Interventional approaches for restoring vision

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。所有策略都以光感受器发育、内稳态和通过其他项目获得的疾病的分子机制的知识为指导(见EY000450-473-475) 结果 1.基因治疗 我们与P.Colosi博士和T.Li博士合作，启动了治疗由CEP290突变引起的Leber先天性黑色素沉着症(LCA)以及由RPGR和RP2基因突变引起的视网膜色素变性(RP)的临床前研究。N-NRL专注于动物模型和基因特征，而Peter Colosi的单位参与了基于AAV的基因治疗(见Colosi报告)。由于CEP290是一个大基因，我们正在与Colosi和LI小组合作测试不同的AAV结构和Lenti病毒。作为一种新的基因治疗工具，我们正在测试包含0.3kb NRL启动子/增强子的AAV载体，它可以特异性地将靶基因定向到发育中的和成熟的杆状感光器。 我们与Sam Jacobson博士、Gus Aguirre博士和Bill Hauswirth博士合作，证明了基因增强疗法在两种致盲犬光感受器疾病模型中的有效性，该疾病模型是由RPGR基因突变引起的常见X连锁形式的视网膜色素变性(1)。我们的结果提供了一条转化为人类治疗的途径。 2.药物发现 我们正在开发用于高通量筛选小分子的试剂和方案，以发现防止光感受器退化的新药。我们正在以斑马鱼为动物模型，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特征良好的视网膜变性小鼠模型进行进一步研究。 我们的小分子筛选器将寻找既能影响光感受器分化又能挽救光感受器退化的化学物质。我们正在使用三个斑马鱼转基因品系。在TalphaCP-GFP和Rho-GFP中，GFP的表达分别局限于锥体和杆状细胞。在使用TalphaCP-GFP报告对NCI多样性II文库进行的约1800种化合物的初步筛选中，我们鉴定了七种候选化合物增强报告荧光，以及两种导致荧光强度降低的化合物，似乎分别促进和阻止了光感受器分化。Pde6cw59株系含有锥体磷酸二酯酶C突变，因此，纯合子胚胎表现出锥体感光细胞在4DPF下的快速退化。荧光标记阳性的纯合子突变胚胎将被纳入NCI多样性II文库。候选化合物将被进一步评估，以确定它们的剂量曲线、IC50和作用机制。其他携带与视网膜退化相关的基因突变的斑马鱼品系正在产生或从合作者那里获得。 我们已经确定胶原蛋白Vitrigel(产自竹泽俊明)是培养发育中的杆状细胞的合适生物材料底物，有利于细胞存活、神经元形态的建立以及包括视紫红质在内的杆状标记物的表达。我们正在开发一种基于生物材料的共培养模型，其中包括IPSC和ESC来源的光感受器与RPE来源的可溶性分子或细胞。使用生物材料来创造一个更接近于体内光感受器-RPE结构的环境是对当前二维或基于溶液的培养系统的重大改进，并为它们在大规模筛选分析中的使用以及作为视网膜细胞替代治疗的可移植支架奠定了基础。 3.利用IPSCs和ESCs开发治疗方法 我们先前的研究表明，在退化的小鼠视网膜中，表达NRL的新生视杆细胞感受器可以分化和整合，从而提示通过光感受器替换来修复视网膜的可行性。从那时起，其他人和我们一直在寻找提高整合效率的方法，并从胚胎干细胞(ESCs)和诱导的多能干细胞(IPSCs)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，我们还成功地开发了培养人类IPSCs和ESCs的方案，并将它们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，现在将使用iggyBac或锌指技术稳定地整合到干细胞中。谱系受限细胞(由特定启动子结构的表达决定)将用于在退化的视网膜中移植。钙成像和膜片钳记录被用来在体外和在受体小鼠移植后对干细胞来源的光感受器进行功能表征。基于对谱系受限细胞的RNA-Seq分析，我们将识别细胞表面标记，使我们能够纯化用于移植的分化细胞亚群，并确定哪种细胞具有最佳的治疗效果。

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.