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

描述（由申请人提供）：Leber先天性黑朦（LCA）是遗传性视网膜退行性疾病中最严重的一种，其特征是早期眼球震颤和失明，通常发生在出生后的第一年。21%的LCA患者携带CEP290基因突变，这意味着它是该疾病的主要致病因素。此外，cep290相关的LCA以常染色体隐性遗传方式遗传，使其成为基因替代治疗的良好候选者。最近LCA I期基因治疗试验的成功也强调了这种治疗对于LCA患者是一种可行的选择。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的大小使当前的载体系统（腺相关病毒- AAV）无法有效地包装该基因。眼球基因转移的一种替代AAV的方法是慢病毒。包装限制远远大于AAV，因此将容纳较大的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细胞-这些细胞类型在LCA的疾病进展中很重要。LCA的临床前动物模型将有助于研究cep290替代疗法的有效性。CEP290rd16小鼠模型在Cep290基因框内携带一个897 bp的纯合子缺失。这些小鼠表现出早期进行性外节变性和外核层厚度减少，这与人类疾病相似。除了CEP290rd16小鼠模型外，从cep290相关LCA患者中产生组织特异性视网膜细胞类型将成为研究cep290替代疗法有效性的良好模型。基于诱导多能干细胞（iPSC）的新技术为研究人员提供了模拟和研究人类疾病和治疗纠正的能力。在这项提议中，我们的目标是从视网膜变性小鼠模型以及cep290相关的LCA视网膜退行性疾病患者中产生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慢病毒载体和诱导多能干细胞技术的治疗性基因校正研究。

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.