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

Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis.

BACKGROUND: RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. METHODS AND FINDINGS: Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. CONCLUSIONS: By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65(-/-) mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose

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

Using magnetic resonance imaging to assess visual deficits : a review

PURPOSE: Over the last two decades, magnetic resonance imaging (MRI) has been widely used in neuroscience research to assess both structure and function in the brain in health and disease. With regard to vision research, prior to the advent of MRI, researchers relied on animal physiology and human post-mortem work to assess the impact of eye disease on visual cortex and connecting structures. Using MRI, researchers can non-invasively examine the effects of eye disease on the whole visual pathway, including the lateral geniculate nucleus, striate and extrastriate cortex. This review aims to summarise research using MRI to investigate structural, chemical and functional effects of eye diseases, including: macular degeneration, retinitis pigmentosa, glaucoma, albinism, and amblyopia. RECENT FINDINGS: Structural MRI has demonstrated significant abnormalities within both grey and white matter densities across both visual and non-visual areas. Functional MRI studies have also provided extensive evidence of functional changes throughout the whole of the visual pathway following visual loss, particularly in amblyopia. MR spectroscopy techniques have also revealed several abnormalities in metabolite concentrations in both glaucoma and age-related macular degeneration. GABA-edited MR spectroscopy on the other hand has identified possible evidence of plasticity within visual cortex. SUMMARY: Collectively, using MRI to investigate the effects on the visual pathway following disease and dysfunction has revealed a rich pattern of results allowing for better characterisation of disease. In the future MRI will likely play an important role in assessing the impact of eye disease on the visual pathway and how it progresses over time

Gene therapy for retinitis pigmentosa and Leber congenital amaurosis caused by defects in AIPL1: effective rescue of mouse models of partial and complete Aipl1 deficiency using AAV2/2 and AAV2/8 vectors

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) are clinically heterogeneous and present as Leber Congenital Amaurosis, the severest form of early-onset retinal dystrophy and milder forms of retinal dystrophies such as juvenile retinitis pigmentosa and dominant cone-rod dystrophy. [Perrault, I., Rozet, J.M., Gerber, S., Ghazi, I., Leowski, C., Ducroq, D., Souied, E., Dufier, J.L., Munnich, A. and Kaplan, J. (1999) Leber congenital amaurosis. Mol. Genet. Metab., 68, 200–208.] Although not yet fully elucidated, AIPL1 is likely to function as a specialized chaperone for rod phosphodiesterase (PDE). We evaluate whether AAV-mediated gene replacement therapy is able to improve photoreceptor function and survival in retinal degeneration associated with AIPL1 defects. We used two mouse models of AIPL1 deficiency simulating three different rates of photoreceptor degeneration. The Aipl1 hypomorphic (h/h) mouse has reduced Aipl1 levels and a relatively slow degeneration. Under light acceleration, the rate of degeneration in the Aipl1 h/h mouse is increased by 2–3-fold. The Aipl1–/– mouse has no functional Aipl1 and has a very rapid retinal degeneration. To treat the different rates of degeneration, two pseudotypes of recombinant adeno-associated virus (AAV) exhibiting different transduction kinetics are used for gene transfer. We demonstrate restoration of cellular function and preservation of photoreceptor cells and retinal function in Aipl1 h/h mice following gene replacement therapy using an AAV2/2 vector and in the light accelerated Aipl1 h/h model and Aipl1–/– mice using an AAV2/8 vector. We have thus established the potential of gene replacement therapy in varying rates of degeneration that reflect the clinical spectrum of disease. This is the first gene replacement study to report long-term rescue of a photoreceptor-specific defect and to demonstrate effective rescue of a rapid photoreceptor degeneration

Quality Management as supportive tool for Controlling, applied to a practical case (biohelp GmbH)

Die folgende Arbeit wurde im Zuge meines Studiums, Bachelor of Arts in Betriebswirtschaftslehre an der Hochschule Mittweida (FH) – Universal of Applied Sciences verfasst. In meiner Bachelorarbeit werden die Begriffe „Qualitätsmanagement“ und „Controlling“ näher erläutert, sowie ausgewählte Methoden aus diesen Themengebieten, die am Unternehmen „biohelp GmbH“ anwendbar sind

Advances in Data-Driven Decision-Making: A Mathematical Optimization Perspective

Data-driven decision-making holds great potential for increasing the productivity of companies and organizations. However, this potential is not yet fully leveraged as there are still certain barriers to implementing such systems in practice. This doctoral thesis presents three research papers following the same purpose: offering solution approaches for common challenges in the field of data-driven decision-making via novel optimization procedures. The three challenges under consideration are the (i) explainability, (ii) scalability, and (iii) accessibility of data-driven decision-making approaches. Besides individual contributions in the respective area of application, each paper targets one of the three challenges in particular. The first paper of this doctoral thesis develops an explainable data-driven algorithm for personalized medicine. In particular for off-policy learning, where the goal is to derive personalized treatment decisions based on individual patient characteristics from observational data, e.g., randomized control trials. The resulting treatment decisions can be presented in disjunctive normal form, i.e., OR-of-ANDs, and fulfill explainability demands from clinical practice. This is shown in a user study, in which we ask actual clinical practitioners to rate the interpretability of our approach. The main contribution, that makes this new algorithm possible, lies in the field of mathematical optimization. That is, a novel formulation of off-policy learning as a mixed-integer linear program, and a tailored column generation procedure within a branch-and-bound framework to solve it. The second paper proposes an efficient Monte Carlo tree search (MCTS) for data-driven dynamic police patrolling. Thereby, the goal is to optimize and dynamically adjust patrol routes of police units through their patrol beats, i.e., predefined patrol areas, with the aim of crime risk reduction. In contrast to state-of-the-art patrol algorithms based on vehicle routing problem formulations, the novel MCTS approach scales to real-world problem instances. This is confirmed in a simulation study using actual crime and operational data from Chicago. Again, the main driving force behind the new approach is a novel problem formulation as a Markov decision process. The latter allows to efficiently guide the optimization process within MCTS by tailored roll-out policies. The third paper is a theoretical contribution to the field of neural learning. Neural networks have shown the potential to create immense value in the area of data-driven decision-making. However, their training usually relies on expert knowledge gained over years. The contribution of this work is to develop a novel optimization approach for learning parameters of neural networks, that allows for general convergence guarantees and refrains from any hyperparameters related to training. Thus, the tuning of hyperparameters, which are only difficult to assess, and the manual inspection of convergence by examining loss curves becomes obsolete. The proposed algorithm is a first step towards turning the training of neural networks into a fully automated process and, thus, making neural networks more accessible to non-experts. This thesis provides solution approaches for three major challenges in data-driven decision-making. The presented results are mainly theoretical contributions and, thus, are likely to generalize to different areas of application