Precision genome editing in vivo to treat retinal diseases

摘要 遗传性视网膜疾病是一组遗传异质性的致盲疾病，具有显著的影响 关于生活质量。治疗方法大大落后于对遗传基因的初步鉴定 这些疾病的根据地。然而，也有一些惊人的成功；例如，RPE65基因增强疗法 是FDA批准的第一种针对任何遗传疾病的基因疗法。CURRENT的临床翻译 CRISPR-CAS9技术由于编辑效率低、容易出错的同源定向修复而受到阻碍 (Hdr)和大量的indel形成。精确基因组编辑是一种先进、创新的CRISPR-Cas9- 相关的基因组编辑工具，解决了典型CRISPR-CAS9实施的局限性。腺嘌呤 碱基编辑(ABES)使点突变的转换独立于Cas9诱导的双链DNA 休息和HDR。当基本编辑不适用时(例如，由于颠换突变、大量删除或 插入)，优质编辑技术提供了可行的替代方案。基因组编辑是高度具体的；然而， 碱基编辑的长时间表达可能导致整个基因组和 转录组。我们假设，通过RNP和合成RNA瞬时传递基因组编辑可以 获得与通过病毒转导提供的基因组编辑相同的高编辑率 非目标和旁观者编辑。因此，我们提出了两个主题相关的目标。 目的1.纠正遗传性视网膜视紫红质基因突变(RhoE150K/E150K) 通过腺嘌呤碱基编辑与常染色体隐性视网膜色素变性(RP)相关。安倍晋三的交付 将在完全特征化的RhoE150K/E150K小鼠模型中进行优化。建议的方法将 为ABES提供一个快速适应任何合适的RPE或视网膜突变的平台。 目的2.对Abca4PV/PV小鼠ABCA4蛋白进行碱基编辑修复。使用PE3b Prime编辑器和两个 同步稳定的编辑引导RNA(EpegRNA)，我们将恢复功能ABCA4蛋白 在携带光感受器和RPE双等位基因突变的Abca4PV/PV小鼠中。使用免疫印迹和 用于检测获救的ABCA4的下一代测序，以及用于检测A2E的双光子成像技术，我们 将在这个动物模型中优化基因组编辑效率，以改进原始编辑技术和它的 应用于治疗遗传性视网膜疾病。 为了达到这两个目标，我们将测试各种方法来暂时向编辑提供：(I)融合到 在纯化的核糖核蛋白(RNP)编辑复合体中的编辑者；(Ii)纯化的RNP-编辑上的考马斯脂标记 (Iii)含有RNP编辑复合体的病毒样颗粒；或(Iv)含有 稳定了基因组编辑材料的mRNAs，以便在细胞内表达。这些交付系统将是 最先在工程显色细胞系中进行优化。在小鼠身上进行碱基和质数编辑的效果将是 以RPE65在Leber先天性黑素瘤RD12动物模型中的表达水平为基准。

SUMMARY Inherited retinal disorders are a genetically heterogeneous group of blinding diseases that have significant impact on quality of life. Therapeutic approaches have lagged significantly behind initial identification of the genetic bases for these diseases. However, there are some striking successes; e.g., RPE65 gene augmentation therapy was the first FDA-approved gene therapy for any genetically inherited disease. Clinical translation of current CRISPR-Cas9 technology has been impeded by its low editing efficiency, error-prone homology-directed repair (HDR), and substantial indel formation. Precision genome editing is an advanced, innovative CRISPR-Cas9- associated genome-editing tool that addresses the limitations of typical CRISPR-Cas9 implementation. Adenine base editors (ABEs) enable conversion of a point mutation independently of Cas9-induced double-stranded DNA breaks and HDR. When base editing is not applicable (e.g., due to transversion mutations, large deletions, or insertions), prime editing technology offers feasible alternatives. Genome editing is highly specific; however, prolonged expression of base editors could lead to undesired off-target alterations throughout the genome and transcriptome. We hypothesize that transient delivery of genome editors via RNPs and synthetic RNAs can achieve the same high editing rates as those for genome editors delivered via viral transduction with reduced off-target and bystander editing. Accordingly, we propose two thematically linked aims. Aim 1. Correct inherited retinal disease-causing mutations in the rhodopsin gene (RhoE150K/E150K) associated with autosomal recessive retinitis pigmentosa (RP) via adenine base editing. Delivery of ABEs will be optimized in the thoroughly characterized RhoE150K/E150K mouse model of RP. Proposed approaches will provide a platform for ABEs to be quickly adapted to any suitable RPE or retinal mutation. Aim 2. Repair the ABCA4 protein in Abca4PV/PV mice by prime editing. Using the PE3b prime editor and two concurrent stabilized engineered prime-editing guide RNAs (epegRNA), we will restore functional ABCA4 protein in Abca4PV/PV mice that carry double allelic mutations in photoreceptors and the RPE. Using immunoblotting and next-generation sequencing for detecting rescued Abca4, and two-photon imaging techniques to detect A2E, we will optimize genome editing efficiency in this animal model to improve prime-editing technology and its application to treat inherited retinal diseases. For both aims, we will test various means to deliver the editors transiently: (i) cell-penetrating peptides fused to editors in purified ribonucleoprotein (RNP)-editing complexes; (ii) Coomassie-lipid tags on purified RNP-editing complexes; (iii) viral-like particles containing RNP-editing complexes; or (iv) lipid nanoparticles containing stabilized mRNAs of genome-editing materials for intracellular expression. These delivery systems will be optimized first in engineered chromogenic cell lines. The efficacy of base and prime editing in mice will be benchmarked against the level of expression of RPE65 in the rd12 animal model of Leber congenital amaurosis.