Silica Nanocapsule-Mediated Nonviral Delivery of CRISPR Base Editor mRNA and Allele Specific sgRNA for Gene Correction in Leber Congenital Amaurosis

项目摘要/摘要-引领开拓者项目1 Leber先天性盲症(LCA)是一种罕见但严重的儿童失明。其中一个亚型LCA16是 由编码内向整流钾的KCNJ13基因的几个单点突变引起的 视网膜中的Kir7.1通道。目前还没有FDA批准的治疗LCA16等超罕见疾病的方法。这个 Lead项目1的目标是利用CRISPR碱基编辑程序(BE)开发一种新的LCA16基因疗法 通过非病毒二氧化硅纳米囊(SNC)输送到视网膜色素上皮(RPE)。SNC拥有 许多理想的性质，包括高传递效率，用于配基共轭的多种表面化学， 颗粒尺寸小，生物相容性好，生产可扩展。在初步研究中，我们表明SNC 可以瞬时输送广泛的生物制品，包括小鼠体内的BE到RPE细胞和LCA16患者来源的LCA16 诱导多能干细胞(IPSC)-RPE。在五年内，我们寻求优化和验证领先候选人， SNC-101，该产品的放大和CGMP生产的进展，以实现非人类灵长类(NHP) 研究并向FDA提交LCA16基础编辑疗法的研究新药(IND)申请。在……里面 目的1，我们将生成一个临床前验证包，其中包含用于W53*KCNJ13矫正的主导SNC配方。 我们将首先优化ATRA靶向配体(RPE细胞特异性)和修饰的KCNJ13 sgRNA的量 在体外通过人类细胞检测核心和在体内的W53*LCA16小鼠模型。然后，我们将开发一种 用于临床前研究的优化SNC(即SNC101)的放大生产工艺。我们计划完成 在这一目标结束时举行一次互动会议。在目标2中，我们将确定基因校正效率、剂量、 以及对小鼠模型和NHP的毒性。我们将综合评估免疫、结构和 通过我们的W53*LCA16小鼠模型视网膜下注射SNC-101的功能后果。使用我们的 除了大型动物核心，我们还将在NHP中进行剂量递增安全性研究。与物种并驾齐驱 相比之下，这项研究将确定储存稳定性、剂量和毒性的详细情况，这是一个重要的里程碑 与FDA一起支持IND。在目标3中，我们将为W53*KCNJ13进行SNC-101的IND启用研究 更正。IND前会议包将在我们监管核心的帮助下提交给FDA CBER。 韦斯曼生物制造公司将开发一种全面的工程运行，用于大鼠的GLP毒理学研究 和国家卫生保健计划。在毒理学研究的同时，将生产一整批CGMP。我们将提交 在最后一年向FDA提交IND申请。到目前为止，还没有一种非病毒基因组编辑疗法达到 印在眼睛里。SNC-101的成功将为这种新的治疗方式铺平道路。最后， 在这条发展道路上获得的知识将加速其他非病毒基因组编辑的翻译 在CRISPR愿景方案的跟随者项目中，包括SNC-201和TACGE-101在内的线索。

PROJECT SUMMARY/ABSTRACT - LEAD TRAILBLAZER PROJECT 1 Leber Congenital Amaurosis (LCA) is a rare but severe form of pediatric blindness. One subtype, LCA16, is caused by several single-point mutations in the KCNJ13 gene, which encodes the inwardly-rectifying potassium channel Kir7.1 in the retina. There is no FDA-approved treatment for ultra-rare conditions such as LCA16. The objective of Lead Project 1 is to develop a new LCA16 gene therapy utilizing a CRISPR base editor (BE) delivered to the retinal pigment epithelium (RPE) via a nonviral silica nanocapsule (SNC). The SNCs possess many desirable properties, including high delivery efficiency, versatile surface chemistry for ligand conjugation, small particle sizes, good biocompatibility, and scalable production. In preliminary studies, we show that the SNC can transiently deliver a wide range of biologics, including a BE to RPE cells in mice and LCA16 patient-derived induced pluripotent stem cell (iPSC)-RPE. Within five years, we seek to optimize and validate a lead candidate, SNC-101, progress to scaled-up and CGMP production of the product to enable non-human primate (NHP) studies and file an Investigational New Drug (IND) application to the FDA for base editing therapy of LCA16. In Aim 1, we will generate a preclinical validation package with a lead SNC formulation for W53* KCNJ13 correction. We will first optimize the amount of ATRA targeting ligand (RPE cell-specific) and the modified KCNJ13 sgRNA in vitro via the Human Cell Assays Core and in vivo in a W53* LCA16 mouse model. We will then develop a scale-up production process for the optimized SNC (i.e., SNC101) for preclinical studies. We plan to complete one INTERACT meeting at the end of this aim. In Aim 2, we will determine the gene correction efficiency, dosing, and toxicity in mouse models and NHPs. We will comprehensively evaluate the immune, structural, and functional consequences of subretinal delivery of SNC-101 through our W53* LCA16 mouse model. Using our Large Animal Core, we will also perform dose-escalation safety studies in NHPs. Alongside the species comparison, the study will ascertain storage stability, dosing, and toxicity profiles in detail, an important milestone to support an IND with the FDA. In Aim 3, we will conduct IND enabling studies of SNC-101 for W53* KCNJ13 correction. A pre-IND meeting package will be submitted to FDA CBER with the help of our Regulatory Core. Waisman Biomanufacturing will develop a full-scale engineering run to be used in GLP toxicology study in rats and NHPs. Concurrent with the toxicology study, one entire CGMP batch will be manufactured. We will submit an IND application to the FDA in the final year. To date, no nonviral genome editing therapeutic has reached an IND in the eye. Success here with SNC-101 would pave the way for this new therapeutic modality. Finally, the knowledge gained along this development path will accelerate the translation of other nonviral genome editing leads, including SNC-201 and TAGE-101, in the Follower Projects of the CRISPR Vision Program.