Demonstration of Safety in Wild Type Mice of npFOXF1, a Novel Nanoparticle-Based Gene Therapy for Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins

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Open Access Full Text Article ORIGINAL RESEARCH Demonstration of Safety in Wild Type Mice of npFOXF1, a Novel Nanoparticle-Based Gene Therapy for Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins Fatemeh Kohram 1–3 , Zicheng Deng 1–4 , Yufang Zhang 1–3 , Abid Al Reza 1–3 , Enhong Li 1–3 , Olena A Kolesnichenko 1–3 , Samriddhi Shukla 1–3 , Vladimir Ustiyan 1–3 , Jose Gomez-Arroyo 1–3 , Anusha Acharya 1–3 , Donglu Shi 4 , Vladimir V Kalinichenko 1–3 , Alan P Kenny 1,2,5,6 1 Department of Pediatrics, University of Cincinnati and Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 2Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 3Center for Lung Regenerative Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 4The Materials Science and Engineering Program, College of Engineering and Applied Science, University of Cincinnati, Cincinnati, OH, USA; 5Division of Neonatology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USA; 6Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA Correspondence: Alan P Kenny, Divisions of Pulmonary Biology and Neonatology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH, 45229, Tel +1 513-803-2224, Email Introduction: Alveolar Capillary Dysplasia with Misaligned Pulmonary Veins (ACDMPV) is a fatal congenital disease resulting from a pulmonary vascular endothelial deficiency of FOXF1, producing abnormal morphogenesis of alveolar capillaries, malposi tioned pulmonary veins and disordered development of lung lobes. Affected neonates suffer from cyanosis, severe breathing insufficiency, pulmonary hypertension, and death typically within days to weeks after birth. Currently, no treatment exists for ACDMPV, although recent murine research in the Kalinichenko lab demonstrates nanoparticle delivery improves survival and reconstitutes normal alveolar-capillary architecture. The aim of the present study is to investigate the safety of intravenous adminis tration of FOXF1-expressing PEI-PEG nanoparticles (npFOXF1), our pioneering treatment for ACDMPV. Methods: npFOXF1 was constructed, validated, and subsequently administered in a single dose to postnatal day 14 (P14) mice via retroorbital injection. Biochemical, serologic, and histologic safety were monitored at postnatal day 16 (P16) and postnatal day 21 (P21). Results: With treatment we observed no lethality, and the general condition of mice revealed no obvious abnormalities. Serum chemistry, whole blood, and histologic toxicity was assayed on P16 and P21 and revealed no abnormality. Discussion: In conclusion, npFOXF1 has a very good safety profile and combined with preceding studies showing therapeutic efficacy, npFOXF1 can be considered as a good candidate therapy for ACDMPV in human neonates. Keywords: nanoparticle, FOXF1, ACDMPV, mouse, toxicity, safety Introduction ACDMPV is a deadly neonatal condition, typified by defects in coordinated development of alveolar capillaries, malpositioning of lung veins and abnormal formation of lung lobules, causing profound hypoxemia, catastrophic breathing insufficiency and pulmonary hypertension ensuing rapidly after delivery. Due to the severity of the anomalies and breathing failure in ACDMPV newborns, death comes less than one month postnatally despite maximal support.1 Despite incomplete genetic characterization, heterozygous copy-number variant (CNV) deletions and point mutations involving the Forkhead Box F1 (FOXF1) gene locus account for the majority of ACDMPV cases.2 To date, over 70 unique FOXF1 point mutations in FOXF1 are associated with ACDMPV.2 FOX proteins constitute a grand family of winged helix transcription factors that mediate multiple molecular signaling pathways, (eg, VEGF pathway).3,4 Foxf1-null mice have reduced lung endothelial cell number both in lung Biologics: Targets and Therapy 2023:17 43–55 Received: 2 December 2022 Accepted: 14 March 2023 Published: 20 March 2023 43 © 2023 Kohram et al. This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms. php and incorporate the Creative Commons Attribution – Non Commercial (unported, v3.0) License (http://creativecommons.org/licenses/by-nc/3.0/). By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms (https://www.dovepress.com/terms.php). Kohram et al Dovepress formation and post-damage pulmonary repair.5 FOXF1 induces VEGF receptor 2, stimulating vascular endothelial growth factor signals in model organisms and systems.6 Research has shown nanoparticles can serve therapeutic purposes to deliver both biologic (DNA, mRNA, et cet. For gene expression) and inorganic compounds to the cytoplasm. They can be utilized as vaccines and to treat cancer, immune disorders, and diabetes. They have tremendous potential for use in tissue regeneration.7 Recent research in the Kalinichenko lab used newly developed polyethylenimine-(5) myristic acid/ poly(ethylene glycol)-oleic acid/cholesterol (PEI600-MA5/PEG-OA/Cho) nanoparticle8 to deliver non-integrating angiogenic cDNAexpressing plasmids into the neonatal pulmonary bloodstream in order to improve pulmonary capillary formation and alveolarization in diseases like ACDMPV. The Kalinichenko laboratory also recently generated Foxf1WT/S52F mice containing the S52F FOXF1 mutation in the conserved serine-52. This mutation was generated in the endogenous mouse Foxf1 locus via CRISPR/Cas9 genome editing.9 Foxf1WT/S52F mutant mice exhibited all the key features of alveolar capillary dysplasia, including fused lung lobes, misalignment of pulmonary veins and increased perinatal mortality.1,9 These mice were phenotypically rescued with the lab’s aforementioned PEI-PEG nanoparticle therapy carrying STAT3, a key downstream target of FOXF1. Nanoparticles containing Foxf1-expressing plasmids have been used to treat another much more common severe respiratory failure disease, bronchopulmonary dysplasia, in mouse models.10 Unfortunately, the plasmid used in this research is less than ideal for human use as it retains all sequence necessary for plasmid replication in bacteria. Furthermore, an untested hypothesis in ACDMPV research is whether FoxF1 delivered to affected mice causes the same or better rescue previously observed with pCMV-STAT3 expression plasmid. Therefore, we designed npFOXF1, a Minicircle FOXF1-expressing plasmid driven by an EF1α p (...truncated)