Engineering of Nanoparticles for prenatal applications

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 haematopoietic

stem 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.