Gene Therapy of Corneal Dystrophy: Lysosomal Storage Diseases

摘要 溶酶体贮积病(LSD)是一种罕见的遗传性疾病家族，由基因突变引起 溶酶体酶和蛋白质，导致代谢物过度积累，缺乏营养 动态平衡。个别LSD的患病率较低，但总体而言，它们的患病率合计为 1：8000。酶替代疗法和骨髓移植是两种常见的治疗方法，但 中和抗体和移植物抗宿主病的产生阻碍了治疗。使用基因疗法 慢病毒产生了令人鼓舞的结果，但可以诱导肿瘤形成。因此，需要新的治疗方法。 溶酶体酶/蛋白存在于细胞外小泡(EV)中，介导细胞间的通讯。 对患者体细胞进行CRISPR基因编辑将导致在体内产生功能性酶/蛋白质 通过EV和/或造血细胞进行循环，并改善症状。提出了三个目标来建立 有效的CRISPR治疗策略并阐明指导基因组编辑的机制 体细胞或移植CRISPR编辑的造血干细胞/造血祖细胞可以 治疗MPS小鼠模型的研究(7).具体目标1：确定MPS的最佳状态(S)和非靶点事件 CRISPR治疗GUSB/MPS的研究VII目标1A：验证β-Glu的编辑效率、合成和分泌 并在基因组编辑后关闭针对事件的目标。目的1B：确定AAV2DJ的最佳给药途径。这个 将分析AAV2DJ-Sa-CRISPR病毒载体多次给药的治疗效果。老鼠会 进行1)HRTII活体共聚焦成像以减少角膜混浊：2)测定存活率； 3)活体三维CT扫描确定肝脏大小；4)β-Glu活性。目的1C：AAV2DJ-Sa-Sa间质内注射 CRISPR检查基因编辑治疗角膜混浊的疗效。具体目标2：确定 造血干细胞及干祖细胞基因编辑治疗的疗效观察 GUSB小鼠Lin-Sca1 HSC/HSPC将从供体GUSB小鼠中分离出来，并进行CRISPR编辑和 扩大了。然后，CRISPR编辑的HSC/HSPC将被移植到伽玛射线照射的受体小鼠身上 ROIV。治疗效果将按照目标1所述进行评估。具体目标3：确定疗效 GUSB/MPS的同源介导的基于末端连接的CRISPR(HMEJ)作为原理证明 LSD。目标3A：GUSB MEF将用于验证由以下组成的二进制AAV的基因组编辑效率 含选择性转基因的AAV2DJ-SpCas9和AAV2DJ-sgRNA/供体DNA模板目标3B：Will 确定双元AAV2载体对GUSB小鼠的给药效果。目标3C：移植 CRISPR编辑的GUSB LIN-Sca1 HSC/HSPC给接受检查的GUSB小鼠，如中所述 具体目标1.拟议的研究将导致制定有效的治疗策略 MPS VII和其他类型的LSD，最终可以转换到床边。

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.