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

预先存在的母体体液免疫是 CRISPR-Cas9 子宫内基因编辑治疗遗传性代谢性肝病的障碍

• 批准号: 10516721
• 负责人: John Samuel Riley
• 金额: $ 7.69万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10516721
• 关键词: Adult; Affect; Animals; Antibodies; Area; Bacteria; Birth; CRISPR/Cas technology; Cells; Childhood; Clinical; Clinical Research; Clinical Trials; Dependovirus; Disease; Fetus; Fluorescence; Genes; Gestational Age; Harvest; Health; Hepatocyte; Human; Humoral Immunities; Immune; Immune system; Immunoglobulin G; Immunoglobulin M; Immunologics; Immunology; Impairment; Inherited; Intramuscular Injections; Investigation; Investigational Therapies; Journals; Knowledge; Liver diseases; Maternal antibody; Maternal-Fetal Transmission; Maternally-Acquired Immunity; Mediating; Mendelian disorder; Metabolic; Mission; Morbidity - disease rate; Mothers; Mus; National Institute of Diabetes and Digestive and Kidney Diseases; Orthologous Gene; Partner in relationship; Pathology; Patients; Pediatric Hospitals; Philadelphia; Population; Proliferating; Property; Publishing; Research; Risk; Safety; Second Pregnancy Trimester; Serum; Technology; Testing; Time; Transfusion; Translations; Tyrosinemias; Umbilical Cord Blood; United States National Institutes of Health; Work; adaptive immunity; adeno-associated viral vector; allograft rejection; antibody transfer; cross reactivity; donor-specific antibody; early childhood; fetal; fetal diagnosis; first-in-human; hematopoietic cell transplantation; improved; in utero; innovation; liver transplantation; mortality; mouse model; pathogen; peripheral blood; postnatal; pre-clinical; pregnant; prenatal; prenatal therapy; success; theories; transmission process

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.

与出生后基因编辑相比，子宫内 CRISPR-Cas9 基因编辑 (IUGE) 具有多种潜在优势。它通过利用胎儿特性（体积小、免疫不成熟和增殖细胞丰富）来提高编辑效率，并在不可逆病理发生之前治疗疾病 (1)。这使得 IUGE 成为治疗儿童早期遗传性代谢性肝病 (IMLD) 的理想方法，最近在遗传性酪氨酸血症 1 型 (HT1) 小鼠模型中证明了这一概念 (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源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年人群中很常见（20-60%，因 AAV 亚型而异）(3,4)，针对细菌衍生的 Cas9 直向同源物的抗体也是如此（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)。理论上，由于胎儿可以抵御外部病原体和不成熟的免疫系统，因此受这种潜在屏障的影响应该较小。然而，我们最近对宫内造血细胞移植（IUHCT）的研究表明，还必须考虑母体免疫力。预先存在的母体供体特异性抗体被转移到子宫内的胎儿，导致 IUHCT 后快速、完全的同种异体移植排斥 (9)。先前存在的 AAV 和/或 Cas9 母体抗体可能会在 IUGE 后类似地转移到胎儿体内，从而对子宫内成功进行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并解决由预先存在的 AAV 和 Cas9 母体抗体对 IUGE 造成的潜在免疫屏障。我们的中心假设是母体 AAV 和 Cas9 亚型特异性 IgG 抗体从母亲转移到胎儿，并会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，它们将在首次人体临床试验之前解决有关 IUGE 免疫学的重要未解答问题，提高 IUGE 的安全性和有效性，作为治疗儿童早期 IMLD 的潜在革命性疗法。为了实现我们的目标，我们将追求以下具体目标：1) 证明 AAV 和 Cas9 抗体在小鼠体内的母胎传播，2) 证明并规避母体抗体介导的小鼠胎儿基因编辑损伤，3) 评估 AAV 和 Cas9 抗体在人类母胎双体中的传播，作为临床研究的桥梁。我们的研究具有创新性，研究了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。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，从而最终治愈 IMLD。