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 ba...