Client Challenge
A biopharmaceutical company developing a lipid nanoparticle (LNP)-formulated gene-editing drug product needed a cell-based assay to measure editing at the intended target site versusan off-target locus of interest, and to understand how editing at each site changed after a single exposure at several dose levels and after repeated low-dose exposure, to support its nonclinical characterization program.
Avance Biosciences’ Solution
Avance Biosciences developed a cell-based assay to quantify on- and off-target editing activity for the LNP drug product.
The approach:
- Designed a targeted NGS primer panel spanning both the intended on-target locus and the candidate off-target locus identified by the client, allowing both sites to be read from the same sequencing run.
- Established an in vitro dosing model in a cell line, evaluating a single exposure at several dose levels and a separate repeated low-dose exposure regimen delivered across multiple sequential treatments.
- Extracted genomic DNA from treated and untreated control cells at defined timepoints and generated targeted sequencing libraries for next-generation sequencing
- Quantified editing frequency at each locus and compared on-target and off-target signals across dose levels and exposure regimens.
Assay Findings
- On-Target Dose Response: Did editing at the intended sitevary with dose? A clear, dose-dependent increase in on-target editing was observed following single-dose exposure, with editing approaching a peak at an intermediate dose.Editing was reduced at the highest dose, with qualitative signs of reduced cell health observed at that exposure level.
- Off-Target Signal: Could the assay detect editing at the off-target locus? The NGS panel detected editing at the off-target locus, but was consistently lower than on-target editing across every dose and exposure condition tested, supporting preferential activity at the intended site under the conditions evaluated.
- Repeated Low-Dose Exposure: How did the editing change over repeated low dose exposure? Repeated low-dose administration produced a progressive increase in on-target editing with each additional treatment, as measured by sequential NGS timepoints, with the incremental gain diminishing at the higher cumulative exposures tested. Off-target editing also increased with cumulative exposure without plateauing and stayed below the corresponding on-target signal at every tested timepoint.
- Assay Background: Did untreated controls show a clean baseline? Untreated negative controls showed low background signal at both the on-target and off-target loci in both experiments, supporting the specificity of the sequencing-based readout.
Customer Outcome
- A custom targeted NGS sequencing panel and bioinformatics pipeline capable of simultaneously quantifying on-target and off-target editing from a single workflow
- Characterization of on- and off- target editing across dose levels and exposure regimens for its LNP-formulated gene-editing drug product in a cell-based model
- Side-by-side on-target/off-target editing data under both single exposure (several dose levels) and repeated low-dose exposure conditions.
Client Impact
The client now has characterization data on its drug product’s editing specificity and dose behavior to help inform its nonclinical program and future off-target risk assessment.
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