Development and characterization of mouse models of RP59 DHDDS deficiency

RP59 DHDDS 缺陷小鼠模型的开发和表征

• 批准号: 10200065
• 负责人: Steven J. Fliesler
• 金额: $ 53.63万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-30 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10200065
• 关键词: Ablation; Address; Alleles; Animal Model; Biochemical; Blindness; CRISPR/Cas technology; Cells; Complex; Congenital disorders of glycosylation; Defect; Development; Diphosphates; Disease; Electron Microscopy; Electroretinography; Embryo; Enzymes; Exhibits; Family; Future; Generations; Genes; Hand; High Pressure Liquid Chromatography; Histocytochemistry; Human; Image; Immunohistochemistry; Intervention; Knock-in; Knock-out; Lead; Lectin; LoxP-flanked allele; Mass Spectrum Analysis; Mediating; Methodology; Modeling; Muller' s cell; Mus; Mutation; Neural Retina; Ocular Pathology; Online Mendelian Inheritance In Man; Opsin; Optical Coherence Tomography; Pathologic; Pathology; Patients; Phenotype; Positioning Attribute; Prevention; Proteins; Reflex action; Reporting; Resolution; Retina; Retinal Degeneration; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Rod; Scheme; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Structure of retinal pigment epithelium; Suggestion; Therapeutic Intervention; Vertebrate Photoreceptors; Western Blotting; cell type; disease mechanisms study; enzyme deficiency; gene therapy; genome editing; glycosylation; insight; light microscopy; macula; mouse model; mutant; novel; postnatal development; pre-clinical; prevent; promoter; recombinase-mediated cassette exchange; retinal rods; therapeutically effective; therapy design; Ablation; Address; Alleles; Animal Model; Biochemical; Blindness; CRISPR/Cas technology; Cells; Complex; Congenital disorders of glycosylation; Defect; Development; Diphosphates; Disease; Electron Microscopy; Electroretinography; Embryo; Enzymes; Exhibits; Family; Future; Generations; Genes; Hand; High Pressure Liquid Chromatography; Histocytochemistry; Human; Image; Immunohistochemistry; Intervention; Knock-in; Knock-out; Lead; Lectin; LoxP-flanked allele; Mass Spectrum Analysis; Mediating; Methodology; Modeling; Muller' s cell; Mus; Mutation; Neural Retina; Ocular Pathology; Online Mendelian Inheritance In Man; Opsin; Optical Coherence Tomography; Pathologic; Pathology; Patients; Phenotype; Positioning Attribute; Prevention; Proteins; Reflex action; Reporting; Resolution; Retina; Retinal Degeneration; Retinal Pigments; Retinitis Pigmentosa; Rhodopsin; Rod; Scheme; Site; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Structure; Structure of retinal pigment epithelium; Suggestion; Therapeutic Intervention; Vertebrate Photoreceptors; Western Blotting; cell type; disease mechanisms study; enzyme deficiency; gene therapy; genome editing; glycosylation; insight; light microscopy; macula; mouse model; mutant; novel; postnatal development; pre-clinical; prevent; promoter; recombinase-mediated cassette exchange; retinal rods; therapeutically effective; therapy design

