Nonviral delivery techniques for in vivo prime editing

基因编辑是治疗甚至永久治愈遗传疾病的一种有前途的策略。在 特别是，一种名为prime editing的新技术有可能使小目标 插入、缺失和替换，具有非常高的已知疾病覆盖率- 导致突变，同时最大限度地减少DNA中危险的双链断裂。为了 要认识到这一潜力，就必须制定强有力的交付战略，以交付主要的编辑工具 有效地转移到疾病相关的器官。一种这样的递送策略是脂质纳米颗粒递送。 基于RNA和/或蛋白质的引物编辑组分。LNP是非病毒的，无毒的，临床上 经过验证的交付工具。然而，可能的LNP存在极其多样化的空间， 成千上万的潜在的脂质结构，可能是有用的LNP交付。选择 因此，用于主要编辑应用的最佳可能的LNP是具有挑战性的，因为在体外 测试通常是不可靠的，并且一次对一个LNP进行体内测试的通量极低。 在这里，我们提出了联合收割机两个可扩展的技术，以产生和测试安全，有效的LNP 用于执行主要编辑的制剂。首先，我们将使用组合化学 技术来产生用于包含到LNP中的可生物降解的脂质的大文库。第二、 我们将介绍一种新技术，我们称之为pegRNA条形码， 数百个LNP在单个小鼠中进行成功的引物编辑。我们将采用这种技术 鉴定用于编辑多个器官的最佳可生物降解的LNP，特别包括 肺和肝。在确定了最佳候选人后，我们将着手使用我们的LNP， 应用prime editing治疗两种不同遗传性遗传疾病的小鼠模型：遗传性 酪氨酸血症I型（HTI），一种肝脏疾病，和囊性纤维化（CF），主要是一种肺部疾病。我们 将评估主要编辑的效率，不希望的编辑事件的水平， 这些小鼠的表型校正。结果可能会确定有前途的临床前候选人 用于治疗HTI、CF和许多其他肺部和肝脏疾病。

Gene editing is a promising strategy for treating or even permanently curing genetic diseases. In particular, a new technique called prime editing has the potential to make small targeted insertions, deletions, and substitutions with very high potential coverage of known disease- causing mutations, and while minimizing dangerous double-stranded breaks in DNA. In order to realize this potential, robust delivery strategies must be developed to deliver prime editing tools efficiently to disease-relevant organs. One such delivery strategy is lipid nanoparticle delivery of RNA and/or protein-based prime editing components. LNPs are nonviral, nontoxic, and clinically validated delivery tools. However, there is an extremely diverse space of possible LNPs, with tens of thousands of potential lipid structures that may be useful for LNP delivery. Selecting the best possible LNP for a prime editing application, therefore, is challenging because in vitro testing is often unreliable and in vivo testing of one LNP at a time is extremely low throughput. Here, we propose to combine two scalable techniques to generate and test safe, potent LNP formulations for performing prime editing. First, we will employ combinatorial chemistry techniques to generate large libraries of biodegradable lipids for inclusion into LNPs. Second, we will introduce a new technique which we term pegRNA barcoding to screen dozens to hundreds of LNPs for successful prime editing in a single mouse. We will employ this technique to identify the best biodegradable LNPs for editing of multiple organs, including in particular the lung and the liver. Having identified the top candidates, we will proceed to use our LNPs to apply prime editing to treat mouse models of two different inherited genetic diseases: hereditary tyrosinemia type I (HTI), a liver disease, and cystic fibrosis (CF), primarily a lung disease. We will evaluate the efficiency of prime editing, the levels of undesired editing events, and phenotypic correction of these mice. The results may identify promising preclinical candidates for the treatment of HTI, CF, and many other lung and liver diseases.