Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)–lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus–mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment. Glaucoma, a common cause of blindness, is currently treated by intraocular pressure (IOP)–lowering interventions. However, this approach is insufficient to completely prevent vision loss. Here, we evaluate an IOP-independent gene therapy strategy using a modified erythropoietin, EPO-R76E, which has reduced erythropoietic function. We used two models of glaucoma, the murine microbead occlusion model and the DBA/2J mouse. Systemic recombinant adeno-associated virus–mediated gene delivery of EpoR76E (rAAV.EpoR76E) was performed concurrent with elevation of IOP. Axon structure and active anterograde transport were preserved in both models. Vision, as determined by the flash visual evoked potential, was preserved in the DBA/2J. These results show that systemic EpoR76E gene therapy protects retinal ganglion cells from glaucomatous degeneration in two different models. This suggests that EPO targets a component of the neurodegenerative pathway that is common to both models. The efficacy of rAAV.EpoR76E delivered at onset of IOP elevation supports clinical relevance of this treatment.
Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. Wesley S. Bond and Lalita Wadhwa are co-first authors.
Glaucoma is a complex neurodegeneration and a leading cause of blindness worldwide. Current therapeutic strategies, which are all directed towards lowering the intraocular pressure (IOP), do not stop progression of the disease. We have demonstrated that recombinant adeno-associated virus (rAAV) gene delivery of a form of erythropoietin with attenuated erythropoietic activity (EpoR76E) can preserve retinal ganglion cells, their axons, and vision without decreasing IOP. The goal of this study was to determine if modulation of neuroinflammation or oxidative stress played a role in the neuroprotective activity of EPO.R76E. Five-month-old DBA/2J mice were treated with either rAAV.EpoR76E or a control vector and collected at 8 months of age. Neuroprotection was assessed by quantification of axon transport and visual evoked potentials. Microglia number and morphology and cytokine and chemokine levels were quantified. Message levels of oxidative stress-related proteins were assessed. Axon transport and visual evoked potentials were preserved in rAAV.EpoR76E-treated mice. The number of microglia was decreased in retinas from 8-month-old rAAV.EpoR76E-treated mice, but proliferation was unaffected. The blood-retina barrier was also unaffected by treatment. Levels of some pro-inflammatory cytokines were decreased in retinas from rAAV.EpoR76E-treated mice including IL-1, IL-12, IL-13, IL-17, CCL4, and CCL5. TNFα messenger RNA (mRNA) was increased in retinas from 8-month-old mice compared to 3-month-old controls regardless of treatment. Expression of several antioxidant proteins was increased in retinas of rAAV.EpoR76E-treated 8-month-old mice. Treatment with rAAV.EpoR76E preserves vision in the DBA/2J model of glaucoma at least in part by decreasing infiltration of peripheral immune cells, modulating microglial reactivity, and decreasing oxidative stress.
Our goal is to investigate the neuroprotective efficacy of systemic rAAV gene delivery of erythropoiesis-attenuated erythropoietin-R76E (EPO-R76E) in a mouse model of experimental glaucoma. We investigated the efficacy of EPO-R76E gene therapy commenced before and after induction of ocular hypertension. C57BL/6 mice were injected intramuscularly with 109 vgc rAAV delivering eGFP or Epo-R76E behind a CMV promoter. To induce ocular hypertension, anterior chambers were injected with 15-μm diameter microbeads or saline, and elevation of intraocular pressure (IOP) was verified by tonometry. For pre-treatment, gene delivery was performed using rAAV2/8 serotype one month prior to IOP elevation. For delayed treatment, gene therapy was administered using rAAV2/1 serotype concurrently with IOP elevation. At 1 month post-IOP elevation, tissue was collected and retinal ganglion cell (RGC) axon function was assessed by optic nerve histology and fluorescent cholera toxin B anterograde transport. Hematocrit was also assessed at collection. Hematocrit was significantly elevated in mice pre-treated with rAAV2/8.CMV.Epo-R76E (59.6±9.1%) and in mice given rAAV2/1.CMV.Epo-R76E delayed (55.0±3.6%) compared to eGFP controls (42.7±3.2%). Mean IOP of 20.3±2.9 mmHg was observed in microbead-injected eyes 1 week post-induction compared to 15.4±2.1 mmHg in saline-injected controls. At 1 month post-induction, microbead-injected eGFP controls developed 20% reduction of RGC axons and 22% impairment of RGC anterograde transport compared to saline-injected controls. Microbead-injected mice pre-treated with rAAV2/8.CMV.Epo-R76E showed 4% reduction of RGC axons and 5% impairment in RGC anterograde transport. Microbead-injected mice given delayed treatment with rAAV2/1.CMV.Epo-R76E showed 18% reduction of RGC axons and 6% impairment of RGC anterograde transport. Gene delivery of modified EPO attenuates glaucomatous pathology of retinal ganglion cells in the induced model of ocular hypertension. Pre-treatment with EPO gene therapy provides more robust preservation of RGC axons than EPO gene therapy commenced at onset of hypertension. Delayed gene therapy preserves axon transport significantly, however, which may be due to delayed therapy preserving normal function of surviving RGC axons while failing to attenuate early axon loss.
Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. Wesley S. Bond and Lalita Wadhwa are co-first authors.
Verification that cell lines used for cancer research are derived from malignant cells in primary tumors is imperative to avoid invalidation of study results. Retinoblastoma is a childhood ocular tumor that develops from loss of functional retinoblastoma protein (pRb) as a result of genetic or epigenetic changes that affect both alleles of the RB1 gene. These patients contain unique identifiable genetic signatures specifically present in malignant cells. Primary cultures derived from retinoblastoma tumors can be established as non-adherent tumorspheres when grown in defined media or as attached monolayers when grown in serum-containing media. While the RB1 genotypes of tumorspheres match those of the primary tumor, adherent cultures have the germline RB1 genotype. Tumorspheres derived from pRb-negative tumors do not express pRb and express the neuroendocrine tumor markers synaptophysin and microtubule-associated protein 2 (MAP2). Adherent cells are synaptophysin-negative and express pRb, the epithelial cell marker cytokeratin that is expressed in the retinal pigmented epithelium and the vascular endothelial cell marker CD34. While tumorspheres are of malignant origin, our results cast doubt on the assumption that adherent tumor-derived cultures are always valid in vitro models of malignant cells and emphasize the need for validation of primary tumor cultures.
The success of gene therapy in the ocular environment is partly due to the presence of hyaluronan in vitreous. Here we explore the mechanism of hyaluronan-mediated enhancement of adenoviral vector transgene expression. Introduction of hyaluronan receptor CD44 into CD44-negative cells followed by transduction in the presence of vitreous with an adenoviral vector containing an IL-12-coding transgene increases IL-12 secretion. We demonstrate that sequential CD44 proteolysis is responsible for hyaluronan-mediated enhancement. Metalloproteinase or γ-secretase inhibitors decrease adenoviral-mediated transgene expression. Deletion of these proteolytic sites in CD44 also inhibits transgene expression. Expression of CD44 with a mutation to prevent phosphorylation of serine 325 inhibits the response to vitreous. Expression of the CD44 intracellular domain enhances transgene expression in the absence of vitreous. CD44-mediated enhancement of gene expression was observed with vectors using different promoters and appears because of an increase in mRNA production, not because of an increase in vector transduction as determined by quantitative RT-PCR and quantitative PCR, respectively. These data fit a model where the interaction of hyaluronan in vitreous and CD44 modulates transgene expression by initiating CD44 proteolysis and release of the cytoplasmic domain, resulting in increased transgene transcription.
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