Poster X2 - Lung SORT LNPs Enable Precise Homology-Directed Repair Mediated CRISPR/Cas Genome Correction in Cystic Fibrosis Models

Introduction: Cystic Fibrosis (CF) is one of the most common and often fatal genetic diseases. It is caused by mutations in the Transmembrane Conductance Regulator (CFTR) protein, which is a chloride channel located in the surface membrane of epithelial cells. Currently, there is no cure for this disease, the majority of the death is caused by progressive lung damage. Patients with CF have abnormal fluid flow across the epithelial cells and thick mucus in lungs leading to infection, and limited ability to breathe over time. Although treatments using small-molecule CFTR modulators can manage the symptoms of the disease, they are only prescribed for people with certain CFTR mutations. CRISPR/Cas9-based gene editing approaches has great potential to be a long-lasting therapy for all cystic fibrosis patients, especially for those carrying currently non-treatable mutations.
To advance the gene editing technology translating into clinical use for CF patients, we focus on an modular lipid nanoparticle (LNP) platform using the Selective ORgan Targeting (SORT) methodology, where we could selectively delivery mRNA to lung after intravenous administration. In doing so, we aim to use SORT LNP-mediated gene editing tools to improve quality of life and expand the life span of CF patients by restoring CFTR function in CF patients’ lungs.

Materials and

Methods: We optimized SORT LNP formulations by adjusting the internal ratio of lipid components for messenger RNA (mRNA) delivery using an in vivo approach. C57BL/6 mice were administrated with Luciferase (Luc) mRNA encapsulated lung SORT formulations prepared by incorporating different cationic lipids as lung-SORT molecules into our base formulation to identify improved lung-specific SORT formulations in considerations of potency, specificity, and toxicity. Further we investigated whether Lung-SORT LNP can achieve delivery to therapeutically relevant cell types by carrying Cre recombinase mRNA to Ai9 tdTomato reporter mice. 48 hours after the injection, mice lung was digested for quantitative flowcytometry analysis cytometry by using surface markers.
For precise DNA correction, a homologous piece of DNA template is required for DNA repair via homology directed repair (HDR) pathway after Cas9 nuclease-mediated DNA double-stranded break (DSB). After identifying the optimal stoichiometry of CRISPR-HDR components (Cas9 mRNA, sgRNA, and donor DNA template) by in vitro screening using HEK293-BGFP cell model, we co-delivered three CRISPR-HDR components by using SORT LNPs to primary CF F508del/F508del human bronchial epithelial (hBE) cells, CF G542X mice intestinal organoids, as well as in the lungs of a CF G542X mouse model. LNP-HDR-mediated gene editing was quantified by Sanger sequencing, CFTR protein, and function restoration measured by western blotting and Ussing chamber respectively.

Results, Conclusions, and Discussions: In vivo, optimized lung SORT LNPs increased lung specificity and efficacy as well as tolerability. The data from flow cytometry analysis showed over 50% of all lung basal cells from Ai9 tdTomato reporter mice was edited after two administration of 2 mg/kg Cre mRNA using optimized lung SORT LNP. Successfully editing in lung base cells can potentially maintain the pool of edited cells for a long term and can potentially reverse progression of the disease.
In vitro screening suggested the optimal Cas9:sgRNA:HDR ratio was 0.5:1:6 (wt/wt/wt) for highest level of gene correction. By treating primary CF F508del/F508del HBEs with LNP-HDR, we observed approximately 20% of correction which leaded to restoration of CFTR protein and Cl- channel function. Similarly in CF G542X mice intestinal organoids, we observed 20% CFTR-dependent forskolin induced swelling after LNP-HDR treatment. In the G542X mice model, 2% of corrected DNA was detected by whole lung sequencing after 3 injections of 2 mg/kg LNP-HDR.
The finding from this work demonstrated the potential of using lung-specific SORT LNPs to advance gene editing technology translating into clinical use for CF patients.

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