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

在子宫内，CRISPR-Cas9基因编辑(IUGE)比出生后基因编辑有几个潜在的优势。它通过利用胎儿的特性--体积小、免疫不成熟和大量的增殖细胞--提高编辑效率，并在不可逆转的病理开始之前治疗疾病(1)。这使得IUGE成为治疗儿童早期表现的遗传性代谢性肝病(IMLD)的理想方法，最近在遗传性酪氨酸血症1型(HT1)(2)的小鼠模型中证实了这一概念。先前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件来自自然发生的病原体。抗腺相关病毒(AAV)载体的抗体在普通成人人群中很常见(20-60%，因AAV亚型而异)(3，4)，以及针对细菌来源的Cas9同源基因的抗体(SaCas9，78%；SpCas9，58%)(5，6)。因此，这些抗体对出生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，胎儿应该不会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免疫系统的影响。然而，我们最近在子宫内造血细胞移植(IUHCT)方面的工作表明，母体免疫也必须被考虑。先前存在的母体供者特异性抗体在子宫内转移到胎儿身上，导致IUHCT后快速、完全的同种异体移植排斥反应(9)。先前存在的抗AAV和/或Cas9的母体抗体可能在IUGE后类似地转移到胎儿，为在子宫内成功地进行基因编辑提供了免疫屏障。在将IUGE从临床前动物研究转化为首个人类临床试验之前，必须研究这一潜在的障碍。我们的总体目标是确定和解决现有的AAV和Cas9母体抗体对IUGE的潜在免疫屏障。我们的中心假设是，母体AAV和Cas9亚型特异性抗体从母亲传给胎儿，并将抑制子宫内的基因编辑，而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。我们进行这些研究的理由是，它们将在人类首次临床试验之前解决有关IUGE免疫学的重要悬而未决的问题，增加IUGE作为一种潜在的革命性治疗儿童早期IMLDs的安全性和有效性。为了达到我们的目标，我们将追求这些特定的目标：1)证明AAV和Cas9抗体在小鼠体内的母胎传播，2)证明和避免母体抗体介导的小鼠胚胎基因编辑的损害，3)评估AAV和Cas9抗体在人类母胎二联体之间的传播，作为通往临床研究的桥梁。我们的研究在宫内治疗背景下，将先前存在的获得性免疫作为基因编辑的障碍进行了研究，这一研究具有创新性。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，这种方法可能产生对IMLDs的最终治愈。

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.