Interventional approaches for restoring vision

恢复视力的介入方法

• 批准号: 8556848
• 负责人: ANAND SWAROOP
• 金额: $ 162.66万
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8556848
• 关键词: Animal Genetics; Animal Model; Architecture; Biocompatible Materials; Biological Assay; Calcium; Canis familiaris; Cell Culture Techniques; Cell Survival; Cell Transplantation; Cell surface; Cells; Chemicals; Coculture Techniques; Collaborations; Collagen; Development; Disease; Disease model; Dose; Embryo; Enhancers; Environment; Exhibits; Fluorescence; Gene Targeting; Genes; Goals; Homeostasis; Human; Image; In Vitro; Inhibitory Concentration 50; Knowledge; Leber' s amaurosis; Libraries; Link; Modality; Modeling; Molecular; Morphology; Mus; Mutation; Neurons; Newborn Infant; Pathway interactions; Pharmaceutical Preparations; Photoreceptors; Population; Protocols documentation; RNA; Reagent; Replacement Therapy; Reporter; Reporting; Research; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rhodopsin; Screening procedure; Solutions; Source; Staging; Stem cells; Subfamily lentivirinae; System; Technology; Testing; Therapeutic Effect; Transgenic Organisms; Translations; Transplantation; Vision; Work; Zebrafish; Zinc Fingers; adeno-associated viral vector; base; drug discovery; embryonic stem cell; gene discovery; gene therapy; high throughput screening; human embryonic stem cell; in vitro Model; in vivo; induced pluripotent stem cell; mouse model; mutant; novel; patch clamp; phosphoric diester hydrolase; photoreceptor degeneration; preclinical study; prevent; promoter; reconstruction; repaired; retinal neuron; retinal rods; rho; scaffold; small molecule; therapy development; tool; two-dimensional

Background We are testing several approaches, including gene therapy, small molecule screening for drug discovery, and retinal reconstruction. All strategies are guided by the knowledge of molecular mechanisms of photoreceptor development, homeostasis, and disease acquired through other projects (see EY000450-473-475) Results 1. Gene therapy In collaboration with Drs. P. Colosi and T. Li, we have initiated pre-clinical studies for the treatment of Leber congenital amaurosis (LCA) caused by CEP290 mutations and of retinitis pigmentosa (RP) caused by mutations in the RPGR and RP2 genes. N-NRL has focused on animal models and genetic characterizations, whereas Peter Colosi's unit is involved in AAV-based gene therapy (see Colosi report). As CEP290 is a large gene, we are working with Colosi and Li groups to test different AAV constructs and lenti-viruses. As a novel tool for gene therapy, we are testing AAV vectors containing 0.3 kb of Nrl promoter/enhancer that can specifically direct the target gene to both developing and mature rod photoreceptors. In collaboration with Drs. Sam Jacobson, Gus Aguirre and Bill Hauswirth, we have proved the efficacy of gene augmentation therapy in two blinding canine photoreceptor disease models for the common X-linked form of retinitis pigmentosa, caused by mutations in the RPGR gene (1). Our results provide a path for translation to human treatment. 2. Drug discovery We are developing reagents and protocols for high throughput screens of small molecules, to discover new drugs to prevent photoreceptor degeneration. We are using zebrafish as animal model, developing in vitro biomaterial-based cell cultures, and plan to use iPS cells for small molecule testing. Candidate molecules will be further investigated in preclinical studies using our well-characterized mouse models of retinal degeneration. Our small molecule screens will search for chemicals that can either influence photoreceptor differentiation o rescue photoreceptor degeneration. We are using three zebrafish transgenic lines. In TalphaCP-GFP and Rho-GFP lines, GFP expression is restricted to cones or rods, respectively. In a pilot screen of the NCI Diversity II library of about 1800 compounds using the TalphaCP-GFP reporter, we identified seven candidate compounds that increase the reporter fluorescence and two compounds that cause a decrease in fluorescence intensity, seemingly promoting and preventing photoreceptor differentiation, respectively. The Pde6cw59 line contains a mutation in the cone phosphodiesterase C. As a result, homozygous embryos exhibit rapid degeneration of cone photoreceptors by 4 dpf. Homozygous mutant embryos, which are positive for the fluorescent marker, will be subjected to the NCI Diversity II library. Candidate compounds will be evaluated further to determine their dose curve, IC50 and mechanism of action. Other zebrafish lines carrying mutations in genes that are associated with retinal degeneration are being generated or acquired from collaborators. We have established that Collagen Vitrigel (from Toshiaki Takezawa) is a suitable biomaterial substrate for culturing developing rods and favors cell survival, establishment of neuronal morphology, and expression of rod markers, including rhodopsin. We are developing a biomaterial-based co-culture model that includes iPSC- and ESC-derived photoreceptors with RPE-derived soluble molecules or cells. The use of biomaterials to create an environment that more closely mimics in vivo photoreceptor-RPE architecture is a significant improvement over current two-dimensional or solution-based culture systems and sets the stage for their use in large scale screening assays and as transplantable scaffolds for retinal cell replacement therapies. 3. Use of iPSCs and ESCs to develop therapies We previously showed that Nrl-expressing newborn rod photoreceptors can differentiate and integrate in degenerating mouse retina, thus suggesting the feasibility of retinal repair by photoreceptor replacement. Since then, others and we have been pursuing avenues to increase the efficiency of integration and to generate photoreceptors from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). More recently, we tested the potential of human ESCs to produce retinal neurons in vivo (2). Furthermore, we have successfully developed protocols to grow human iPSCs and ESCs and differentiated these into retinal neurons. We have generated several fluorescence reporter constructs that will now be stably integrated into stem cells using PiggyBac or zinc-finger technology. Lineage-restricted cells (as determined by the expression of specific promoter construct) will be used for transplantation in the degenerating retina. Calcium imaging and patch clamp recording are being used to functionally characterize the stem cell-derived photoreceptors in vitro and after transplantation in the recipient mouse. Based on RNA-Seq analysis of lineage-restricted cells, we will identify cell surface markers that will allow us to purify sub-populations of differentiating cells for transplantation and to establish which one has the best therapeutic effect.

