Resolving the Unresolvable: Nanopore Sequencing as a Comprehensive Quality Control Platform for Gene Therapy Vectors

Area of Science:

• Biotechnology
• Genomics
• Molecular Biology

Background:

• Cell and gene therapy (CGT) manufacturing faces challenges with traditional quality control (QC) methods, creating a "black box" for vector characterization.
• Legacy assays like Sanger and short-read sequencing struggle with complex nucleic acid structures crucial for CGT efficacy.
• Adeno-associated virus (AAV) inverted terminal repeats, lentiviral recombination, and mRNA poly(A) tails require advanced analytical techniques.

Purpose of the Study:

• To review the application of Oxford Nanopore Technologies' long-read, native single-molecule sequencing across the CGT lifecycle.
• To highlight how nanopore sequencing overcomes limitations of traditional assays for vector quality control.
• To assess the potential of nanopore sequencing as a validated lot-release method in CGT manufacturing.

Main Methods:

• Review of existing literature and applications of nanopore sequencing in CGT.
• Analysis of nanopore sequencing capabilities for plasmid DNA, viral vectors (AAV, lentivirus), and mRNA therapeutics.
• Discussion of advanced techniques like adaptive sampling for impurity enrichment and epigenetic profiling.

Main Results:

• Nanopore sequencing confirms plasmid DNA identity and reveals structural heterogeneity.
• It serves as an integrity assay for viral vectors, distinguishing full genomes and detecting sequence-resolved impurities.
• Direct RNA sequencing profiles mRNA poly(A) tail length and base modifications in a single assay.

Conclusions:

• Nanopore sequencing provides high-resolution, risk-based QC for CGT, directly addressing complex structural attributes.
• It has the potential to transition from an orthogonal tool to a validated lot-release method, improving CGT manufacturing efficiency.
• Further assessment of accuracy, compliance, and regulatory pathways is needed for widespread adoption.