the treatment. A2E, a major component of lipofuscin, was lower in the treated eyes than in the control eyes. Molecular analysis showed that AAV5.sFLT01 lowered retinal extracellular signal-regulated kinase (ERK) phosphorylation and inducible nitric oxide synthetase expression, which suggested the involvement of reactive nitrogen species in the retinal lesions of Ccl2(-/-)/Cx3cr1(-/-). We concluded that local delivery of AAV5.sFLT01 can stabilize retinal lesions in Ccl2(-/-)/Cx3cr1(-/-) mice. The findings provide further support for the potential beneficial effects of sFLT01 gene therapy for age-related macular degeneration.
    Neurobiology of aging 03/2011; 33(2):433.e1-10. · 5.94 Impact Factor
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    ABSTRACT: The retinal degeneration 10 (rd10) mouse is a well-characterized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β subunit of rod cGMP-phosphodiesterase (PDEβ). Rd10 mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degeneration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDEβ was subretinally delivered to postnatal day 14 (P14) rd10 mice in one eye only. Six months after injection, spectral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)-mediated PDEβ expression restored retinal function and visual behavior and preserved retinal structure in treated rd10 eyes for at least 6 months. This is the first demonstration of long-term phenotypic rescue by gene therapy in an animal model of PDEβ-RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)-mediated correction of a retinal phenotype. These results lay the groundwork for the development of PDEβ-RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration.
    Molecular Therapy 02/2011; 19(2):234-42. · 7.04 Impact Factor
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    ABSTRACT: RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued. Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin-specific antibodies. Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin-positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin-positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths. The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials.
    Investigative ophthalmology & visual science 01/2011; 52(1):7-15. · 3.43 Impact Factor
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    ABSTRACT: Vectors based on adeno-associated virus serotype 2 (AAV2) have been used extensively in many gene-delivery applications, including several successful clinical trials for one type of Leber congenital amaurosis in the retina. Many studies have focused on improving AAV2 transduction efficiency and cellular specificity by genetically engineering its capsid. We have previously shown that vectors-containing single-point mutations of capsid surface tyrosines in serotypes AAV2, AAV8, and AAV9 displayed significantly increased transduction efficiency in the retina compared with their wild-type counterparts. In the present study, we evaluated the transduction characteristics of AAV2 vectors containing combinations of multiple tyrosine to phenylalanine mutations in seven highly conserved surface-exposed capsid tyrosine residues following subretinal or intravitreal delivery in adult mice. The multiply mutated vectors exhibited different in vivo transduction properties, with some having a unique ability of transgene expression in all retinal layers. Such novel vectors may be useful in developing valuable new therapeutic strategies for the treatment of many genetic diseases.
    Molecular Therapy 11/2010; 19(2):293-301. · 7.04 Impact Factor
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    ABSTRACT: To clarify whether transduction efficiency and cell type specificity of self-complementary (sc) AAV5 vectors are similar to those of standard, single-stranded AAV5 vectors in normal retina, one micro liter of scAAV5-smCBA-GFP vector (1 x 10(12) genome-containing particles/ml) and AAV5-smCBA-GFP vector (1 x 10(12) genome-containing particles/ml) were subretinally or intravitreally (in both cases through the cornea) injected into the right and left eyes of adult C57BL/6J mice, respectively. On post-injection day (PID) 1, 2, 5, 7, 10, 14, 21, 28 and 35, eyes were enucleated; retinal pigment epithelium (RPE) wholemounts, neuroretinal wholemounts and eyecup sections were prepared to evaluate green fluorescent protein (GFP) expression by fluorescent microscopy. GFP expression following trans-cornea subretinal injection of scAAV5-smCBA-GFP vector was first detected in RPE wholemounts around PID 1 and in neuroretinal wholemounts between PID 2 and 5; GFP expression peaked and