Direct RNA sequencing for therapeutic RNA quality
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As RNA-based therapeutics continue to transform cell and gene therapy, ensuring the quality and integrity of both mRNA and single-guide RNA (sgRNA) has become critical for safety, efficacy, and regulatory compliance. However, current analytical workflows rely on multiple orthogonal methods, often providing only partial insight into critical quality attributes (CQAs).
In this webinar, we explore how nanopore direct RNA sequencing enables a unified, single-molecule approach to RNA characterisation, delivering comprehensive, full-length insight into sequence identity, integrity, and structural features in a single assay.
Drawing on recent research, the Cell Therapy Engineering and Development team at Genentech demonstrate how Oxford Nanopore direct RNA sequencing simultaneously assesses key CQAs such as sequence integrity, 5′ capping efficiency, poly(A) tail length, and sgRNA structure. The approach also reveals impurities such as truncations and deletions that directly impact gene-editing performance.
Importantly, the team at Genentech connect these analytical insights to functional outcomes, showing how RNA quality attributes, including degradation and sgRNA impurities, can significantly influence editing efficiency and variability in cell-based systems.
Attendees will gain a clear understanding of how Oxford Nanopore sequencing can support end-to-end RNA therapeutic development.
The webinar will conclude with a live Q&A session, providing an opportunity to engage directly with the speaker.
Learning objectives:
- Understand the critical quality attributes (CQAs) of RNA therapeutics
- Recognise the limitations of traditional RNA analytical methods
- Learn how Oxford Nanopore direct RNA sequencing enables multi-attribute RNA analysis
- Explore detection and characterisation of sgRNA impurities
- Connect RNA quality to functional performance in gene editing
- Discover how nanopore sequencing supports future-ready quality control (QC) workflows
Meet the speakers
Abstract As RNA-based vaccines and CRISPR-Cas9 gene-editing therapeutics advance toward commercial manufacturing, robust analytical strategies are required to monitor Critical Quality Attributes (CQAs). Conventional USP-recommended workflows rely on multiple low-throughput, orthogonal assays for identity, integrity, 5′ capping, and poly(A) tail characterization, introducing significant operational complexity in CMC environments. Here, we evaluated Nanopore Direct RNA Sequencing (NDRS) as a single, consolidated Multi-Attribute Method (MAM) to streamline these measurements within cell and gene therapy (CGT) workflows.
We cross-validated NDRS against established orthogonal methods (CGE, IP-RP-HPLC, and MS) using therapeutic mRNA and 100-nt sgRNAs to evaluate sequence accuracy, truncation species, poly(A) tail heterogeneity, and CQA concordance. To enable this, we implemented an enzymatic workflow for direct 5′ cap quantification alongside optimised ligation strategies for full-length sgRNA identity.
NDRS demonstrated strong concordance with orthogonal methods while offering superior molecular resolution. Unlike bulk-averaging techniques, NDRS resolved poly(A) tail length distributions and 5′ truncation species at single-molecule depth. Consensus-based bioinformatic corrections mitigated homopolymer errors, achieving >99% sequence accuracy. Crucially, analytical integrity metrics correlated directly with biological function: a reduction in intact mRNA content corresponded to a ~50% decrease in Cas9-mediated gene knockout efficiency.
By consolidating identity, integrity, 5′ capping, and poly(A) characterization into a single 4–8-hour assay, this NDRS-based MAM workflow establishes a clear analytical functional link while significantly reducing analytical burden. These findings support the platform's implementation for in-process monitoring, comparability assessments, and QC release testing of next-generation RNA therapeutics.
Abstract As RNA-based vaccines and CRISPR-Cas9 gene-editing therapeutics advance toward commercial manufacturing, robust analytical strategies are required to monitor Critical Quality Attributes (CQAs). Conventional USP-recommended workflows rely on multiple low-throughput, orthogonal assays for identity, integrity, 5′ capping, and poly(A) tail characterization, introducing significant operational complexity in CMC environments. Here, we evaluated Nanopore Direct RNA Sequencing (NDRS) as a single, consolidated Multi-Attribute Method (MAM) to streamline these measurements within cell and gene therapy (CGT) workflows.
We cross-validated NDRS against established orthogonal methods (CGE, IP-RP-HPLC, and MS) using therapeutic mRNA and 100-nt sgRNAs to evaluate sequence accuracy, truncation species, poly(A) tail heterogeneity, and CQA concordance. To enable this, we implemented an enzymatic workflow for direct 5′ cap quantification alongside optimised ligation strategies for full-length sgRNA identity.
NDRS demonstrated strong concordance with orthogonal methods while offering superior molecular resolution. Unlike bulk-averaging techniques, NDRS resolved poly(A) tail length distributions and 5′ truncation species at single-molecule depth. Consensus-based bioinformatic corrections mitigated homopolymer errors, achieving >99% sequence accuracy. Crucially, analytical integrity metrics correlated directly with biological function: a reduction in intact mRNA content corresponded to a ~50% decrease in Cas9-mediated gene knockout efficiency.
By consolidating identity, integrity, 5′ capping, and poly(A) characterization into a single 4–8-hour assay, this NDRS-based MAM workflow establishes a clear analytical functional link while significantly reducing analytical burden. These findings support the platform's implementation for in-process monitoring, comparability assessments, and QC release testing of next-generation RNA therapeutics.
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Kamalakar Chatla, Technical Development Principal Scientist, Genentech:quality(80))
Tina de los Reyes, Associate Director, Biopharma Specialists, Oxford Nanopore TechnologiesTina de los Reyes is a biopharma commercial leader with over a decade of experience advancing genomics technologies across the life sciences industry. As Associate Director of Biopharma Sales, Americas at Oxford Nanopore Technologies, she partners with pharmaceutical and biotechnology organisations to accelerate innovation through next-generation sequencing and multiomics solutions. Her background combines scientific expertise with a track record of driving growth and technology adoption.
