Development of novel therapeutic approaches for treatment of Alveolar Capillary Dysplasia

University Of Arizona, Tucson AZ

Investigators

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Abstract

PROJECT SUMMARY. Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a severe congenital disorder associated with neonatal pulmonary hypertension (PH). Despite all available therapies and respiratory support, the vast majority of ACDMPV patients die from respiratory failure in the first month after birth. There is an urgent need for innovative therapeutic approaches for ACDMPV newborns. ACDMPV is associated with heterozygous loss-of-function mutations in the Forkhead Box F1 (FOXF1) gene encoding a transcription factor critical for lung angiogenesis. Based on human genetic data, there are 3 types of ACDMPV: type 1, with loss of coding FOXF1 sequences; type 2, with point mutations in FOXF1 exons; type 3, with loss of non-coding (regulatory) sequences. During previously funded periods, my laboratory generated the mouse models of type 1 (Foxf1wt/-) and type 2 ACDMPV (Foxf1wt/S52F) that recapitulate main features of the human disease and can be used for preclinical testing of potential ACDMPV therapeutics. However, the animal model of type 3 ACDMPV has not been generated. We recently used single-cell RNA and ATAC sequencing of mouse and human ACDMPV lungs to identify 4 evolutionarily conserved FOXF1 enhancers (FELs) that are often lost in type 3 ACDMPV. In Aim 1, we will focus on the FEL1 enhancer. In our preliminary data, the loss of the FEL1 enhancer is found in 9 out of 10 patients with type 3 ACDMPV. CRISPR/Cas9-mediated deletion of FEL1 in mouse embryonic stem cells (ESCs) disrupts differentiation of lung ECs in vivo, decreasing the numbers of capillary, arterial and venous ECs, and causing aberrant accumulation of non-mature “transitional” ECs characterized by decreased EC markers and upregulation of fibroblast markers. In Aim 1, we will test the hypothesis that the FEL1 enhancer is required for differentiation of lung ECs and if its loss causes ACDMPV. We will generate a novel mouse model of type 3 ACDMPV and identify molecular mechanisms through which FEL1 regulates EC differentiation. In the previously funded period, we used a high throughput screen to identify the TanFe small molecule compound, which prevents protein-protein interaction of FOXF1 protein with HECTD1 E3 ubiquitin ligase, and therefore, stabilizes the FOXF1 protein. In preliminary data for Aim 2, we chemically modified TanFe to produce a non-toxic and stable derivative, TanFe[F], which can be encapsulated into the P22- F1 PBAE/PEI/PEG nanoparticles and specifically delivered to lung capillary ECs in vivo. We also discovered that nanoparticle delivery of a known HECTD1 inhibitor, I3A, a plant-derived small molecule compound which is currently in clinical trials for cancer patients, increases lung FOXF1 protein levels and prevents neonatal PH caused by prolonged neonatal hyperoxia. In Aim 2, we will test the hypothesis that nanoparticle delivery of FOXF1-stabilizing compounds into lung capillary ECs will increase neonatal angiogenesis, improve respiratory function and protect from PH in mouse ACDMPV models. Altogether, we will develop a novel mouse model of type 3 ACDMPV, and test promising ACDMPV therapies based on stabilization of the FOXF1 protein in ECs.