背景 我们正在测试几种方法，包括基因治疗，药物发现的小分子筛选和视网膜重建。所有策略均以通过其他项目获得的光感受器发育、稳态和疾病的分子机制知识为指导（参见EY 000450 -473-475） 结果 1. 基因治疗 与P. Colosi和T. Li，我们已经启动了治疗由CEP 290突变引起的Leber先天性黑蒙（LCA）和由RPGR和RP 2基因突变引起的视网膜色素变性（RP）的临床前研究。N-NRL专注于动物模型和遗传特征，而Peter Colosi的单位参与了基于AAV的基因治疗（见Colosi报告）。由于CEP 290是一个大基因，我们正在与Colosi和Li团队合作测试不同的AAV构建体和慢病毒。作为基因治疗的新工具，我们正在测试含有0.3kb的Nrl启动子/增强子的AAV载体，其可以特异性地将靶基因引导至发育和成熟的视杆细胞。 在与Sam Jacobson、Gus Aguirre和Bill Hausperth博士的合作中，我们已经证明了基因增强疗法在两种致盲犬感光细胞疾病模型中对常见的X连锁形式的视网膜色素变性的有效性，这种疾病是由RPGR基因突变引起的（1）。我们的研究结果为人类治疗提供了一条途径。 2. 药物发现 我们正在开发用于小分子高通量筛选的试剂和方案，以发现预防感光细胞变性的新药。我们正在使用斑马鱼作为动物模型，开发基于生物材料的体外细胞培养，并计划使用iPS细胞进行小分子测试。候选分子将在临床前研究中使用我们充分表征的视网膜变性小鼠模型进行进一步研究。 我们的小分子筛选将寻找可以影响感光细胞分化或挽救感光细胞变性的化学物质。 我们正在使用三种斑马鱼转基因品系。 在TalphaCP-GFP和Rho-GFP系中，GFP表达分别限于视锥或视杆。在使用TalphaCP-GFP报告基因对NCI Diversity II文库的约1800种化合物进行的中试筛选中，我们确定了7种增加报告基因荧光的候选化合物和2种导致荧光强度降低的化合物，似乎分别促进和阻止感光细胞分化。Pde 6cw 59系在视锥磷酸二酯酶C中含有突变。 结果，纯合子胚胎在4 dpf时表现出视锥光感受器的快速退化。荧光标记阳性的纯合突变胚胎将进入NCI Diversity II文库。将进一步评价候选化合物，以确定其剂量曲线、IC 50和作用机制。其他携带视网膜变性相关基因突变的斑马鱼品系正在从合作者那里产生或获得。 我们已经确定胶原Vitrigel（来自Toshiaki Takezawa）是用于培养发育中的视杆的合适的生物材料基质，并且有利于细胞存活、神经元形态的建立和视杆标志物（包括视紫红质）的表达。我们正在开发一种基于生物材料的共培养模型，包括iPSC和ESC衍生的光感受器与RPE衍生的可溶性分子或细胞。使用生物材料来创建更紧密地模拟体内光感受器-RPE结构的环境是对当前二维或基于溶液的培养系统的显著改进，并且为其在大规模筛选测定中的使用以及作为视网膜细胞替代疗法的可移植支架奠定了基础。 3. 使用iPSC和ESC开发疗法 我们先前表明，表达Nrl的新生视杆细胞光感受器可以在退化的小鼠视网膜中分化和整合，从而表明通过光感受器替换进行视网膜修复的可行性。从那时起，其他人和我们一直在寻求提高整合效率的途径，并从胚胎干细胞（ESC）和诱导多能干细胞（iPSC）中产生光感受器。 最近，我们测试了人类胚胎干细胞在体内产生视网膜神经元的潜力（2）。此外，我们已经成功开发了培养人类iPSC和ESC的方案，并将其分化为视网膜神经元。我们已经产生了几个荧光报告构建体，现在将稳定地整合到干细胞使用PiggyBac或锌指技术。谱系限制性细胞（如通过特异性启动子构建体的表达所确定的）将用于在变性视网膜中移植。钙成像和膜片钳记录被用来功能特性的干细胞衍生的光感受器在体外和移植后在受体小鼠。基于对谱系限制性细胞的RNA-Seq分析，我们将鉴定细胞表面标记，这将使我们能够纯化用于移植的分化细胞亚群，并确定哪一种具有最佳治疗效果。