Abstract Details

Read the abstract and find the presentation below

Full text and presentation details

B - Gene Targeting and Gene Correction -> B1 – Gene Targeting and Gene Correction – In Vivo Studies (Basic development of novel technologies for genome editing, with or without site-specific endonuclease.

977: Gene Editing in the Lung Using Polymeric Vehicles

Type: Poster Session

Poster Board Number: 977
Presentation Details
Session Title: Thursday Poster Session
Start Time: 5/18/2023 12:00
End Time: 5/18/2023 14:00

The lungs remain a key organ of interest for the treatment of myriad diseases, such as cystic fibrosis, with gene therapy and gene editing therapeutics. Non-viral lipid- and polymer-based vehicles have shown promise for nucleic acid delivery in vitro and in vivo. However, the primary barrier for clinical translation remains delivery to target tissues in vivo. Local deposition throughout the airway can be achieved through inhalation and facilitates targeting of the epithelium. Inhaled mRNA delivery has been achieved through a variety of administration routes, including: intranasal, oropharyngeal, nebulization chamber, and intratracheal (IT) delivery. We have utilized IT administration to deliver both Cre mRNA (1) as well as CRISPR/Cas9-based gene editing agents using polymeric vehicles in the Ai14 reporter mouse model. We demonstrate that nucleic acid-based therapeutics can be effectively delivered in vivo using a class of polymeric vehicles consisting of members from a family of mildly cationic poly(amine-co-ester) (PACE) biodegradable polymers (2,3) that are designed for gene delivery and result in robust encapsulation of mRNA and sgRNA.
Our initial studies focused on Cre mRNA delivery by PACE polyplexes via IT administration in the Ai14 mouse model. By flow cytometry, IT delivery of Cre mRNA results in ~10% tdTomato expression in bulk lung and ~30% expression in cells from bronchoalveolar lavage fluid (BALF) of treated animals. We further assessed which specific cell types in the lung were transfected: CD31+ endothelial cells, EpCAM+ epithelial cells, and CD45+ leukocytes. We observed ~20% of epithelial cells and leukocytes expressing tdTomato, with no detectable expression in endothelial cells. We next assessed the ability of PACE polyplexes to deliver SpCas9 mRNA and sgRNA to edit Ai14 mice. We measured tdTomato expression levels in the lung by flow cytometry 3 or 7 days after delivering one dose of gene editing PACE NPs. At day 3 post-treatment, the highest editing levels were observed in epithelial cells, though with less than 1% of cells exhibiting tdTomato expression by flow cytometry. Editing levels were significantly boosted when lungs were evaluated at day 7 post-dosing. In this case, we observed in ~0.5% of bulk lung cells, ~1.5% of leukocytes, and ~3% of epithelial cells, again by flow cytometry.
We also imaged frozen lung sections and performed quantitative microscopy analysis of treated tissues. In this case, we quantified editing in small airways positive for tdTomato, by thresholding this signal based on the background signal from untreated control lung images. We then quantified the number of edited cells as a percentage of total cells in the airway. Using this analysis method, we observed ~60% editing in small airways on average. This number was similar among different lung samples analyzed from distinct animals. To obtain a broad sense of editing across a larger area closer to the size of a lobe, we tiled images of untreated control lungs as well as lungs from animals treated with IT-administered PACE NPs encapsulating Cas9 mRNA and sgRNA. We observe broad distribution/diffuse signal of tdTomato signal across the lung. On the whole, our findings demonstrate the promise of nucleic acid delivery mediated by inhalable polymeric carriers for the treatment of genetic lung disorders.
References (1) Suberi A, et al. bioRxiv. 2022. (2) Grun M, et al. Biomaterials. 2021. (3) Kauffman A and Piotrowski-Daspit A, et al. Biomacromolecules. 2018.

Alexandra S. Piotrowski-Daspit1, Alexandra Suberi2, Laura G. Bracaglia3, Laiba Akhtar2, Madalina Ene2, Hee Won Suh2, Peter M. Glazer4, W. Mark Saltzman2

1Biomedical Engineering, University of Michigan, Ann Arbor, MI,2Biomedical Engineering, Yale University, New Haven, CT,3Chemical and Biological Engineering, Villanova University, Villanova, PA,4Yale University School of Medicine, New Haven, CT
  A.S. Piotrowski-Daspit: 1; Commercial Interest i.e. Company X; Xanadu Bio, Inc.. 1; What was received? i.e. Honorarium; Consulting Fees, Equity. 1; For what role? i.e. Speaker; Consulting, Cofounder.

Find This Presentation

This site uses cookies to offer you a better user experience and to analyze site traffic. By continuing to use this website, you accept our use of cookies.