(612) Optimized nanoparticle-mediated base editing of CF-causing variants

Background: Next-generation SARS-CoV-2 vaccines demonstrated that nanoparticle mRNA delivery is effective and safe for in vivodelivery in humans. Current treatments for CF primarily focus on modulator drug therapies designed to correct malfunctioning CFTR protein. However, these modulators are ineffective for the 10% of CF individuals with variants that do not allow protein production. Among these is the splice variant 3120+1G >A, the most common CF-causing mutation in native Africans. Gene editing would allow production of CFTR protein and enhancement of function using available CFTR modulators. We have demonstrated that electroporation of a modified CRISPR-Cas9 base editor to primary human bronchial epithelial (HBE) cells carrying 3120+1G >A and F508del mutant alleles achieved 75%genome editing of the splice variant, resulting in ~40%WT CFTR function (see Joynt A et al., this meeting). Here, we evaluate the effectiveness of several new nanoparticle formulations at delivering green fluorescent protein (GFP) mRNA to CF bronchial epithelial (CFBE41o-) cells. Using the optimal formulation, we then tested the efficacy correction of the 3120+1G >A variant in a CFTR expression minigene (EMG) integrated in the genome of isogenic CFBE cells using mRNA and plasmid DNA encoding adenine base editor (ABE) and guide RNA.

Methods: GFP served as a reporter to evaluate transfection efficiency, cell viability and mean fluorescence intensity (MFI) across three dosages (150, 75, 32.5ng mRNA), four polymer-to-mRNA to weight (w/w) ratios (60, 40, 30, 20) and four different polymers (polymer R, Y, G, B). 7-AAD served as a live/dead stain to quantify viability, with flow cytometry results being analyzed using FlowJo software. CFBE cells stably expressing the 3120+1G >A EMG were transfected with the optimized nanoparticle formulation to deliver ABE and guide RNA at two dosages (150, 75ng) of mRNA and DNA. CFTR function in CFBE cells was measured by short circuit current, forskolin stimulation and inh-172 inhibition as a measure of editing efficiency.

Results: Flow cytometry showed polymer R achieved >85% GFP transfection compared to a maximum of ~35% across the other three polymers at the maximum 150ng dosage with ~80% viability normalized to untreated cells. In addition, Polymer R achieved GFP MFI over one order of magnitude higher compared to other formulations (~30,000 vs 2,700 MFI respectively) across the other three polymers at 150 ng dosage and 40 w/w ratio. CFBE cells transfected with polymer R nanoparticles containing ABE and guide RNA at 75 and 150ng showed CFTR function increase to 10.6±1.1μA (mean±SEM; ~10% of WT) and 6.3±0.9 μA; ~6% of WT), respectively. Increased toxicity at the higher dosage could explain the larger increase in CFTR current at the lower dosage. DNA encoded ABE plasmid and guide RNA showed a less robust increase in CFTR function (2.9±0.4 μA and 3.0±0.4 μA for 75 and 150ng doses respectively), likely a result of the nanoparticle formulation being optimized for RNA instead of DNA cargo or the additional intracellular barriers that must be overcome for successful DNA delivery.

Conclusions: We demonstrated that an optimized nanoparticle formulation containing ABE and guide RNA can correct splicing of isogenic cells bearing the 3120+1G >A CFTR variant, resulting in recovery of CFTR function. In ongoing work, we are adapting these nanoparticles for RNA and DNA encoded ABE and guide RNA delivery to primary HBE cells.

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