M. Tschernutter

AAV-Mediated gene transfer slows photoreceptor loss in the RCS rat model of retinitis pigmentosa

In the Royal College of Surgeons (RCS) rat, the retinal pigment epithelium (RPE) cannot phagocytose the outer segment discs that are continually shed from photoreceptors. The resulting accumulation of debris in the subretinal space leads to a progressive loss of photoreceptors. The defect results from a mutation in the Mertk gene, which is normally expressed in the RPE. Mertk is a receptor tyrosine kinase, involved in the binding of photoreceptor debris. Mutations in MERTK have also been described in patients with retinitis pigmentosa (RP). Here we demonstrate that subretinal injection of recombinant adeno-associated virus (AAV) expressing the murine Mertk gene can significantly prolong photoreceptor cell survival in the RCS rat. Electroretinographic analysis of treated eyes showed that functional photoreceptors were still present at 9 weeks, when there is virtually no activity in untreated control eyes. Histological analysis of treated eyes revealed a decrease in the amount of debris in the subretinal space, suggesting that RPE function was restored. Moreover, 9 weeks after treatment the number of photoreceptors was 2.5-fold higher in treated than in control eyes. This study provides strong support for the development of AAV-mediated gene therapy for RP caused by mutations in the MERTK gene.

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

Intraocular delivery of a variety of neurotrophic factors has been widely investigated as a potential treatment for retinal dystrophy (RD). The most commonly studied factor, ciliary neurotrophic factor (CNTF), has been shown to preserve retinal morphology and to promote cell survival in a variety of models of RD. In order to evaluate CNTF as a potential treatment for RD, we used the Prph2Rd2/Rd2 mouse. CNTF was expressed intraocularly using AAV-mediated gene delivery either by itself or, in a second treatment group, combined with AAV-mediated gene replacement therapy of peripherin2, which we have previously shown to improve photoreceptor structure and function. We confirmed in both groups of animals that CNTF reduces the loss of photoreceptor cells. Visual function, however, as assessed over a time course by electroretinography (ERG), was significantly reduced compared with untreated controls. Furthermore, CNTF gene expression negated the effects on function of gene replacement therapy. In order to test whether this deleterious effect is only seen when degenerating retina is treated, we recorded ERGs from wild-type mice following intraocular injection of AAV expressing CNTF. Here a marked deleterious effect was noted, in which the b-wave amplitude was reduced by at least 50%. Our results demonstrate that intraocular CNTF gene delivery may have a deleterious effect on the retina and caution against its application in clinical trials.

Long-term preservation of retinal function in the RCS rat model of retinitis pigmentosa following lentivirus-mediated gene therapy

The Royal College of Surgeons (RCS) rat is a well-characterized 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 , a receptor tyrosine kinase found in RPE cells, that 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. In order to evaluate the efficacy of lentiviral-mediated gene replacement therapy in the RCS rat, we produced recombinant VSV-G pseudotyped HIV-1-based lentiviruses containing a murine Mertk cDNA driven by a spleen focus forming virus (SFFV) promoter. The vector was subretinally injected into the right eye of 10-day-old RCS rats; the left eye was left untreated as an internal control. Here, we present a detailed assessment of the duration and extent of the morphological rescue and the resulting functional benefits. We examined animals at various time points over a period of 7 months by light and electron microscopy, and electroretinography. We observed correction of the phagocytic defect, slowing of photoreceptor cell loss and preservation of retinal function for up to 7 months. This study demonstrates the potential of gene therapy approaches for the treatment of retinal degenerations caused by defects specific to the RPE and supports the use of lentiviral vectors for the treatment of such disorders.

Stable and efficient intraocular gene transfer using pseudotyped EIAV lentiviral vectors.

We have developed minimal non‐primate lentiviral vectors based on the equine infectious anaemia virus (EIAV). We evaluated the in vivo expression profiles of these vectors delivered regionally to ocular tissues to define their potential utility in ocular gene therapy.

Clinical characterisation of a family with retinal dystrophy caused by mutation in the Mertk gene

Background/aim: MERTK, a tyrosine kinase receptor protein expressed by the retinal pigment epithelium (RPE), is mutated in both rodent models and humans affected by retinal disease. This study reports a survey of families for Mertk mutations and describes the phenotype exhibited by one family. Methods: 96 probands with retinal dystrophy, consistent with autosomal recessive segregation, were screened by direct sequencing. A family homozygous for a likely null allele was investigated clinically. Results: A novel frame shifting deletion was identified in one of 96 probands. Other polymorphisms were detected. The deletion allele occurred on both chromosomes of four affected family members. Electrophysiology demonstrated early loss of scotopic and macular function with later loss of photopic function. Visual acuities and visual fields were preserved into the second decade. Perception of light vision was present in a patient in the fourth decade. A “bull’s eye” appearance and a hyperautofluorescent lesion at the central macula were consistent clinical findings. Conclusions: Mutations in Mertk are a rare cause of ARRP in humans. The study extends the phenotypic characteristics of this retinal dystrophy and shows distinctive clinical signs that may improve its clinical identification. The moderate severity and presence of autofluorescence implies that outer segment phagocytosis is not entirely absent.

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.

AAV-mediated knockdown of Peripherin-2 in vivo using miRNA-based hairpins

Gene therapy for inherited retinal degeneration in which expression of a mutant allele has a gain-of-function effect on photoreceptor cells is likely to depend on efficient silencing of the mutated allele. Peripherin-2 (Prph2, also known as peripherin/RDS) is an abundantly expressed photoreceptor-specific gene. In humans, gain-of-function mutations in PRPH2 result in both autosomal dominant retinitis pigmentosa and dominant maculopathies. Gene-silencing strategies for these conditions include RNA interference by short hairpin RNAs (shRNAs). Recent evidence suggests that microRNA (miRNA)-based hairpins may offer a safer and more effective alternative. In this study, we used for the first time a virally transferred miRNA-based hairpin to silence Prph2 in the murine retina. The results show that an miRNA-based shRNA can efficiently and specifically silence Prph2 in vivo as early as 3 weeks after AAV2/8-mediated subretinal delivery, leading to a nearly 50% reduction of photoreceptor cells after 5 weeks. We conclude that miRNA-based hairpins can achieve rapid and robust gene silencing after efficient vector-mediated delivery to the retina. The rationale of using an miRNA-based template to improve the silencing efficiency of a hairpin may prove valuable for allele-specific silencing in which the choice for an RNAi target is limited and offers an alternative therapeutic strategy for the treatment of dominant retinopathies.