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Background: 

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A key sequela is abnormally viscous mucous in the airway, resulting in frequent infection and inflammation, which can lead to subsequent respiratory failure and early death. Currently, CFTR modulators and molecules have improved quality of life for some patients. However, treatment is lifelong, expensive, and not effective for cases where CFTR expression is obliterated or very low. Therefore, gene therapy to replace the mutant CFTR gene with a functional one may fill this gap in care. Challenges in the development of a successful CF gene therapy includes the inability to target relevant stem cells residing in airway submucosal glands (SMGs). In this study, our approach is to enable systemic delivery of gene editing reagents to relevant stem cells by functionalizing lipid nanoparticles (LNPs) to cross the endothelial barrier utilizing a targeting peptide. 

Objective: 

Our study explores the knowledge gaps in endothelial extravasation that will be foundational for the development of a systemic gene therapy. We also explore methods to functionalize LNPs to increase targeting of SMG stem cells. 

Methods:

We use click chemistry and post-insertion methods to optimize the stability and density of a targeting peptide RPARPAR (RPAR) on our LNPs. Functionalized LNPs are characterized by assessing size, polydispersity index (PDI), and encapsulation efficiency. Specific peptide binding and stability of LNPs are confirmed through binding assays.

To assess RPARPAR-mediated transcytosis, we co-culture human umbilical vein endothelial cells (HUVECs) pre-treated with fluorescently labelled LNPs along with untreated human bronchial epithelial (HBE) cells. Microscopy and flow cytometry were used to quantify transcytosis of LNPs. We then develop a novel 2D in vitro system in which HUVECs are grown in the apical portion of an insert of a transwell cell culture plate and HBE cells are grown directly on the underside of the same insert. The apical portion are then loaded with RPAR-functionalized LNPs, and transcytosis is assessed.

Results: 

We found that functionalizing LNPs with RPARPAR using post-insertion and click chemistry preserved LNP size and homogeneity at targeting moiety density up to 5x higher than the traditional in-line formulation method. Functionalized LNPs showed specific binding to purified RPARPAR target receptor, NRP1, in an in vitro binding assay. We also illustrate that the type of pegylated lipid used in our LNP formulation affects the stability and transfection efficiency of our LNPs. As a preliminary model, we show that HUVECs treated with RPAR-functionalized silver nanoparticles result in higher uptake and transfer of nanoparticles to HBE cells in co-culture compared nanoparticles functionalized with control peptides. 

Conclusions:

Thus far, we demonstrate a consistent and efficient method to functionalize LNPs that is superior to traditional in-line formulation methods. We confirm that RPAR specifically binds the proposed target receptor that induces transcytosis. We illustrate that composition of our LNP formulation also has a role in targeting ability. Lastly, the increased transfer of silver nanoparticle displaying RPAR to HBEs in co-culture compared to control peptide shows that RPARPAR confers a specific targeting advantage and is a potential method to increase gene therapy delivery across the endothelial barrier.