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Biomaterials

Biomaterials - Poster Session E

Poster O10 - Novel Biodegradable Ionizable Lipids for Genome Editing in Lung Epithelia

Saturday, October 26, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Messenger RNA(mRNA)-lipid nanoparticles have emerged as a new platform for various applications, such as protein replacement, Cancer, and genetic diseases. Pfizer-BioNTech and Moderna Therapeutics used mRNA-lipid nanoparticles for COVID-19 vaccines and are the first mRNA vaccines clinically approved. Inspired by these, we focused our aim on mRNA delivery to the liver [1] and Lungs; previously, our group delivered nucleic acids to both lung endothelium and pulmonary immune cells but could not deliver them to epithelia [2]. Epithelial cells are the targets for most congenital lung diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and alpha-1 antitrypsin (AAT) deficiency.

Materials and

Methods:  We report here from a three-component system activated nitro Ricinoleic acrylate (NRA), alcohol, and amines; we synthesized novel 720 biodegradable lipids and screened them in vitro and in vivo for mRNA delivery. We screened different formulations for efficient mRNA delivery to Lung Cells. LNPs were formulated with a classical four-component system of ionizable lipid, DOPE, cholesterol, and PEG2k to identify lead candidates and loaded with firefly luciferase mRNA. To expedite the screening process for in vivo, batch phase testing was performed and intramuscularly injected into mice. Lead lipid formulations are amenable to multiple intratracheal dosing and can efficiently deliver Cre or Cas9 mRNA to mouse airway epithelium. The top-performing ionizable lipid is formulated with Cas 9 mRNA/sgRNA, and gene editing efficacy is performed in vivo [3].

Results, Conclusions, and Discussions: First tested, these novel 720 biodegradable Lipids on A549 Lung epithelial cells were treated with mLuc-LNPs, and luciferase expression was measured after overnight incubation with Bright-Glo-Assay. The hit rate of each amine head group was calculated by the % of LNPs with luciferase expression > 50,000, and a hit rate of at least 80% across lipid variants was chosen for further screening. For the in vivo, the transfection potency was examined by intramuscular injection in mice, followed by intratracheal (IT) injection. The top nine lipids from IT administration were investigated for gene editing on HEK-GFP cell lines, and six of the nine LNPs knocked out GFP in >80% of cells. Notably, the RCB-4-8 LNP displayed the highest GFP knockout efficiency in vitro by >95%. When loaded once or thrice with Cre-recombinase mRNA, the top-performing lipid RCB-4-8 could result in tdTomato+ expression in 42 and 53% of total lung cells, respectively, which is twofold higher than Cre-mRNA delivered by polymer polyplexes. Interestingly, these Cre-mRNA LNPs were found to perform gene editing in both club cells and ciliated cells, which are major cell subtypes of lung airway epithelial cells, by 13 and 10%, respectively. Finally, RCB-4-8 LNPs, when loaded with Cas9 mRNA/sgRNA at low and high doses and quantified gene-editing, could result in 3 and 7% tdTomato+ cells, respectively.

Conclusions:
In Summary, we identified RCB-4-8 by rational and high-throughput screening as an ionizable lipid to enable highly efficient pulmonary mRNA delivery and gene editing in the lung. We have shown that RCB-4-8 can deliver to both ciliated and club epithelial cells in the lung, suggesting its potential to enable gene editing for various airway diseases, including CF (ciliated epithelial cells). The RCB-4-8 LNP is an efficient nonviral delivery system for gene editing in the lung epithelium and opens new avenues for the treatment of congenital lung diseases. Applications of these novel Biodegradable lipids for Cancer Therapy and Vaccines are ongoing.

Acknowledgements (Optional):  

References:
[1]. Rhym, L. H.; Manan, R. S.; Koller, A.; Stephanie, G.; Anderson, D. G., Peptide-encoding mRNA barcodes for the high-throughput in vivo screening of libraries of lipid nanoparticles for mRNA delivery. Nat. Biomed. Eng. 2023, 7 (7), 901-910.
[2]. Patel, A. K.; Kaczmarek, J. C.; Bose, S.; Kauffman, K. J.; Mir, F.; Heartlein, M. W.; DeRosa, F.; Langer, R.; Anderson, D. G., Gene Delivery: Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium Adv. Mater. 2019, 31 (8), 1970053.
[3]. Li, B.; Manan, R. S.; Liang, S.-Q.; Gordon, A.; Jiang, A.; Varley, A.; Gao, G.; Langer, R.; Xue, W.; Anderson, D., Combinatorial design of nanoparticles for pulmonary mRNA delivery and genome editing. Nat. Biotechnol. 2023, 41 (10), 1410-1415.