Gene Therapy of Corneal Dystrophy: Lysosomal Storage Diseases

角膜营养不良的基因治疗：溶酶体贮积病

• 批准号: 10203999
• 负责人: WINSTON W KAO
• 金额: $ 37.96万
• 依托单位: UNIVERSITY OF CINCINNATI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-30 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10203999
• 关键词: 3-Dimensional; Adult; Beta-glucuronidase; Blood Circulation; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; CRISPR/Cas technology; Cells; Cessation of life; Clustered Regularly Interspaced Short Palindromic Repeats; Complementary DNA; Cornea; Corneal dystrophy; DNA; Development; Disease; Early treatment; Embryo; Enzymes; Event; Extracellular Matrix; Family; Fibroblasts; Genes; Genetic; Hematopoietic; Hematopoietic stem cells; Hepatocyte; Hereditary Disease; Histologic; Homeostasis; Individual; Injections; Internal Ribosome Entry Site; Intravenous; Left; Length; Lentivirus; Lipids; Liver; Longevity; Low Prevalence; Lysosomal Storage Diseases; Lysosomes; Mediating; Mediator of activation protein; Mesenchymal Stem Cells; Modeling; Mucopolysaccharidosis VII; Mus; Mutation; Neurons; Nutrient; Outcome; Patients; Play; Prevalence; Production; Proteins; Role; Route; Somatic Cell; Stem cell transplant; Survival Rate; Transgenes; Translating; Transplantation; Treatment Efficacy; Treatment Protocols; Viral Vector; Virus; Western Blotting; X-Ray Computed Tomography; allotransplant; arm; base; common treatment; confocal imaging; efficacy evaluation; efficacy validation; enzyme replacement therapy; extracellular vesicles; gene therapy; genome editing; genome-wide; graft vs host disease; improved; in vivo; intercellular communication; intrahepatic; intravenous injection; lysosomal proteins; magnetic beads; mouse model; neonate; neutralizing antibody; novel; reduce symptoms; repaired; somatic cell gene editing; stem; stem cells; therapeutic genome editing; therapeutically effective; treatment strategy; tumorigenesis; vector

Summary Lysosomal storage diseases (LSDs) are a family of rare inherited diseases caused by a mutation in genes of lysosomal enzymes and proteins, resulting in excessive accumulation of metabolites and lack of nutrients for homeostasis. Individual LSDs have a low prevalence, but collectively they have a combined prevalence of 1:8000. Enzyme replacement therapy and bone marrow transplantation are two common treatments, but production of neutralizing antibodies and graft versus host disease hampers treatment. Gene therapy using lentivirus yields encouraging outcomes, but can induce tumorigenesis. Thus, novel treatments are needed. Lysosomal enzymes/proteins are found in extracellular vesicles (EV) that mediate intercellular communication. CRISPR gene editing of a patient's somatic cells will lead to production of functional enzymes/proteins in circulation via EV and/or hematopoietic cells and ameliorate symptoms. Three aims are proposed to establish efficacious CRISPR treatment strategies and to elucidate the mechanism in which direct genome editing of somatic cells or transplantation of CRISPR-edited hematopoietic stem/hematopoietic stem progenitor cells can treat a mouse model of MPS VII. Specific Aim 1: Define Optimal Condition(s) and Off-target events of CRISPR in Treating Gusb/MPS VII Aim 1A: To validate the editing efficiency, synthesis and secretion of β-Glu and off targeting events following genome editing. Aim 1B: Determine the best route of AAV2DJ delivery. The treatment efficacy of multiple administrations with AAV2DJ-Sa-CRISPR viral vectors will be analyzed. Mice will be subjected to 1) HRTII in vivo confocal imaging for reduction of corneal haze; 2) Survival rate determination; 3) In vivo 3D CT scan to determine liver size; 4) β-Glu activity. Aim 1C: Intrastromal injection of AAV2DJ-Sa- CRISPR to examine the efficacy of gene editing in treating corneal haze. Specific Aim 2: To Determine the Efficacy of Gene Editing Therapy of Hematopoietic Stem and Stem Progenitor Cells (HSC/HSPC) for Gusb mice Lin-Sca1+ HSC/HSPC will be isolated from donor Gusb mice and subjected to CRISPR editing and expanded. The CRISPR-edited HSC/HSPC will then be transplanted to gamma-irradiated recipient mice via ROIV. The treatment efficacy will be assessed as described in Aim 1. Specific Aim 3: To Determine Efficacy of Homology Mediated End Joining-based CRISPR (HMEJ) for Gusb/MPS VII as a Proof of Principle for LSDs. Aim 3A: Gusb MEF will be used to validate the genome editing efficiency of a binary AAV consisting of AAV2DJ-SpCas9 and AAV2DJ-sgRNA/donor DNA template containing selective transgenes Aim 3B: will determine the efficacy of administration of the binary AAV2 vectors for Gusb mice. Aim 3C: Transplantation of CRISPR edited Gusb Lin-Sca1+ HSC/HSPC to recipient Gusb mice that will be examined as described in Specific Aim 1. The proposed studies will lead to the development of effective therapeutic strategies for MPS VII and other types of LSDs, which can ultimately be translated to the bedside.

