Showing papers on "MERTK published in 2006"

Gene therapy progress and prospects: the eye.

Abstract: The eye has unique advantages as a target organ for gene therapy of both inherited and acquired ocular disorders and offers a valuable model system for gene therapy. The eye is readily accessible to phenotypic examination and investigation of therapeutic effects in vivo by fundus imaging and electrophysiological techniques. Considerable progress has been made in the development of gene replacement therapies for retinal degenerations resulting from gene defects in photoreceptor cells (rds, RPGRIP, RS-1) and in retinal pigment epithelial cells (MerTK, RPE65, OA1) using recombinant adeno-associated virus and lentivirus-based vectors. Gene therapy also offers a potentially powerful approach to the treatment of complex acquired disorders such as those involving angiogenesis, inflammation and degeneration, by the targeted sustained intraocular delivery of therapeutic proteins. Proposals for clinical trials of gene therapy for early-onset retinal degeneration owing to defects in the gene encoding the visual cycle protein RPE65 have recently received ethical approval.

Lymphoblastic leukemia/lymphoma in mice overexpressing the Mer (MerTK) receptor tyrosine kinase.

Abstract: Mer (MerTK) is a receptor tyrosine kinase important in platelet aggregation, as well as macrophage cytokine secretion and clearance of apoptotic cells. Mer is not normally expressed in thymocytes or lymphocytes; however, ectopic Mer RNA transcript and protein expression is found in a subset of acute lymphoblastic leukemia cell lines and patient samples, suggesting a role in leukemogenesis. To investigate the oncogenic potential of Mer in vivo, we created a transgenic mouse line (Mer(Tg)) that expresses Mer in the hematopoietic lineage under control of the Vav promoter. Ectopic expression and activation of the transgenic Mer protein was demonstrated in lymphocytes and thymocytes of the Mer(Tg) mice. At 12-24 months of age, greater than 55% of the Mer(Tg) mice, compared to 12% of the wild type, developed adenopathy, hepatosplenomegaly, and circulating lymphoblasts. Histopathological analysis and flow cytometry were consistent with T-cell lymphoblastic leukemia/lymphoma. Mer may contribute to leukemogenesis by activation of Akt and ERK1/2 anti-apoptotic signals, which were upregulated in Mer(Tg) mice. Additionally, a significant survival advantage was noted in Mer(Tg) lymphocytes compared to wild-type lymphocytes after dexamethasone treatment. These data suggest that Mer plays a cooperative role in leukemogenesis and may be an effective target for biologically based leukemia/lymphoma therapy.

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

Abstract: . 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.

Novel role for αvβ5-integrin in retinal adhesion and its diurnal peak

Abstract: αvβ5-Integrin is the sole integrin receptor at the retinal pigment epithelium (RPE)-photoreceptor interface and promotes RPE phagocytic signaling to the tyrosine kinase Mer tyrosine kinase (MerTK) ...

Mertk Activation During RPE Phagocytosis in Vivo Requires αVβ5 Integrin

Abstract: Daily phagocytosis of shed photoreceptor outer segment fragments (POS) is a key task of the retinal pigment epithelium (RPE) in the retina. Lack or inefficiency of daily POS clearance causes early onset, rapid, and complete retinal degeneration in experimental animals and likely contributes to human blinding diseases such as retinitis pigmentosa and age-related macular degeneration (Dowling and Sidman, 1962, Gal et al., 2000). The phagocytic mechanism of the RPE belongs to a group of conserved non-inflammatory clearance pathways that mediate recognition and engulfment of apoptotic cells in both non-professional and professional phagocytic cells, such as fibroblasts and macrophages, respectively (Finnemann and Rodriguez-Boulan, 1999). These pathways share the use of phagocyte cell surface receptors such as the lipid scavenger receptor CD36 (Ryeom et al., 1996), the integrin adhesion receptor αv β5 (Finnemann et al., 1997; Miceli et al., 1997; Lin and Clegg, 1998) and the receptor tyrosine kinase Mer (MerTK) (D’Cruz et al., 2000; Nandrot et al., 2000). In vitro phagocytosis assays studying primary or permanent RPE in culture fed with isolated POS suggest that CD36 and MerTK participate in the engulfment step of the phagocytic process (Chaitin and Hall, 1983; Finnemann and Silverstein, 2001), while αv β5 integrin promotes POS recognition/binding and initiates a downstream cytoplasmic signaling cascade in the RPE (Finnemann et al., 1997). However, the precise function of these receptors and their roles in the intact retina are so far only poorly understood. Most recently, we have begun to study phagocytosis and receptor activity in animal models that lack αv β5 integrin or MerTK to determine how these different plasma membrane receptors of the RPE functionally interact to coordinate particle uptake.

