Client Objective
To support a preclinical study, a biotechnology company advancing a novel gene-editing therapy required confirmation of in vivo editing efficiency in rat tissue samples. The objective was to accurately quantify the percentage of gene-edited cells in multiple tissues, including blood, spleen, and bone marrow, using a validated method with high sensitivity and reproducibility.
Our Solution
Avance Biosciences used a validated NGS Amplicon Sequencing method to detect and quantify targeted genomic edits across preclinical rat tissues. Our method used Illumina’s MiSeq sequencing system in combination with custom-designed primers to amplify the region of interest, enabling accurate measurement of editing frequencies.
Project Overview
Submitted rat tissue samples contained defined single-nucleotide substitutions introduced by the client’s gene-editing therapeutic. Using a combination of synthetic DNA controls and test samples, we developed and validated a customized NGS assay capable of detecting editing frequencies as low as 1%.
Key steps in our workflow included:
- Genomic DNA extraction from diverse tissue types using optimized, tissue-specific protocols
- DNA quality and concentration assessment via fluorescence-based methods (e.g., PicoGreen, Qubit) and spectrophotometry (e.g., Nanodrop or equivalent)
- Targeted PCR amplification and sample indexing optimized for high-throughput sequencing NGS workflows
- Next-generation sequencing on Illumina platforms with appropriate read length and depth for variant detection
- Custom, validated bioinformatics pipelines for variant detection and quantitative analysis of editing efficiency
Assay Validation
Custom synthetic controls spanning 1%–100% edited DNA mixtures enabled a comprehensive assay validation, supporting regulatory-grade confidence in:
- Specificity to distinguish edited from wild-type sequences
- Accuracy and precision, including inter-operator reproducibility
- Assay linearity and dynamic range across relevant editing frequencies
- Lower limit of detection (LOD) for edited sequences down to 0.02%
- Lower limit of quantification (LLOQ) established at 1%
- Stability assessment of QC samples over defined storage intervals
Outcome
We successfully delivered a full editing profile across all preclinical samples within 12 weeks, meeting the client’s deadline and scientific requirements. Our data enabled the client to:
- Precisely determine percent editing per tissue type
- Identify variability between tissues and animals
- Justify dosing strategies and selection criteria for future IND-enabling studies
The validated data package included a complete bioanalytical report, raw and processed sequencing data, and comprehensive documentation of assay performance metrics.
Client Impact
By partnering with Avance Biosciences, the client gained critical insight into the in vivo performance of their gene-editing therapy. Our results informed key go/no-go decisions during preclinical development and laid the foundation for regulatory submissions.
Related Services / Technologies