Development of Safe in Utero Gene Editing Technology in Mice

摘要 自从人类基因组的开创性测序以来的近20年里，精确度的潜力 基因医学至今仍未实现。测序技术的进步极大地提高了我们的筛查能力 患者(包括父母和胎儿)基因异常，极大地扩大了对 产前中的遗传性疾病。这为胎儿分子疗法打开了大门，以解决关键的 子宫内的遗传条件，防止早孕终止、新生儿死亡或不可逆转的组织 造成终身残疾的损害。CRISPR系统的发现及其非凡的能力 快速进行精确的基因编辑展示了临床应用于治疗遗传疾病的可能性。 然而，由于突变和DNA重组的引入，CRISPR的临床应用受到限制。 基因组中意想不到的脱靶位置，这可能导致毒性和癌症。这种缺陷无处不在 比CRISPR在子宫内基因编辑中的应用更严重，在那里不受控制的编辑会产生副作用 可能会影响患者的整个生活。控制CRISPR基因编辑是确保 宫内基因治疗的安全性和有效性。阿克利根生物科学公司正在将技术商业化，以带来 安全体内CRISPR为基础的基因编辑疗法到临床。Acrigen利用抗CRISPR(ACR)蛋白作为 强大的CaS核酸酶抑制剂，为基因编辑提供关闭开关，防止脱靶效应。这个 加州大学旧金山分校的麦肯齐实验室专门从事胎儿手术和应用分子疗法纠正新生儿基因 疾病。受控CRISPR基因编辑与精确宫内分娩的结合将使我们能够 解决以前无法治疗的遗传性疾病，包括阿尔法地中海贫血和 神经系统疾病，如神经性高谢病。我们建议开发一种安全有效的子宫内 CRISPR基因编辑递送系统在胎鼠模型中靶向造血干细胞和神经元。 这项第一阶段的STTR项目有三个目标。目的1：设计针对小鼠报告基因的SaCas9指南。 里程碑1：选择在报告鼠细胞系中显示70%编辑的向导。目标2：构建单个载体 (Cas9-sgRNA-ACR)子宫内受控编辑的传递系统。里程碑2：演示维护的目标 在ACR控制下，通过与AAV兼容的单载体系统，活性和&gt；90%的脱靶活性减少。 目的3：演示在子宫内安全有效地编辑报告小鼠模型。里程碑3：展示&gt；50%- 使用AAV6靶向造血干细胞和AAV9靶向进行靶向编辑和5%的非靶向编辑 神经元。

Abstract In the nearly 20 years since the groundbreaking sequencing of the human genome, the potential for precision genetic medicine has still not been realized. Advances in sequencing have vastly increased our ability to screen patients (including parents and fetuses) for genetic abnormalities, dramatically expanding identification of hereditary diseases in the prenatal period. This opens the door for fetal molecular therapies to address critical genetic conditions in utero, preventing preterm pregnancy termination, newborn death, or irreversible tissue damage resulting in life-long disabilities. The discovery of CRISPR systems and their remarkable ability to perform precision gene editing quickly showed possibilities for clinical applications to treat genetic disorders. However, clinical application of CRISPR is limited due to the introduction of mutations and DNA restructuring at unintended off-target sites within the genome, which can lead to toxicity and cancer. Nowhere is this deficiency more acute than in the application of CRISPR for in utero gene editing, where uncontrolled editing side effects could affect the patient for the entirety of their lives. Controlling CRISPR gene editing is critical to ensure the safety and efficacy of in utero gene therapies. Acrigen Biosciences is commercializing technology to bring safe in vivo CRISPR-based gene editing therapies to the clinic. Acrigen utilizes anti-CRISPR (Acr) proteins as robust inhibitors of Cas nuclease, providing an off-switch for gene editing and preventing off-target effects. The MacKenzie lab at UCSF specializes in fetal surgery and applying molecular therapies to correct neonatal genetic diseases. The combination of controlled CRISPR gene editing with precise in utero delivery will allow us to address previously untreatable genetic disorders, including hematopoietic disorders like alpha thalassemia and neurologic diseases such as neuropathic Gaucher disease. We propose to develop a safe and effective in utero CRISPR gene editing delivery system targeting hematopoietic stem cells and neurons in a fetal mouse model. This Phase I STTR project has three aims. Aim 1: Design SaCas9 guides targeting a mouse reporter gene. Milestone 1: Select guides showing >70% editing in reporter mouse cell line. Aim 2: Construct a single vector (Cas9-sgRNA-Acr) delivery system for controlled in utero editing. Milestone 2: Demonstrate maintained on-target activity and >90% reduction of off-target activity with an AAV compatible single vector system under Acr control. Aim 3: Demonstrate safe and effective editing of a reporter mouse model in utero. Milestone 3: Show >50% on- target editing and <5% off-target editing with AAV6 targeting hematopoietic stem cells and AAV9 targeting neurons.