stabilized between PID 10-14 in RPE wholemounts and between P14 and P21 in neuroretinal wholemounts with strong, homogeneous green fluorescence covering the entire wholemounts. The frozen sections supported the following findings from the wholemounts: GFP expression appeared first in RPE around PID 1-2 and soon spread to photoreceptors (PR) cells; by PID 7, moderate GFP expression was found mainly in PR and RPE layers; between PID 14 and 21, strong and homogenous GFP expression was observed in RPE and PR cells. GFP expression following subretinal injection of AAV5-smCBA-GFP was first detected in RPE wholemounts around PID 5-7 and in neuroretinal wholemounts around PID 7-10; ssAAV5-mediated GFP expression peaked at PID 21 in RPE wholemounts and around PID 28 in neuroretinal wholemounts; sections from AAV5 treated eyes also supported findings obtained from wholemounts: GFP expression was first detected in RPE and then spread to the PR cells. Peak GFP expression in RPE mediated by scAAV5 was similar to that mediated by AAV5. However, peak GFP expression mediated by scAAV5 in PR cells was stronger than that mediated by AAV5. No GFP fluorescence was detected in any retinal cells (RPE wholemounts, neuroretinal wholemounts and retinal sections) after trans-cornea intravitreal delivery of either scAAV5-GFP or AAV5-GFP. Neither scAAV5 nor AAV5 can transduce retinal cells following trans-cornea intravitreal injection. The scAAV5 vector used in this study directs an earlier onset of transgene expression than the matched AAV5 vector, and has stronger transgene expression in PR cells following subretinal injection. Our data confirm the previous reports that scAAV vectors have an earlier onset than the standard, single strand AAV vectors (Natkunarajah et al., 2008; Yokoi et al., 2007). scAAV5 vectors may be more useful than standard, single-stranded AAV vector when addressing certain RPE and/or PR cell-related models of retinal dystrophy, particularly for mouse models of human retinitis pigmentosa that require rapid and robust transgene expression to prevent early degeneration in PR cells.
    Experimental Eye Research 02/2010; 90(5):546-54. · 3.03 Impact Factor
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    ABSTRACT: Achromatopsia is an autosomal recessive retinal disease involving loss of cone function that afflicts approximately 1 in 30,000 individuals. Patients with achromatopsia usually have visual acuities lower than 20/200 because of the central vision loss, photophobia, complete color blindness and reduced cone-mediated electroretinographic (ERG) amplitudes. Mutations in three genes have been found to be the primary causes of achromatopsia, including CNGB3 (beta subunit of the cone cyclic nucleotide-gated cation channel), CNGA3 (alpha subunit of the cone cyclic nucleotide-gated cation channel), and GNAT2 (cone specific alpha subunit of transducin). Naturally occurring mouse models with mutations in Cnga3 (cpfl5 mice) and Gnat2 (cpfl3 mice) were discovered at The Jackson Laboratory. A natural occurring canine model with CNGB3 mutations has also been found. These animal models have many of the central phenotypic features of the corresponding human diseases. Using adeno-associated virus (AAV)-mediated gene therapy, we and others show that cone function can be restored in all three models. These data suggest that human achromatopsia may be a good candidate for corrective gene therapy.
    Advances in experimental medicine and biology 01/2010; 664:639-46. · 1.83 Impact Factor
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    ABSTRACT: Human gene therapy with rAAV2-vector was performed for the RPE65 form of childhood blindness called Leber congenital amaurosis. In three contemporaneous studies by independent groups, the procedure was deemed safe and there was evidence of visual gain in the short term. At 12 months after treatment, our young adult subjects remained healthy and without vector-related serious adverse events. Results of immunological assays to identify reaction to AAV serotype 2 capsid were unchanged from baseline measurements. Results of clinical eye examinations of study and control eyes, including visual acuities and central retinal structure by in vivo microscopy, were not different from those at the 3-month time point. The remarkable improvements in visual sensitivity we reported by 3 months were unchanged at 12 months. The retinal extent and magnitude of rod and cone components of the visual sensitivity between 3 and 12 months were also the same. The safety and efficacy of human retinal gene transfer with rAAV2-RPE65 vector extends to at least 1 year posttreatment.
