Interventional approaches for restoring vision

恢复视力的介入方法

• 批准号: 8938332
• 负责人: ANAND SWAROOP
• 金额: $ 147.41万
• 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/8938332
• 关键词: Affect; Age-Months; Aging; Animal Genetics; Animal Model; Architecture; Biocompatible Materials; Biological Assay; Cell Line; Cell Therapy; Cells; Collaborations; Collection; Complementary DNA; Cone; Development; Disease; Embryo; Gene Expression; Gene Expression Profile; Gene Expression Profiling; Gene Mutation; Generations; Genes; Goals; Hip region structure; Homeobox; Homeobox Genes; Homeostasis; Human; In Vitro; Injection of therapeutic agent; Intervention; Kinetics; Knockout Mice; Knowledge; Leber' s amaurosis; Legal patent; Modality; Modeling; Molecular; Monitor; Mus; Mutant Strains Mice; Mutation; Neuroglia; Organoids; Pathogenesis; Pathway interactions; Patients; Pattern; Phenotype; Photoreceptors; Pluripotent Stem Cells; Protocols documentation; Reporter; Research; Resources; Retina; Retinal; Retinal Cone; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Screening Result; Sorting - Cell Movement; Source; Staging; Structure; System; Testing; Therapeutic; Time; Transgenic Organisms; Vertebrate Photoreceptors; Vision; Zebrafish; adeno-associated viral vector; base; cost; efficacy testing; fetal; gene discovery; gene therapy; high throughput screening; human embryonic stem cell; in vitro Model; induced pluripotent stem cell; mouse model; mutant mouse model; novel; preclinical study; promoter; reconstruction; research clinical testing; retinal neuron; retinal rods; retinogenesis; rho; screening; small molecule; small molecule libraries; therapy development; tool; transcriptome sequencing; vector; working group

Background Retinal degenerative diseases manifest with heterogeneous phenotypes, suggesting that one single therapeutic approach may not be applicable to all. Different modalities of intervention (in some cases in combination) could be targeted to specific retinal diseases based on known mechanisms of pathogenesis. We are testing gene therapy, small molecules, and retinal reconstruction to identify novel modalities to treat retinal degenerative diseases. All strategies are guided by the knowledge acquired through other projects of the molecular mechanisms of photoreceptor development, homeostasis, and disease (see EY000450-73-75). - Gene therapy Pre-clinical studies in collaboration with Z. Wu and T. Li (NNRL, NEI) are well underway for the treatment of retinitis pigmentosa (RP) caused by mutations in the RPGR and RP2 genes and of Leber congenital amaurosis (LCA) caused by CEP290 mutations. We focused on animal models and genetic characterizations, and Z. Wu's group worked on AAV-vector generation and gene therapy. Mouse and human RPGR-AAV vectors were tested in the RPGR-/- and rd9 mutant mouse models. A third mouse model with faster degeneration was also generated by crossing to a strain with a different background. Human and mouse vectors showed an effect in the RPGR-/- model at 18-24 months post-administration. Upon injection of the AAV8 human RP2 vector in RP2-null mice, cone function was rescued at 4 months post-administration and persisted up to 18 months. AAV8-RP2 effect on rod function could not be evaluated because of the rod degeneration kinetics with stabilization at one month of age. The large size of CEP290 cDNA exceeds the packaging limit of AAV vector. Among several AAV vectors that we tested, we recently identified one vector that is able to preserve retinal structure and function of the Cep290 mutant mouse model. A patent application has been filed (1). - Small molecule screening We are using zebrafish, embryonic and induced pluripotent stem cells (ESCs and iPSCs) and in vitro cell-based cultures to develop protocols for high throughput screens (HTS) of small molecules. One of our aims is to identify small molecules that facilitate the generation of rod and cone photoreceptors in zebrafish or from hESCs and hiPSCs expressing rod- and cone-specific transgenic reporter lines. We selected one cone-specific candidate molecule resulting from screening of the NCI Diversity II set (2000 compounds) and The Spectrum Collections (3000 compounds) chemical libraries using two zebrafish transgenic reporter lines (Rho-GFP and TalphaCP-GFP). We are further investigating the effects of the candidate compound and the developmental and/or homeostatic pathways affected. Rod or cone photoreceptors produced from hESCs or hiPSCs are a potential renewable source for cell-based therapy. However, lengthy differentiation protocols and low yield make their use currently unpractical. We are testing small molecule libraries using hES and hiPS cell lines with fluorescent reporter constructs driven by photoreceptor-specific promoters to identify compounds that facilitate the differentiation of high quality and pure transplantable photoreceptor cells with moderate cost and effort. - Use of iPSCs and ESCs to develop therapies Although animal models are an essential resource to study retinal development, homeostasis, aging/disease and to test novel therapies, they not always recapitulate the features of the human retina. Three-dimentional organoids generated from floating aggregates of hESCs are a novel tool for elucidating the mechanism underlying retinogenesis and disease. We generated a hESC line expressing a reporter GFP in rod and cone photoreceptor precursors under control of the cone-rod homeobox (CRX) gene promoter. GFP pattern of expression recapitulated that of endogenous CRX in the differentiating retinal organoids that also expressed markers of rod and cone photoreceptors and of other retinal neurons and Mller glia. Time-course gene expression profiling by RNA-seq of GFP+ flow sorted photoreceptors was performed to monitor the developmental steps in vitro. The gene expression signature revealed that CRX-GFP+ cells derived from hESCs are similar to human fetal photoreceptors, validating the organoids as in vitro models for studies of the human retina. Several patient and control iPSCs have been generated and are being characterized to be used to study the effect of gene mutation and of targeted therapies on photoreceptors. The lengthy differentiation protocols to generate photoreceptors from human pluripotent stem cells pose a limitation also to their use as pre-clinical testing systems. To overcome this limitation, we focused on mouse induced pluripotent stem cells for proof-of-concept preclinical studies that can be further evaluated on human cells at a later stage of therapy development. We generated iPSCs from the Cep290/rd16 mutant mouse and differentiated them into retinal organoids. We found that the architecture and gene expression of the Cep290/rd16 iPSC-derived retina were abnormal compared to WT controls. The retinal differentiation assay will be employed to test the efficacy of AAV vectors encoding different domains of CEP290 for gene therapy.

