Pre-existing Maternal Humoral Immunity as a Barrier to CRISPR-Cas9 In Utero Gene Editing for Inherited Metabolic Liver Diseases

与产后基因编辑相比，子宫内CRISPR-Cas9基因编辑（IUGE）具有几个潜在的优势。它利用胎儿的特性——小体积、免疫不成熟和增殖细胞丰富——提高了编辑效率，并在不可逆病理发生之前治疗疾病(1)。这使得IUGE成为儿童早期表现的遗传性代谢性肝病（imld）的理想治疗方法，最近在遗传性酪氨酸血症1型（HT1）小鼠模型中证实了这一概念(2)。预先存在的体液免疫是出生后CRISPR-Cas9基因编辑的潜在障碍，因为该技术的几个组成部分来自自然存在的病原体。针对腺相关病毒（AAV）载体的抗体在一般成人人群中很常见（20-60%，因AAV亚型而异）（3,4），针对细菌衍生的Cas9同源物的抗体也很常见（SaCas9, 78%; SpCas9, 58%）（5,6）。因此，这些抗体对产后基因编辑效率的临床影响是一个活跃的研究领域（7,8）。从理论上讲，胎儿应该较少受到这种潜在屏障的影响，因为它可以保护胎儿免受外部病原体和不成熟的免疫系统的侵害。然而，我们最近在子宫内造血细胞移植（IUHCT）方面的工作表明，母体免疫也必须考虑在内。母体先前存在的供体特异性抗体在子宫内被转移到胎儿体内，导致IUHCT后快速、完全的同种异体移植排斥反应(9)。存在的母体AAV和/或Cas9抗体可能在IUGE后同样转移到胎儿身上，对子宫内成功的基因编辑构成免疫屏障。在将IUGE从临床前动物研究转化为首次人体临床试验之前，必须对这一潜在障碍进行调查。我们的总体目标是表征和解决由预先存在的AAV和Cas9母源抗体构成的潜在免疫屏障。我们的中心假设是，母体AAV和Cas9亚型特异性IgG抗体从母体转移到胎儿，并将抑制子宫内的基因编辑，并且选择不存在母体体液免疫的替代AAV或Cas9亚型将恢复成功的基因编辑。我们进行这项研究的基本原理是，它们将在首次人体临床试验之前解决关于IUGE免疫学的重要未解问题，提高IUGE作为儿童早期表现的imld的潜在革命性治疗方法的安全性和有效性。为了实现我们的目标，我们将追求以下具体目标：1)在小鼠中证明AAV和Cas9抗体的母胎传播，2)在小鼠胎儿中证明并规避母胎抗体介导的基因编辑损伤，以及3)评估人类母胎双体之间的AAV和Cas9抗体传播，作为临床研究的桥梁。我们的研究在研究预先存在的适应性免疫作为子宫内治疗背景下基因编辑的障碍方面具有创新性。这项工作的重大贡献将是消除产前基因编辑方法的临床障碍，这种方法可能产生最终治愈imld的方法。

In utero CRISPR-Cas9 gene editing (IUGE) has several potential advantages over postnatal gene editing. It increases editing efficiency by taking advantage of fetal properties – small size, immunologic immaturity, and abundance of proliferating cells – and treats disease prior to the onset of irreversible pathology (1). This makes IUGE an ideal treatment approach for inherited metabolic liver diseases (IMLDs) manifesting in early childhood, with proof of concept recently demonstrated in the murine model of Hereditary Tyrosinemia Type 1 (HT1) (2). Pre-existing humoral immunity is a potential barrier to postnatal CRISPR-Cas9 gene editing, as several components of the technology are derived from naturally occurring pathogens. Antibodies against adeno-associated virus (AAV) vectors are common in the general adult population (20-60%, varying by AAV subtype) (3,4), as are antibodies to bacteria-derived Cas9 orthologs (SaCas9, 78%; SpCas9, 58%) (5,6). The clinical impact of these antibodies on postnatal gene editing efficiency is therefore an active area of investigation (7,8). In theory, the fetus should be less affected by this potential barrier due to its protection from external pathogens and immature immune system. However, our recent work on in utero hematopoietic cell transplantation (IUHCT) has shown that maternal immunity must also be considered. Pre-existing maternal donor-specific antibodies are transferred to the fetus in utero, causing rapid, complete allograft rejection after IUHCT (9). Pre-existing maternal antibodies to AAV and/or Cas9 may be similarly transferred to the fetus following IUGE, posing an immune barrier to successful gene editing in utero. This potential barrier must be investigated prior to translation of IUGE from preclinical animal studies to first-in-human clinical trials. Our overall objective is to characterize and resolve the potential immune barrier to IUGE posed by pre-existing maternal antibodies to AAV and Cas9. Our central hypotheses are that maternal AAV and Cas9 subtype-specific IgG antibodies are transferred from mother to fetus and will inhibit gene editing in utero, and that selection of alternative AAV or Cas9 subtypes for which no maternal humoral immunity exists will restore successful gene editing. Our rationale for the studies is they will resolve important unanswered questions about the immunology of IUGE prior to first-in-human clinical trials, increasing the safety and efficacy of IUGE as a potentially revolutionary treatment for IMLDs manifesting in early childhood. To attain our objective, we will pursue these specific aims: 1) demonstrate maternal-fetal transmission of AAV and Cas9 antibodies in mice, 2) demonstrate and circumvent maternal antibody-mediated impairment of gene editing in the murine fetus, and 3) assess AAV and Cas9 antibody transmission among human maternal-fetal dyads as a bridge to clinical studies. Our research is innovative in its study of pre-existing adaptive immunity as a barrier to gene editing in the context of in utero therapy. The significant contribution of this work will be to remove clinical barriers to a prenatal gene editing approach that could yield a definitive cure for IMLDs.