7. Project Summary/Abstract Within the family of Congenital Disorders of Glycosylation (CDGs), defects in the dehydrodolichyl diphosphate synthase (DHDDS) gene cause a recessive form of retinitis pigmentosa (RP59; OMIM #613861). Patients with a K42E mutation in DHDDS exhibit progressive loss of both rod and cone function, as well as macular changes, suggestive of RPE involvement. The DHDDS enzyme is ubiquitously required in all cells for protein N- glycosylation. We wish to understand the basis for selective ocular pathology associated with ubiquitous DHDDS mutation and the contribution of specific ocular cell types to the pathology of mutant Dhdds-mediated retinal degeneration. As a first step, we devised a selective knockout scheme to study the importance of the enzyme in specific retinal cell types. To circumvent known embryonic lethality associated with DHDDS knockout, we generated a Cre-dependent knockout allele of murine Dhdds (Dhddsflx/flx). Additionally, we used CRISPR/Cas technology to generate a knock-in K42E mouse model of RP59. We propose to use these novel mouse lines to examine the mechanism of disease induced by DHDDS enzyme deficiency and to identify the primary site(s) of ocular pathology. In Aim 1, using rod-specific Cre expression, we will address the prevailing hypothesis that defective rhodopsin glycosylation is the major cause of Dhdds mutation-mediated pathology. In Aim 2, using retinal pigment epithelium (RPE)-specific Cre expression, we will examine the effects of perturbation of DHDDS activity on RPE structure and function. In Aim 3, we will selectively perturb DHDDS activity in Müller glia, and examine their contribution to Dhdds-dependent retinal degeneration. In Aim 4, we will characterize a newly generated K42E knock-in RP mouse model and compare the resulting structural and functional changes with those observed in the three knockout models to assess the pathological importance of each cell type. These four Aims will utilize a combination of state-of-the-art methodologies, including Cre-lox technology, CRISPR-Cas9- mediated genome editing, electroretinography (ERG), ultrahigh resolution spectral domain-optical coherence tomography (UHR SD-OCT), optokinetic reflex (OKR), light and electron microscopy, immunohistochemistry and lectin histochemistry, Western blot analysis, HPLC, and MALDI imaging mass spectrometry (IMS). Each stand- alone aim will provide significant new insights into the importance of DHDDS activity in the neural retina and RPE, and collectively will provide information that can guide rational treatment design for specific intervention in DHDDS-mediated RP. The overarching hypothesis is that, contrary to conclusions from initial reports, RP59 is not a simple disorder of altered rhodopsin glycosylation; rather, it is a complex disorder involving primary pathology in more than one cell type that may require a more global approach for effective therapeutic intervention. Upon completion of the proposed studies, we will be well positioned to pursue future proof-of- principle preclinical gene therapy studies to correct the genetic defect that otherwise would result in blindness.

7。项目摘要/摘要 在糖基化（CDG）的先天性疾病家族中，脱氢二醇二磷酸的缺陷 合成酶（DHDDS）基因引起色素性视网膜炎的隐性形式（RP59； OMIM＃613861）。患者 DHDD中的K42E突变暴露了杆和锥功能的进行性逐渐丧失，以及黄斑变化， 暗示RPE参与。在所有细胞中，DHDDS酶无处不在蛋白质N- 糖基化。我们希望理解与普遍存在DHDD相关的选择性眼病理学的基础 突变和特定的眼细胞类型对突变DHDDS介导的残留的病理的贡献 退化。作为第一步，我们设计了一个选择性敲除方案，以研究该酶在 特定的视网膜细胞类型。为了绕过与DHDDS敲除相关的已知胚胎致死性，我们 产生了鼠DHD（DHDDSFLX/FLX）的CRE依赖性敲除等位基因。此外，我们使用了CRISPR/CAS 生成RP59的Knock-In k42e鼠标模型的技术。我们建议将这些新颖的鼠标线 检查DHDDS酶缺乏诱导的疾病机制，并确定主要部位 眼病理学。在AIM 1中，使用杆特异性的CRE表达，我们将解决以下假设 有缺陷的视紫红质糖基化是DHDDS突变介导的病理学的主要原因。在AIM 2中，使用 视网膜色素上皮（RPE）特异性CRE表达，我们将检查DHDDS扰动的影响 RPE结构和功能的活动。在AIM 3中，我们将在MüllerGlia中有选择地扰动DHDDS的活动，并且 检查它们对DHDDS依赖性残余变性的贡献。在AIM 4中，我们将描述一个新的 生成的K42E敲入RP小鼠模型，并将所得的结构和功能变化与 在三个基因敲除模型中观察到的那些，以评估每种细胞类型的病理重要性。这四个 Aims将利用最新方法的结合，包括Cre-Lox技术，CRISPR-CAS9- 介导的基因组编辑，电视图（ERG），超高分辨率光谱域 - 光学连贯性 断层扫描（UHR SD-OCT），光（OKR），光和电子显微镜，免疫组织化学和 凝集素组织化学，Western印迹分析，HPLC和MALDI成像质谱法（IMS）。每个待机 单独的AIM将为DHDD活动在神经视网膜中的重要性和 RPE，并将共同提供可以指导理性治疗设计的信息 DHDDS介导的RP。总体假设是，与初始报告的结论相反，RP59是 不是简单的变化的视紫红质糖基化的疾病；相反，这是一种涉及主要的复杂疾病 可能需要更全球的方法进行有效治疗的多种细胞类型的病理学 干涉。拟议的研究完成后，我们将有能力寻求未来的证明 原理临床前基因治疗研究以纠正遗传缺陷，否则会导致失明。