背景 视网膜退行性疾病表现为异质性表型，表明单一的治疗方法可能不适用于所有疾病。基于已知的发病机制，不同的干预方式（在某些情况下组合）可以针对特定的视网膜疾病。我们正在测试基因治疗，小分子和视网膜重建，以确定治疗视网膜退行性疾病的新方法。所有策略都是由通过其他项目获得的感光细胞发育、稳态和疾病的分子机制的知识指导的（参见EY 000450 -73-75）。 - 基因治疗 与Z. Wu和T. Li（NNRL，NEI）正在顺利进行中，用于治疗由RPGR和RP 2基因突变引起的视网膜色素变性（RP）和由CEP 290突变引起的Leber先天性黑蒙（LCA）。我们集中在动物模型和遗传特征，Z。Wu的团队致力于AAV载体的产生和基因治疗。 在RPGR-/-和rd 9突变小鼠模型中测试小鼠和人RPGR-AAV载体。通过与具有不同背景的品系杂交，还产生了具有更快退化的第三种小鼠模型。人和小鼠载体在施用后18-24个月在RPGR-/-模型中显示出效果。 在RP 2缺失小鼠中注射AAV 8人RP 2载体后，视锥细胞功能在施用后4个月得到拯救，并持续长达18个月。AAV 8-RP 2对视杆功能的影响无法评估，因为视杆变性动力学在一个月龄时稳定。 CEP 290 cDNA的大尺寸超过了AAV载体的包装极限。在我们测试的几种AAV载体中，我们最近鉴定了一种能够保留Cep 290突变小鼠模型的视网膜结构和功能的载体。已提交专利申请（1）。 - 小分子筛选 我们正在使用斑马鱼，胚胎和诱导多能干细胞（ESC和iPSC）和体外细胞培养来开发小分子高通量筛选（HTS）的方案。我们的目标之一是确定小分子，促进产生的杆和锥光感受器在斑马鱼或从hESC和hiPSC表达杆和锥特异性转基因报告细胞系。 我们选择了一个锥特异性的候选分子，从筛选的NCI多样性II集（2000化合物）和光谱收集（3000化合物）的化学库使用两个斑马鱼转基因报告线（Rho-GFP和TalphaCP-GFP）。我们正在进一步研究候选化合物的影响以及受影响的发育和/或稳态途径。 由hESC或hiPSC产生的视杆或视锥光感受器是基于细胞的治疗的潜在可再生来源。然而，冗长的分化方案和低产率使得它们的使用目前不切实际。我们正在使用hES和hiPS细胞系测试小分子文库，这些细胞系具有由光感受器特异性启动子驱动的荧光报告构建体，以鉴定促进高质量和纯的可移植光感受器细胞分化的化合物，其成本和努力适中。 - 使用iPSC和ESC开发疗法 虽然动物模型是研究视网膜发育、稳态、衰老/疾病和测试新疗法的重要资源，但它们并不总是概括人类视网膜的特征。 由hESC的漂浮聚集体产生的三维类器官是用于阐明视网膜发生和疾病的潜在机制的新工具。我们产生了一个人胚胎干细胞系，在视锥-视杆同源框（CRX）基因启动子的控制下，在视杆和视锥光感受器前体中表达报告基因GFP。GFP的表达模式概括了内源性CRX在分化中的视网膜类器官中的表达模式，所述视网膜类器官还表达视杆和视锥光感受器以及其他视网膜神经元和Mller神经胶质的标志物。通过GFP+流式分选的光感受器的RNA-seq进行时程基因表达谱分析以监测体外发育步骤。基因表达特征揭示了源自hESC的CRX-GFP+细胞与人胎儿光感受器相似，验证了类器官作为研究人视网膜的体外模型。 已经产生了几种患者和对照iPSC，并且正在对其进行表征以用于研究基因突变和靶向治疗对光感受器的影响。 从人多能干细胞产生光感受器的漫长分化方案也限制了其作为临床前测试系统的用途。为了克服这一限制，我们专注于小鼠诱导多能干细胞的概念验证临床前研究，可以在治疗开发的后期阶段对人类细胞进行进一步评估。我们从Cep 290/rd 16突变小鼠中产生了iPSC，并将其分化为视网膜类器官。我们发现Cep 290/rd 16 iPSC衍生的视网膜的结构和基因表达与WT对照相比是异常的。将采用视网膜分化测定来测试编码CEP 290的不同结构域的AAV载体用于基因治疗的功效。