Congenital monogenic lung diseases, including cystic fibrosis, surfactant protein disorders, and alpha-1 antitrypsin deficiency (A1ATD), can cause perinatal respiratory failure and death or chronic lung disease. Despite medical advances, therapy options are limited, often focused on treating disease complications, and culminating in the need for a lung transplant for many patients. Thus, there is a critical need for novel therapies for monogenic lung diseases. Many monogenic lung disease-causing mutations are well known, can be diagnosed before birth, and, often, one mutation is responsible for the majority of cases. A GA mutation (Glu342Lys, the PiZ allele) in the SERPINA1 gene accounts for 90% of A1ATD mutations and results in severe disease, increasing the risk of developing chronic obstructive pulmonary disease. Advances in CRISPR gene editing technology provide an unprecedented opportunity to permanently correct disease-causing mutations in monogenic lung diseases after a single treatment. Although encouraging, in vivo CRISPR gene editing studies targeting other organs in adult mouse models of human diseases highlight limitations to the postnatal approach including low-levels of homology directed repair (HDR) due to inaccessible and nonproliferative target cells and a mature immune barrier. These obstacles are even more daunting in the postnatal lung, a barrier organ with immune and physical barriers in which only 1% of epithelial progenitor cells, the target cell population for most lung diseases, are cycling at homeostasis. In utero gene editing has the potential to overcome these barriers and treat perinatal lethal diseases before the onset of irreversible pathology. The fetus is immunologically tolerant and progenitor cells of multiple organs, including the lung, are highly proliferative and accessible during development. The objective of this proposal is to establish the feasibility of prenatal lung gene editing and use prenatal gene editing to treat a mouse A1ATD model as a model for monogenic lung diseases. Our central hypotheses are that prenatal pulmonary cell gene editing is more efficient than postnatal editing and prenatal gene editing will not have a detrimental effect on edited pulmonary progenitor cell fate. We hypothesize that prenatal HDR can provide therapeutic levels of circulating alpha-1 antitrypsin protein and pulmonary cell gene correction in the A1ATD mouse model. Our hypotheses are based on our published data demonstrating efficient liver and pulmonary epithelial cell editing via prenatal CRISPR-nonhomologous end joining (NHEJ). To attain our objective, we will pursue the following aims: 1) evaluate prenatal pulmonary cell gene editing in normal and neonatal injury states, 2) evaluate prenatal HDR targeting the lung and compare it to postnatal HDR, and 3) correct A1ATD by prenatal HDR and compare it to postnatal HDR. Our research is innovative in the prenatal timing and targeting of the lung for therapeutic gene editing. The significant contribution of this work will be to provide the foundation for a one- shot, long-term therapy that cures A1ATD and is applicable to other monogenic lung diseases.
The proposed research is relevant to public health because monogenic lung diseases result in significant morbidity and mortality, including some diseases which are lethal at birth, and the only cure for many patients is a lung transplant which is limited by organ availability and complications associated with the transplant. Thus, there is an unmet need for new and better approaches to treat congenital monogenic lung diseases. The proposed research, which evaluates prenatal CRISPR gene editing as a novel treatment for monogenic lung diseases, is relevant to the part of the NIH?s mission that seeks to develop fundamental knowledge to enhance health, lengthen life and reduce illness and disability.
