Monogenic disorders, many of which are prenatally diagnosed, are responsible for significant morbidity and mor- tality. Some have limited or no postnatal therapies and result in death shortly after birth. Advances in CRISPR- Cas9 technology present a unique opportunity for therapeutic gene editing. Mouse studies of postnatal in vivo CRISPR-Cas9 editing highlight limitations of this approach including low editing efficiency, a potential immune response to the viral vector/Cas9 protein, and initiation of treatment after disease onset. Thus, there is a critical need to develop novel approaches to increase the efficiency and safety of gene editing. In utero gene editing (IUGE) is an innovative approach to in vivo gene editing that has the potential to overcome these limitations and cure perinatal lethal monogenic disorders before birth. The normal developmental properties of the fetus includ- ing the abundance of proliferative/accessible progenitor cells of multiple organs and the tolerant immune system suggest the hypothesis that prenatal in vivo gene editing can be more efficient than postnatal editing, result in long-term persistence of edited cells and can rescue the lethal phenotype in animal models of congenital mono- genic human diseases. This hypothesis is based on our preliminary data which support our ability to robustly transduce target cells and perform CRISPR-Cas9 gene editing in multiple organs following in utero AAV and adenovirus injection in normal mouse models. The preliminary studies will be extended and hypothesis ad- dressed in normal and disease mouse and preclinical large animal models. The efficiency and persistence of editing in different target cell populations including pulmonary progenitor cells will be assessed following prenatal and postnatal editing. Furthermore, in light of studies suggesting genetic instability associated with postnatal gene editing, the presence and functional consequences of unwanted genetic rearrangements following prenatal editing will be compared to postnatal editing. These studies will include IUGE of a cell population that is tradi- tionally difficult to target after birth and which is responsible for diseases causing neonatal death. Specifically, in utero CRISPR-mediated gene editing of pulmonary epithelial cells will be assessed in a perinatal lethal surfactant protein deficient mouse model and the preclinical fetal sheep model. ?Standard? CRISPR-Cas9 editing (HDR and NHEJ) requires forming double strand DNA breaks (DSBs) and has an inherent risk of unwanted mutations at on- and off-target sites. Base editing can change adenine to guanine bases in a site-specific fashion. It does not require DSBs and thus is potentially safer. In utero base editing will be evaluated in the mouse and preclinical canine model of Hurler syndrome, a lysosomal storage disease in which a GA change is a common disease- causing mutation in humans and animal models. These studies are expected to demonstrate that IUGE results in efficient and persistent editing and can correct the phenotype in small and large animal models of monogenic human diseases. This research is significant because it is expected to support an innovative treatment for dis- eases that currently have no effective therapy and result in significant morbidity and mortality.
The proposed research investigates in utero CRISPR-mediated gene editing in clinically relevant small and large animal models of human diseases. This research is relevant to the public health because prenatal in vivo gene editing has the potential to cure, before birth, perinatal lethal or morbid congenital monogenic diseases which currently have no adequate treatment. Thus, the proposed research is relevant to the part of the NIH?s mission that pertains to developing fundamental knowledge that will help to enhance health, reduce illness and disability, and lengthen life.