Gene therapy for retinal degeneration due to a defect in the retinal pigment epithelium.

The Royal College of Surgeons (RCS) rat is a well characterised model of autosomal recessive retinitis pigmentosa (RP) due to a defect in the retinal pigment epithelium (RPE). It is homozygous for a null mutation in the gene encoding Mertk, a receptor tyrosine kinase found in RPE cells, which is required for phagocytosis of shed photoreceptor outer segments. The absence of Mertk results in accumulation of outer segment debris. This subsequently leads to progressive loss of photoreceptor cells. Recently, MERTK has been established as a human retinal dystrophy gene. Retinal dystrophies are the most common cause of visual impairment in the Western World, for which no effective treatment exists. In order to evaluate the efficacy of virus mediated gene replacement therapy in the RCS rat, we produced recombinant adeno-associated viruses (AAV) and lentiviruses containing murine Mertk cDNA. Vectors were subretinally injected into the right eye of 10 day old RCS rats the left eye was left untreated as an internal control. Animals were examined at various time points by light and electron microscopy, electroretinography, and ophthalmoscopy. A detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits is presented in this thesis. AAV-2-mediated gene therapy resulted in preservation of retinal function for more than 9 weeks, when there is no activity in untreated eyes. Photoreceptors were still present at this time point and debris layer thickness was reduced. After subretinal delivery of human immunodeficiency virus type 1 (HIV-1) based lentiviral vectors carrying a functional copy of Mertk to the RCS rat eye, correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function was observed for up to 7 months, the latest time point evaluated. Whilst this was an improvement of the rescue compared to that achieved with AAV-2, lentiviral vectors raise more safety concerns regarding clinical application. Due to these biosafety issues, gene therapy vectors based on non-human lentiviruses and integration-deficient vectors have been developed. As part of this project, the potential of equine infectious anemia virus (EIAV) and non-integrating HIV-1-based vectors for the management of retinal degenerative disorders has been evaluated. The results presented in this thesis support the use of viral vectors for the treatment of retinal dystrophies. However, the development of an efficient therapy depends on the identification of patients and characterisation of pathological changes. Therefore, a panel of DNA samples from patients with autosomal recessive and sporadic forms of RP was screened for mutations in the MERTK gene. A new homozygous frame- shifting deletion was identified in four affected members of a family with RP. Clinical examination of these patients showed distinctive clinical signs that may improve the chances of identifying further patients and families in the future

The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice

Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration

The Tight Junction Associated Signalling Proteins ZO-1 and ZONAB Regulate Retinal Pigment Epithelium Homeostasis in Mice

Cell-cell adhesion regulates the development and function of epithelia by providing mechanical support and by guiding cell proliferation and differentiation. The tight junction (TJ) protein zonula occludens (ZO)-1 regulates cell proliferation and gene expression by inhibiting the activity of the Y-box transcription factor ZONAB in cultured epithelial cells. We investigated the role of this TJ-associated signalling pathway in the retinal pigment epithelium (RPE) in vivo by lentivirally-mediated overexpression of ZONAB, and knockdown of its cellular inhibitor ZO-1. Both overexpression of ZONAB or knockdown of ZO-1 resulted in increased RPE proliferation, and induced ultrastructural changes of an epithelial-mesenchymal transition (EMT)-like phenotype. Electron microscopy analysis revealed that transduced RPE monolayers were disorganised with increased pyknosis and monolayer breaks, correlating with increased expression of several EMT markers. Moreover, fluorescein angiography analysis demonstrated that the increased proliferation and EMT-like phenotype induced by overexpression of ZONAB or downregulation of ZO-1 resulted in RPE dysfunction. These findings demonstrate that ZO-1 and ZONAB are critical for differentiation and homeostasis of the RPE monolayer and may be involved in RPE disorders such as proliferative vitroretinopathy and atrophic age-related macular degeneration

Retinal Degeneration Progression Changes Lentiviral Vector Cell Targeting in the Retina

In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb) with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina

Pharmacological disruption of the outer limiting membrane leads to increased retinal integration of transplanted photoreceptor precursors

AbstractRetinal degeneration is the leading cause of untreatable blindness in the developed world. Cell transplantation strategies provide a novel therapeutic approach to repair the retina and restore sight. Previously, we have shown that photoreceptor precursor cells can integrate and form functional photoreceptors after transplantation into the subretinal space of the adult mouse. In a clinical setting, however, it is likely that far greater numbers of integrated photoreceptors would be required to restore visual function. We therefore sought to assess whether the outer limiting membrane (OLM), a natural barrier between the subretinal space and the outer nuclear layer (ONL), could be reversibly disrupted and if disruption of this barrier could lead to enhanced numbers of transplanted photoreceptors integrating into the ONL. Transient chemical disruption of the OLM was induced in adult mice using the glial toxin, dl-alpha-aminoadipic acid (AAA). Dissociated early post-natal neural retinal cells were transplanted via subretinal injection at various time-points after AAA administration. At 3weeks post-injection, the number of integrated, differentiated photoreceptor cells was assessed and compared with those found in the PBS-treated contralateral eye. We demonstrate for the first time that the OLM can be reversibly disrupted in adult mice, using a specific dose of AAA administered by intravitreal injection. In this model, OLM disruption is maximal at 72h, and recovers by 2weeks. When combined with cell transplantation, disruption of the OLM leads to a significant increase in the number of photoreceptors integrated within the ONL compared with PBS-treated controls. This effect was only seen in animals in which AAA had been administered 72h prior to transplantation, i.e. when precursor cells were delivered into the subretinal space at a time coincident with maximal OLM disruption. These findings suggest that the OLM presents a physical barrier to photoreceptor integration following transplantation into the subretinal space in the adult mouse. Reversible disruption of the OLM may provide a strategy for increasing cell integration in future therapeutic applications