PMID- 34742747 OWN - NLM STAT- MEDLINE DCOM- 20220322 LR - 20230102 IS - 1873-4995 (Electronic) IS - 0168-3659 (Print) IS - 0168-3659 (Linking) VI - 341 DP - 2022 Jan TI - Amniotic fluid stabilized lipid nanoparticles for in utero intra-amniotic mRNA delivery. PG - 616-633 LID - S0168-3659(21)00584-8 [pii] LID - 10.1016/j.jconrel.2021.10.031 [doi] AB - Congenital disorders resulting in pathological protein deficiencies are most often treated postnatally with protein or enzyme replacement therapies. However, treatment of these disorders in utero before irreversible disease onset could significantly minimize disease burden, morbidity, and mortality. One possible strategy for the prenatal treatment of congenital disorders is in utero delivery of messenger RNA (mRNA). mRNA is a nucleic acid therapeutic that has previously been investigated as a platform for protein replacement therapies and gene editing technologies. While viral vectors have been explored to induce intracellular expression of mRNA, they are limited in their clinical application due to risks associated with immunogenicity and genomic integration. As an alternative to viral vectors, safe and efficient in utero mRNA delivery can be achieved using ionizable lipid nanoparticles (LNPs). While LNPs have demonstrated potent in vivo mRNA delivery to the liver following intravenous administration, intra-amniotic delivery has the potential to deliver mRNA to cells and tissues beyond those in the liver, such as in the skin, lung, and digestive tract. However, LNP stability in fetal amniotic fluid and how this stability affects mRNA delivery has not been previously investigated. Here, we engineered a library of LNPs using orthogonal design of experiments (DOE) to evaluate how LNP structure affects their stability in amniotic fluid ex utero and whether a lead candidate identified from these stability measurements enables intra-amniotic mRNA delivery in utero. We used a combination of techniques including dynamic light scattering (DLS), transmission electron microscopy (TEM), and chromatography followed by protein content quantification to screen LNP stability in amniotic fluids. These results identified multiple lead LNP formulations that are highly stable in amniotic fluids ranging from small animals to humans, including mouse, sheep, pig, and human amniotic fluid samples. We then demonstrate that stable LNPs from the ex utero screen in mouse amniotic fluid enabled potent mRNA delivery in primary fetal lung fibroblasts and in utero following intra-amniotic injection in a murine model. This exploration of ex utero stability in amniotic fluids demonstrates a means by which to identify novel LNP formulations for prenatal treatment of congenital disorders via in utero mRNA delivery. CI - Copyright (c) 2021. Published by Elsevier B.V. FAU - Swingle, Kelsey L AU - Swingle KL AD - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA. FAU - Billingsley, Margaret M AU - Billingsley MM AD - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA. FAU - Bose, Sourav K AU - Bose SK AD - The Center for Fetal Research, Division of Pediatric General, Thoracic, and Fetal Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. FAU - White, Brandon AU - White B AD - The Center for Fetal Research, Division of Pediatric General, Thoracic, and Fetal Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. FAU - Palanki, Rohan AU - Palanki R AD - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; The Center for Fetal Research, Division of Pediatric General, Thoracic, and Fetal Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. FAU - Dave, Apeksha AU - Dave A AD - The Center for Fetal Research, Division of Pediatric General, Thoracic, and Fetal Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. FAU - Patel, Savan K AU - Patel SK AD - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA. FAU - Gong, Ningqiang AU - Gong N AD - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA. FAU - Hamilton, Alex G AU - Hamilton AG AD - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA. FAU - Alameh, Mohamad-Gabriel AU - Alameh MG AD - Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. FAU - Weissman, Drew AU - Weissman D AD - Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. FAU - Peranteau, William H AU - Peranteau WH AD - The Center for Fetal Research, Division of Pediatric General, Thoracic, and Fetal Surgery, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA. Electronic address: peranteauw@emaol.chop.edu. FAU - Mitchell, Michael J AU - Mitchell MJ AD - Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA; Abramson Cancer Center, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Immunology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Cardiovascular Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA; Institute for Regenerative Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Electronic address: mjmitch@seas.upenn.edu. LA - eng GR - DP2 HL152427/HL/NHLBI NIH HHS/United States GR - DP2 TR002776/TR/NCATS NIH HHS/United States GR - R01 DK123049/DK/NIDDK NIH HHS/United States GR - R01 HL151352/HL/NHLBI NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20211103 PL - Netherlands TA - J Control Release JT - Journal of controlled release : official journal of the Controlled Release Society JID - 8607908 RN - 0 (Lipid Nanoparticles) RN - 0 (Liposomes) RN - 0 (RNA, Messenger) SB - IM MH - *Amniotic Fluid MH - Animals MH - Liposomes/chemistry MH - Mice MH - *Nanoparticles/chemistry MH - RNA, Messenger MH - Sheep MH - Swine PMC - PMC8776620 MID - NIHMS1765272 OTO - NOTNLM OT - Gene therapy OT - In utero OT - Lipid nanoparticles OT - Nucleic acid therapeutics OT - mRNA COIS- 6. Declaration of Competing Interests D.W. is an inventor on several patents related to this work filed by the Trustees of the University of Pennsylvania (11/990,646; 13/585,517; 13/839,023; 13/839,155; 14/456,302; 15/339,363; 16/299,202). M.J.M. is an inventor on a patent related to this work filed by the Trustees of the University of Pennsylvania (PCT/US20/56252). The authors declare that they have no other competing interests. EDAT- 2021/11/08 06:00 MHDA- 2022/03/23 06:00 PMCR- 2023/01/01 CRDT- 2021/11/07 20:34 PHST- 2021/07/01 00:00 [received] PHST- 2021/10/03 00:00 [revised] PHST- 2021/10/31 00:00 [accepted] PHST- 2021/11/08 06:00 [pubmed] PHST- 2022/03/23 06:00 [medline] PHST- 2021/11/07 20:34 [entrez] PHST- 2023/01/01 00:00 [pmc-release] AID - S0168-3659(21)00584-8 [pii] AID - 10.1016/j.jconrel.2021.10.031 [doi] PST - ppublish SO - J Control Release. 2022 Jan;341:616-633. doi: 10.1016/j.jconrel.2021.10.031. Epub 2021 Nov 3.