    Human gene therapy 08/2009; 20(9):999-1004. · 4.20 Impact Factor
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    ABSTRACT: In an earlier study we found normal adeno-associated viral vector type 2 (AAV2)-mediated GFP expression after intravitreal injection to one eye of normal C57BL/6J mice. However, GFP expression was very poor in the partner eye of the same mouse if this eye received an intravitreal injection of the same vector one month after the initial intravitreal injection. We also found both injections worked well if they were subretinal. In this study, we tested whether the efficiency of subretinal AAV vector transduction is altered by a previous intravitreal injection in the partner eye and more importantly whether therapeutic efficiency is altered in the rd12 mouse (with a recessive RPE65 mutation) after the same injection series. One microl of scAAV5-smCBA-GFP (1 x 10(13) genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old C57BL/6J mice and 1 microl of scAAV5-smCBA-hRPE65 (1 x 10(13) genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old rd12 mice Four weeks later, the same vectors were subretinally injected into the left eyes of the same C57BL/6J and rd12 mice. Left eyes of another cohort of eight-week-old rd12 mice received a single subretinal injection of the same scAAV5-smCBA-hRPE65 vector as the positive control. Dark-adapted electroretinograms (ERGs) were recorded five months after the subretinal injections. AAV-mediated GFP expression in C57BL/6J mice and RPE65 expression and ERG restoration in rd12 mice were evaluated five months after the second subretinal injection. Frozen section analysis was performed for GFP fluorescence in C57BL/6J mice and immunostaining for RPE65 in rd12 eyes. In rd12 mice, dark-adapted ERGs were minimal following the first intravitreal injection of scAAV5-smCBA-RPE65. Following subsequent subretinal injection in the partner eye, dramatic ERG restoration was recorded in that eye. In fact, ERG b-wave amplitudes were statistically similar to those from the eyes that received the initial subretinal injection at a similar age. In C57BL/6J mice, GFP positive cells were detected in eyes following the first intravitreal injection around the injection site. Strong GFP expression in both the retinal pigment epithelium (RPE) and photoreceptor (PR) cells was detected in the partner eyes following the subsequent subretinal injection. Immunostaining of retinal sections with anti-RPE65 antibody showed strong RPE65 expression mainly in the RPE cells of subretinally injected eyes but not in the intravitreally injected eyes except minimally around the injection site. These results show that an initial intravitreal injection of AAV vectors to one eye of a mouse does not influence AAV-mediated gene expression or related therapeutic effects in the other eye when vectors are administered to the subretinal space. This suggests that the subretinal space possesses a unique immune privilege relative to the vitreous cavity.
    Molecular vision 02/2009; 15:267-75. · 1.99 Impact Factor
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    ABSTRACT: Vectors derived from adeno-associated viruses (AAVs) have become important gene delivery tools for the treatment of many inherited ocular diseases in well-characterized animal models. Previous studies have determined that the viral capsid plays an essential role in the cellular tropism and efficiency of transgene expression. Recently, it was shown that phosphorylation of surface-exposed tyrosine residues from AAV2 capsid targets the viral particles for ubiquitination and proteasome- mediated degradation, and mutations of these tyrosine residues lead to highly efficient vector transduction in vitro and in vivo. Because the tyrosine residues are highly conserved in other AAV serotypes, in this study we evaluated the intraocular transduction characteristics of vectors containing point mutations in surface- exposed capsid tyrosine residues in AAV serotypes 2, 8, and 9. Several of these novel AAV mutants were found to display a strong and widespread transgene expression in many retinal cells after subretinal or intravitreal delivery compared with their wild-type counterparts. For the first time, we show efficient transduction of the ganglion cell layer by AAV serotype 8 or 9 mutant vectors, thus providing additional tools besides AAV2 for targeting these cells. These enhanced AAV vectors have a great potential for future therapeutic applications for retinal degenerations and ocular neovascular diseases.
    Molecular Therapy 01/2009; 17(3):463-71. · 7.04 Impact Factor
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    ABSTRACT: The RPE65 gene encodes the isomerase of the retinoid cycle, the enzymatic pathway that underlies mammalian vision. Mutations in RPE65 disrupt the retinoid cycle and cause a congenital human blindness known as Leber congenital amaurosis (LCA). We used adeno-associated virus-2-based RPE65 gene replacement therapy to treat three young adults with RPE65-LCA and measured their vision before and up to 90 days after the intervention. All three patients showed a statistically significant increase in visual sensitivity at 30 days after treatment localized to retinal areas that had received the vector. There were no changes in the effect between 30 and 90 days. Both cone- and rod-photoreceptor-based vision could be demonstrated in treated areas. For cones, there were increases of up to 1.7 log units (i.e., 50 fold); and for rods, there were gains of up to 4.8 log units (i.e., 63,000 fold). To assess what fraction of full vision potential was restored by gene therapy, we related the degree of light sensitivity to the level of remaining photoreceptors within the treatment area. We found that the intervention could overcome nearly all of the loss of light sensitivity resulting from the biochemical blockade. However, this reconstituted retinoid cycle was not completely normal. Resensitization kinetics of the newly treated rods were remarkably slow and required 8 h or more for the attainment of full sensitivity, compared with <1 h in normal eyes. Cone-sensitivity recovery time was rapid. These results demonstrate dramatic, albeit imperfect, recovery of rod- and cone-photoreceptor-based vision after RPE65 gene therapy.