Varied expression of functionally important genes of RPE and choroid in the macula and in the periphery of normal human eyes.

Abstract: Topographic differences in RPE and choroid between macular and peripheral areas of the eye may predispose to morphologic and cell survival changes with aging. An understanding of the molecular events that distinguish RPE and choroid by their spatial location could give hints for the identification of survival factors and the development of new therapeutic approaches. To determine the mRNA expression of functionally important genes in RPE and choroid of morphologically normal human eyes, tissue patches were dissected from the macula and peripheral locations. The mRNA levels of 29 genes with known functions or expression in the RPE/choroid were quantified in these sections by real time RT-PCR. Variations in the mRNA expression were determined due to differences in the mean normalized expression (MNE) between different peripheral locations, left and right eye of the same donor, and eyes of different donors. In the macula, the lysosomal enzyme cathepsin D (1.27E+00±1.54E-01) and the MERTK ligand Gas6 (1.08E+00±1.60E-01) had the highest MNE, whereas the apoptosis inducer Fas-Ligand (1.41E-04±6.46E-05) and the ROS internalization receptor CD36 (2.15E-04±1.11E-05) demonstrated the lowest expression. Interestingly, the PEDF expression (1.80E-01±4.56E-02) was 10 times higher than the VEGF expression (1.84E-02±2.46E-03) in the macular area. For most of the analyzed genes (52%, e.g. MERTK, integrin αV and β5, RPE65, tyrosinase, VEGF) there was equal gene expression in the macula and in the periphery. For 31% of the genes (e.g. CD36, MAP1B) there was higher expression in the macula and for 17% of the genes (e.g. 11-cis RDH, VEGF-R2, PEDF) there was higher expression in the periphery. Whereas most of the analyzed genes expressed in RPE and choroid had equal mRNA expression levels in the macula and the periphery with donor dependent variations, there are important exceptions in genes that are involved in the maintenance of a specific vascular status in the macula (PEDF, VEGF and VEGR-R2) and in the recycling of rod outer segments (11-cis RDH). Applying this technique to the gene expression analysis of patients with AMD could identify those genes that are involved in molding of the disease.

Indirect exclusion of four candidate genes for generalized progressive retinal atrophy in several breeds of dogs

Abstract: Background: Generalized progressive retinal atrophy (gPRA) is a hereditary ocular disorder with progressive photoreceptor degeneration in dogs. Four retina-specific genes, ATP binding cassette transporter retina (ABCA4), connexin 36 (CX36), c-mer tyrosin kinase receptor (MERTK) and photoreceptor cell retinol dehydrogenase (RDH12) were investigated in order to identify mutations leading to autosomal recessive (ar) gPRA in 29 breeds of dogs. Results: Mutation screening was performed initially by PCR and single strand conformation polymorphism (SSCP) analysis, representing a simple method with comparatively high reliability for identification of sequence variations in many samples. Conspicuous banding patterns were analyzed via sequence analyses in order to detect the underlying nucleotide variations. No pathogenetically relevant mutations were detected in the genes ABCA4, CX36, MERTK and RDH12 in 71 affected dogs of 29 breeds. Yet 30 new sequence variations were identified, both, in the coding regions and intronic sequences. Many of the sequence variations were in heterozygous state in affected dogs. Conclusion: Based on the ar transmittance of gPRA in the breeds investigated, informative sequence variations provide evidence allowing indirect exclusion of pathogenetic mutations in the genes ABCA4 (for 9 breeds), CX36 (for 12 breeds), MERTK (for all 29 breeds) and RDH12 (for 9 breeds).

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

Abstract: 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.

MerTK mediates immune homeostasis: effects upon dendritic cell function and Type 1 diabetes

Abstract: Self-antigens expressed by apoptotic cells may become targets for autoimmunity. Tolerance to these self-antigens is established partly by an ill-defined capacity of apoptotic cells to inhibit the activation and function of antigen presenting cells such as dendritic cells (DC). We present evidence that the receptor tyrosine kinase Mer (MerTK) has a key role in mediating apoptotic cell-induced inhibition of DC. Pretreatment of DC prepared from nonobese diabetic (NOD) mice with apoptotic cells blocked IL-12p70 secretion and upregulation of co-stimulatory molecule expression following stimulation. In contrast, NOD DC treated with a blocking anti-MerTK antibody or lacking MerTK expression (NOD.MerTK) were resistant to apoptotic cell-induced inhibition. Apoptotic cell pretreatment failed to inhibit the capacity of NOD.MerTK but not NOD DC to stimulate CD4 and CD8 T cells in vitro. Similarly, diabetogenic CD4 T cells adoptively transferred into NOD.MerTK mice in which β cell apoptosis was induced, exhibited increased expansion and differentiation into type 1 T effectors. Finally, the development of