Engineering of Nanoparticles for prenatal applications

SCD是一种严重的、进行性衰弱和危及生命的遗传性疾病，其导致频繁的血管闭塞发作和贫血，在儿童和成人中引起慢性器官损伤、感染和中风[1]。在非洲，SCD导致生活质量低下，在许多情况下，寿命显著缩短，占所有五岁以下儿童死亡的9-15%[1]。在全球范围内，预计到2050年，SCD发病率将上升至每年40万例[1]。大多数可用的治疗方法只能控制症状[1]，患者一生的医疗保健费用可能总计460，000美元[2，3]。2010-2011年，SCD相关的入院花费了NHS 1800万英镑[4]。大约三分之一的英国SCD患者在我的机构，伦敦国王学院（KCL）的合作伙伴，NHS信托基金，盖伊和托马斯（GSTT）和国王学院医院（KCH）进行管理。SCD的细胞或基因治疗是一种潜在的治愈性治疗，与终生支持性治疗相比，在成人SCD患者中可能具有成本效益[5]，如果SCD胎儿出生时无病，则可能更具成本效益。使用来自母亲血液的循环胎儿DNA的非侵入性产前诊断的发展允许从妊娠10周开始早期检测先天性胎儿疾病[6，7]。诊断后，目前，两个主要选择是1）终止妊娠或2）仔细监测和分娩，然后在适当的情况下进行产后治疗。该提案涉及关于潜在的第三种选择的知识的开发和生成;通过提供封装了主要编辑系统（NP编辑器）的NP来进行SCD的子宫内治疗[8-10]以及利益相关者对这种疗法的看法。最初尝试用子宫内异基因造血干细胞（HSC）移植治疗先天性血液病，如SCD[11]，但没有成功，尽管供体进行了适当的匹配。最初，这种方法受到来自母亲和胎儿的免疫应答的限制[12-15]。另一种选择是移植已纠正先天性疾病的自体（匹配）祖细胞/干细胞，可以避免阴性母体和胎儿免疫反应。然而，这需要从胎儿中分离细胞（从血液、羊水或胎肝中）[12，16]，然后在子宫内移植之前校正和扩增细胞[16]。这使得这一过程劳动强度大、成本高，中低收入国家无法进行，因为这些国家的SCD负担最重。母体抗体反应可通过将母体骨髓（BM）HSC移植至胎儿来避免[17]。使用这种技术，已经成功地治疗了三名患有α地中海贫血的胎儿[18]。然而，该程序涉及风险，如死胎，流产和感染[19]。此外，如果母亲是SCD纯合子，这种方法是不可行的。另一种选择是使用子宫内基因编辑技术[8，9]靶向胎儿肝脏中造血小生境中的HSC [15，20，21]。使用慢病毒（LV）或其他载体的子宫内基因递送引起了安全性问题，如插入诱变、胎儿免疫应答和胎儿发育的改变[22]。然而，可以安全地使用NP封装PE编辑器以纠正胎儿造血生态位中的基因缺陷[8，23]。这些体内遗传校正的HSC将自然迁移到骨髓，随后提供终身治疗[20]。我们发表了使用LV进行产前HSC基因治疗的原理证明，通过在妊娠中期将β-珠蛋白载体（地球仪LV）递送至胎儿肝脏，证明了在鼠模型系统中对轻度β-地中海贫血的产后挽救[15]。NP编辑器在体外校正人胎儿HSC。

SCD is a severe, progressively debilitating, and life-threatening genetic disease, which results in frequent vaso-occlusive episodes and anaemia, causing chronic organ damage, infection and stroke in children and adults[1]. In Africa, SCD results in poor quality of life and, in many cases, a significantly shortened lifespan and accounts for 9-15% of all deaths of children under five[1]. Worldwide, SCD incidence is expected to rise to 400,000 births per year in 2050[1]. Most available treatments only manage symptoms,[1] and health care costs may total $460,000 over a patient's lifetime[2,3]. SCD associated admissions cost the NHS £18 million in 2010-2011[4]. About a third of all UK SCD patients are managed at my institution, King's College London (KCL) partners, NHS Trusts, Guy's and St Thomas' (GSTT) and King's College Hospitals (KCH). Cell or gene therapy for SCD is a potential curative treatment, is likely to be cost-effective in adult SCD patients compared to supportive treatment throughout life[5] and could be even more cost-effective if a SCD fetus is born disease-free. The development of non-invasive prenatal diagnosis using circulating fetal DNA from the mother's blood allows the early detection of congenital fetal diseases from ten weeks of gestation[6,7]. Following diagnosis, currently, the two main options are 1) termination of pregnancy or 2) careful monitoring and delivery followed by post-natal treatment where appropriate. This proposal addresses the development and generation of knowledge on a potential third option; in utero treatment of SCD by delivering NPs encapsulating a prime editing system (NP-Editor)[8-10] and the stakeholder's views of such a therapy. Initial attempts to treat congenital haematological diseases, such as SCD[11], with in-utero allogeneic haematopoieticstem cells (HSC) transplantation were unsuccessful, despite donors being suitably matched. Initially, this approach was limited by the immune response from both the mother and fetus[12-15]. The alternative, transplantation of autologous (matched) progenitor/stem cells, which have been corrected for congenital disease, could avoid a negative maternal and fetal immune response. However, this requires isolation of the cells from the fetus (either from blood, amniotic fluid or fetal liver)[12,16], and then correction and expansion of the cells[16] before in utero transplantation. This makes the process labour intense, expensive, and inaccessible to low and middle-income countries, where the burden of SCD is the highest. A maternal antibody response, can be avoided by transplantation of maternal bone marrow (BM) HSCs to the fetus[17]. Using this technique, three fetuses with alpha thalassaemia have already been treated successfully[18]. However, the procedure involves risks, such as stillbirth, miscarriage and infection[19]. Also, this approach is not feasible if the mother is homozygous for SCD. Another option is to use in utero gene editing techniques[8,9] targeting HSCs in the haematopoietic niche in the fetal liver[15,20,21]. In utero gene delivery using lentiviral (LV) or other vectors raises safety concerns such as insertional mutagenesis, fetal immune response and alterations in fetal development[22]. However, it is possible to safely use NPs to encapsulate a PE editor to correct the gene defect in the fetal haematopoietic niche[8,23]. These in vivo genetically corrected HSCs will naturally migrate to the bone marrow and subsequently offer life-long therapy [20]. We published proof-of-principle of prenatal HSC gene therapy, using a LV, by demonstrating post-natal rescue of a mild form of beta-thalassaemia in a murine model system by delivering a beta-globin vector (GLOBE LV) to the fetal liver at mid-gestation[15]. Hypothesis The NP-Editor corrects Human Fetal HSCs in vitro.