总结 溶酶体贮积病（LSD）是一种罕见的遗传性疾病家族，由以下基因突变引起： 溶酶体酶和蛋白质，导致代谢物过度积累和缺乏营养， 体内平衡个别LSD的患病率较低，但总体而言， 1：8000。酶替代疗法和骨髓移植是两种常见的治疗方法， 中和抗体和移植物抗宿主病的产生妨碍治疗。基因治疗使用 慢病毒产生令人鼓舞的结果，但可以诱导肿瘤发生。因此，需要新的治疗方法。 溶酶体酶/蛋白质存在于介导细胞间通讯的细胞外囊泡（EV）中。 患者体细胞的CRISPR基因编辑将导致在细胞内产生功能性酶/蛋白质。 通过EV和/或造血细胞循环并改善症状。提出了三个目标， 有效的CRISPR治疗策略，并阐明直接基因组编辑的机制。 CRISPR编辑的造血干细胞/造血干祖细胞的体细胞或移植可 治疗MPS VII的小鼠模型。具体目标1：定义最佳条件和脱靶事件 CRISPR治疗Gusb/MPS VII目的1A：验证β-Glu的编辑效率、合成和分泌 以及基因组编辑后的脱靶事件。目的1B：确定AAV 2DJ递送的最佳途径。的 将分析用AAV 2DJ-Sa-CRISPR病毒载体多次施用的治疗功效。小鼠将 进行1）HRTII体内共焦成像以减少角膜混浊; 2）存活率测定; 3)体内3D CT扫描以确定肝脏大小; 4）β-Glu活性。目的1C：基质内注射AAV 2DJ-Sa- CRISPR检查基因编辑治疗角膜混浊的疗效。具体目标2：确定 造血干细胞和干祖细胞（HSC/HSPC）的基因编辑治疗对 Gusb小鼠Lin-Sca 1 + HSC/HSPC将从供体Gusb小鼠中分离并进行CRISPR编辑， 扩大然后，CRISPR编辑的HSC/HSPC将通过以下方式移植到γ辐照的受体小鼠中： ROIV。将按照目标1所述评估治疗疗效。具体目标3：确定疗效 Gusb/MPS VII的基于同源介导末端连接的CRISPR（HMEJ）作为以下原理的证明： 迷幻药目的3A：Gusb MEF将用于验证由以下组成的二元AAV的基因组编辑效率： AAV 2DJ-SpCas 9和AAV 2DJ-sgRNA/含有选择性转基因Aim 3B的供体DNA模板：将 确定二元AAV 2载体对Gusb小鼠的施用功效。目标3C：移植 将CRISPR编辑的Gusb Lin-Sca 1 + HSC/HSPC转移至受体Gusb小鼠，将如图10中所述进行检查。 具体目标1.拟议的研究将导致有效的治疗策略的发展， MPS VII和其他类型的LSD，最终可以转移到床边。

项目成果

The gene therapy for corneal pathology with novel nonsense cystinosis mouse lines created by CRISPR Gene Editing.

利用 CRISPR 基因编辑创建的新型无意义胱氨酸病小鼠品系进行角膜病理基因治疗。

• DOI: 10.1016/j.jtos.2023.06.002
• 发表时间: 2023
• 期刊: The ocular surface
• 作者: Dong,Fei;Amlal,Hassane;Venkatakrishnan,Jhuwala;Zhang,Jianhua;Fry,Matthew;Yuan,Yong;Cheng,YuChia;Hu,Yueh-Chiang;Kao,WinstonW-Y
• 通讯作者: Kao,WinstonW-Y