LCA Gene Therapy in Somatic Cell-Derived Induced Pluripotent Stem Cells

描述(申请人提供)：Leber先天性青光眼(LCA)是遗传性视网膜退行性疾病中最严重的一种，其特征是早期出现眼球震颤和失明，通常发生在出生后第一年。21%的LCA患者携带CEP290基因突变，这表明它是导致疾病的主要因素。此外，CEP290相关的LCA以常染色体隐性方式遗传，使其成为基因替代治疗的良好候选者。最近LCA的I期基因治疗试验的成功也强调了这种治疗对于LCA患者来说是一个可行的选择。虽然这种疾病可以通过使用病毒载体的基因替代疗法来治疗，但CEP290的大小排除了当前的载体系统(腺相关病毒-AAV)有效包装基因的可能性。用于眼部基因转移的AAV的替代方案是慢病毒。包装极限比AAV大得多，因此可以容纳大的CEP290基因。此外，慢病毒载体可以转导眼睛中的多种细胞类型，包括光感受器和视网膜色素上皮-在LCA的疾病进展中至关重要的细胞类型。临床前的LCA动物模型将有助于研究CEP290替代疗法的有效性。CEP290rd16小鼠模型携带CEP290基因897bp的纯合子框内缺失。这些小鼠表现出外节的早期进行性退化和外核层厚度的减少，这类似于人类疾病。除了CEP290rd16小鼠模型外，CEP290相关LCA患者的组织特异性视网膜细胞类型的产生将为研究CEP290替代疗法的有效性提供一个很好的模型。基于诱导多能干细胞(IPSC)的新技术为研究人员提供了建模、研究人类疾病和治疗纠正的能力。在这项建议中，我们的目标是从视网膜变性的小鼠模型以及CEP290相关的LCA视网膜退行性疾病患者中培养出IPSCs和随后的光感受器前体细胞。这些细胞将在体内和体外用于利用慢病毒载体和诱导多能干细胞技术进行治疗性基因矫正的研究。 公共卫生相关性：Leber先天性黑色素沉着症(LCA)是遗传性视网膜退行性疾病中最严重的形式，目前还没有治愈的方法。我们计划利用患者特有的干细胞和基因治疗的巨大再生能力来纠正遗传缺陷和恢复视网膜功能。

DESCRIPTION (provided by applicant): Leber Congenital Amaurosis (LCA) is the most severe form of inherited retinal degenerative diseases and is characterized by early on-set nystagmus and blindness, generally occurring within the first year of life. 21% of LCA patients carry mutations in the CEP290 gene, implicating it as a major contributor to the disease. Moreover, CEP290-associated LCA is inherited in an autosomal recessive manner, making it a good candidate for gene-replacement therapy. The recent success of a phase I gene therapy trial for LCA also highlights this treatment as a feasible option for LCA patients. Although this disease may be treatable by gene-replacement therapy using viral vectors, the size of CEP290 precludes the current vector system (adeno-associated virus - AAV) from efficiently packaging the gene. An alternative to AAV for ocular gene transfer is lentivirus. The packaging limit is much greater than that of AAV, and thus will accommodate the large CEP290 gene. Moreover, lentiviral vectors can transduce multiple cell types in the eye, including photoreceptors and retinal pigment epithelium - cell types important in the disease progression of LCA. Pre-clinical animal models of LCA will be useful for studying the effectiveness of CEP290-replacement therapy. The CEP290rd16 mouse model carries a homozygous in-frame 897 bp deletion in the Cep290 gene. These mice display early progressive degeneration of the outer segment and reduction in thickness of the outer nuclear layer, which resembles the human disease. In addition to the CEP290rd16 mouse model, generation of tissue specific retinal cell types from patients with CEP290-associated LCA will make a good model for studying the effectiveness of CEP290-replacement therapy. New induced pluripotent stem cell (iPSC)-based technologies are providing researchers with the ability to model and study human disease and therapeutic correction. In this proposal we aim to generate iPSCs and subsequently photoreceptor precursor cells from a mouse model of retinal degeneration as well as patients with CEP290-associated LCA retinal degenerative disorder. These cells will be used both in and ex vivo for the study of therapeutic gene correction using lentiviral vectors and induced pluripotent stem cell technology. PUBLIC HEALTH RELEVANCE: Leber Congenital Amaurosis (LCA) is the most severe form of inherited retinal degenerative diseases for which there is currently no cure. We plan to harness the tremendous regenerative power of patient-specific stem cells and gene therapy to correct the genetic defect and restore retinal function.