Abstract Rationale Bronchopulmonary Dysplasia (BPD) and Alveolar Capillary Dysplasia with Malformation of Pulmonary Veins (ACDMPV) are severe neonatal lung disorders, with ACDMPV carrying a 100% mortality rate. Both conditions involve endothelial dysfunction. FOXF1 mutations are causative for ACDMPV, and FOXF1 expression is lost in both the pulmonary endothelium and mesenchyme in BPD. FENDRR, a long non-coding RNA transcribed antisense to the FOXF1 promoter and is essential for embryonic development. Loss of FENDRR is linked to ACDMPV and embryonic lethality. FOXM1 is a pro-proliferative forkhead transcription factor that becomes activated after injury and promotes endothelial repair and is implicated in embryogenesis. Previous studies show that FOXF1 stimulation improves outcomes in both BPD and ACDMPV, while endothelial delivery of CMV-driven FOXM1 rescues pulmonary function specifically in BPD models. However, these plasmids contain prokaryotic backbones that induce inflammation and prevent FDA approval. To overcome this limitation, we developed two clinically compatible gene therapy platforms: a minicircle FOXM1(mcFOXM1) for BPD, and a native promoter driven FOXF1-FENDRR bidirectional Nanoplasmid for both BPD and ACDMPV. Both vectors are non-integrating, lack bacterial sequences, and are suitable for GMP production and FDA approval. Methods Human FOXM1 cDNA was cloned into a minicircle parental vector and produced using ZYCY10P3S2T bacteria. Human FOXF1 and FENDRR cDNA was cloned from primary pulmonary fibroblasts into a bidirectional expression plasmid. To amplify the promoter, primers specific to the 5’UTR of FOXF1 and FENDRR were designed.The native FOXF1-FENDRR Nanoplasmid was retrofitted from the Aldevron backbone plasmid. Both constructs were tested in MFLM-91U cells for RNA and protein expression. Nanoparticles loaded with mcFOXM1 or Nano-FOXF1-FENDRR were intravenously injected into adult mice for testing before therapeutic use. Flow cytometry quantified endothelial number. mcFOXM1 was tested in a severe BPD model (P0-P14, 75% FiO2), followed by histological and immunofluorescent analyses. Results mcFOXM1 significantly expanded pulmonary endothelium in adult mice and increased Ccnd1 expression. In the severe BPD model, mcFOXM1 attenuated alveolar damage and restored endomucin expression. In vitro, Nano-FOXF1-FENDRR induced robust FOXF1 and FENDRR expression and activated FOXF1-associated pathways. In vivo delivery doubled endothelial populations and increased PECAM-1 mean fluorescence intensity. Conclusions Endothelial delivery of mcFOXM1 mitigates alveolar and vascular injury in BPD. The Nano-FOXF1-FENDRR construct effectively induces pulmonary endothelial expansion and represents a promising therapeutic for both BPD and ACDMPV. Ongoing work will evaluate mcFOXM1 effects on lung function and Nano-FOXF1-FENDRR efficacy in ACDMPV models. This abstract is funded by: HL141174, HL149631, HL152973, HL179683
Do et al. (Fri,) studied this question.