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Published on: February 26, 2019
Endotoxin contamination in commercially available Cas9 proteins potentially induces T-cell mediated responses
Vijaya L Simhadri1, Joseph R McGill1, Zuben E Sauna2
1Hemostasis Branch, Division of Plasma Protein Therapeutics, Office of Tissues and Advanced Therapies, Center for Biologics Evaluation and Research, Food and Drug Administration, Silver Spring, MD, USA.
Abstract:
Immune responses to Cas proteins have been demonstrated recently and these may prove to be an impediment to their clinical use in gene editing. To make meaningful assessments of Cas9 immunogenicity during drug development and licensure it is imperative the reagents are free of impurities that could affect in vitro assessments of immunogenicity. Here we address the issue of endotoxin levels in laboratory grade Cas9 proteins used to measure T-cell memory responses. Many of these reagents have not been developed for immunogenicity assays, are or microbial origin and carry varying levels of endotoxin. The use of these reagents, off the shelf, without measuring endotoxin levels is likely to introduce incorrect estimates of the prevalence of memory T-cell responses in research studies. We demonstrate wide variation in endotoxin levels in Cas9 proteins from seven suppliers. Different lots from the same supplier also contained varying levels of endotoxin. ELISPOT assays showed similar large variations in the interferon-γ signals. Finally, when we carried out endotoxin depletion in four Cas9 proteins with strong signals in the ELISPOT assay, we found dampening of the interferon-γ signals.
Insights
Endotoxin contamination in Cas9 proteins can skew results for immune response assays. Measuring and controlling endotoxin levels is crucial for accurate gene editing immunogenicity studies.
Area of Science:
- Molecular Biology
- Immunology
- Biotechnology
Background:
- Immune responses to Cas proteins pose challenges for clinical gene editing applications.
- Accurate assessment of Cas9 immunogenicity requires high-purity reagents free from contaminants.
- Laboratory-grade Cas9 proteins, often of microbial origin, may contain endotoxins impacting assays.
Purpose of the Study:
- To investigate endotoxin levels in commercially available Cas9 proteins.
- To evaluate the impact of endotoxins on T-cell memory response assays.
- To highlight the necessity of endotoxin control in gene editing drug development.
Main Methods:
- Analysis of endotoxin levels in Cas9 proteins from multiple suppliers and lots.
- Utilizing ELISPOT assays to measure interferon-gamma (IFN-γ) signals.
- Performing endotoxin depletion on selected Cas9 protein samples.
Main Results:
- Significant variations in endotoxin levels were observed across different Cas9 protein suppliers and lots.
- ELISPOT assays revealed substantial variability in IFN-γ responses, correlating with endotoxin levels.
- Endotoxin depletion led to a dampening of IFN-γ signals in tested Cas9 proteins.
Conclusions:
- Endotoxin contamination is a prevalent issue in laboratory-grade Cas9 proteins.
- Uncontrolled endotoxins can lead to inaccurate assessments of T-cell memory responses.
- Standardized endotoxin testing and purification are essential for reliable immunogenicity studies in gene editing research.
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