Development of Safe in Utero Gene Editing Technology in Mice

摘要 在人类基因组突破性测序的近20年里， 基因医学尚未实现。测序技术的进步极大地提高了我们筛选 患者（包括父母和胎儿）的遗传异常，大大扩大了识别 产前遗传性疾病。这为胎儿分子疗法打开了大门， 子宫内遗传疾病，防止早产终止，新生儿死亡或不可逆组织 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显示了临床应用于治疗遗传疾病的可能性。 然而，CRISPR的临床应用受到限制，这是由于在CRISPR中引入突变和DNA重组。 基因组内的非预期脱靶位点，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，在子宫内基因编辑中，不受控制的编辑副作用 会影响病人的一生控制CRISPR基因编辑对于确保 子宫内基因治疗的安全性和有效性。Acrigen Biosciences正在将技术商业化， 安全的基于体内CRISPR的基因编辑疗法应用于临床。Acrigen利用抗CRISPR（Acr）蛋白作为 Cas核酸酶的强大抑制剂，为基因编辑提供关闭开关并防止脱靶效应。的 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遗传 疾病受控CRISPR基因编辑与精确的子宫内递送相结合将使我们能够 解决以前无法治疗的遗传疾病，包括造血系统疾病，如α地中海贫血， 神经系统疾病，如神经性戈谢病。我们建议在子宫内开发一种安全有效的 CRISPR基因编辑递送系统靶向胚胎小鼠模型中的造血干细胞和神经元。 第一阶段STTR项目有三个目标。目的1：设计靶向小鼠报告基因的SaCas 9指导物。 里程碑1：选择在报告小鼠细胞系中显示>70%编辑的指导物。目标2：构建单一载体 （Cas9-sgRNA-Acr）递送系统用于子宫内受控编辑。里程碑2：证明保持在目标上 在Acr控制下，使用AAV相容性单载体系统，具有>90%的脱靶活性和>90%的脱靶活性降低。 目的3：证明子宫内报告小鼠模型的安全和有效编辑。里程碑3：显示>50%- 靶向编辑和<5%脱靶编辑，其中AAV 6靶向造血干细胞，AAV 9靶向造血干细胞 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.