    Proceedings of the National Academy of Sciences 09/2008; 105(39):15112-7. · 9.74 Impact Factor
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    ABSTRACT: To test AAV-mediated gene therapy in the rd10 mouse, a natural model of recessive RP caused by mutation of the beta-subunit of rod photoreceptor cGMP phosphodiesterase. One eye of a cohort of rd10 mice kept in a dark environment was subretinally injected at postnatal day (P) 14 with 1 microL AAV5-smCBA-PDEbeta. The contralateral eye was not injected. The animals were then maintained for 2 weeks in the dark before they were moved to a normal 12-hour light/12-hour dark cycling light environment for visually guided behavioral training. Three weeks after injection, treated rd10 mice were examined by scotopic and photopic electroretinography and then killed for biochemical and morphologic examination. Substantial scotopic ERG signals were maintained in treated rd10 eyes, whereas untreated eyes in the same animals showed minimal signals. Treated eyes showed photopic ERG b-wave amplitudes similar to those of the normal eyes; in untreated partner eyes, only half the normal amplitudes remained. Strong PDEbeta expression was observed in photoreceptor outer segments only in treated eyes. Light microscopy showed a substantial preservation of the outer nuclear layer in most parts of the treated retina only. Electron microscopy showed good outer segment preservation only in treated eyes. A visually guided water maze behavioral test under dim light showed significantly improved performance in one eye-treated rd10 mice compared with untreated mice. These data demonstrate that P14 administration of AAV5-smCBA-PDEbeta can prevent retinal degeneration in rd10 mice, as reflected by significant structural, biochemical, electrophysiological, and behavioral preservation/restoration. These results serve as a baseline for studying long-term retinal rescue in rd10 mice.
    Investigative ophthalmology & visual science 07/2008; 49(10):4278-83. · 3.43 Impact Factor
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    ABSTRACT: The specificity of retinal cells transduced by AAV serotype 1, 2 or 5 vectors was determined in vivo versus in vitro in the normal P7 mouse in order to develop a rapid and accurate way to anticipate the behavior of AAV vectors in the retina. In vivo results confirm that AAV1 transduces retinal pigment epithelial cells, while AAV2 and AAV5 transduce both RPE and photoreceptor cells by subretinal injection. AAV2 was the only serotype to efficiently transduce inner retinal cells by intravitreal injection. Parallel analysis employing in vitro retinal organ culture showed qualitatively similar AAV-mediated GFP expression as seen in vivo suggesting that organ culture substitute is a useful method to screen new vector transduction patterns, particular in retinal cells in neonatal mice.
    Vision Research 03/2008; 48(3):377-85. · 2.14 Impact Factor
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    ABSTRACT: To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD). A ribozyme that targets the protective enzyme manganese superoxide dismutase (MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography (ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas. The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch's membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin. These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.
    Investigative Ophthalmology &amp Visual Science 11/2007; 48(10):4407-20. · 3.44 Impact Factor

Publication Stats

967 Citations
158 Downloads
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108.91 Total Impact Points

Institutions

  • 2005–2013
    • University of Florida
      • • Department of Pediatrics
      • • Department of Ophthalmology
      Gainesville, FL, United States
  • 2009–2012
    • Wenzhou Medical College
      • Eye Hospital
      Yung-chia, Zhejiang Sheng, China
  • 2008–2011
    • University of Pennsylvania
      • • Perelman School of Medicine
      • • Department of Ophthalmology
      Philadelphia, PA, United States
  • 2007
    • Treatment Research Institute, Philadelphia PA
      Philadelphia, Pennsylvania, United States
    • Columbia University
      • Department of Ophthalmology
      New York City, New York, United States