Using next-generation sequencing to strengthen AAV genome integrity, identity, and impurity analysis

As gene therapy continues to advance, adeno-associated virus (AAV) has become one of the most important platforms for in vivo gene delivery. Because AAV vectors are complex biological products, their quality control requires more than traditional release testing. In addition to titer, purity, potency, and safety testing, developers increasingly need deeper information about vector genome integrity, sequence identity, and packaged nucleic acid impurities.

Next-generation sequencing, or NGS, offers a powerful tool for AAV characterization. It can provide high-resolution information on the intended vector genome, sequence variants, truncations, rearrangements, and unintended DNA species packaged into AAV particles. While NGS does not replace routine assays such as qPCR/ddPCR, ELISA, HPLC, capillary electrophoresis, or cell-based potency testing, it can serve as an important orthogonal method for advanced AAV quality assessment. Regulatory expectations for gene therapy CMC information emphasize the need to demonstrate product safety, identity, quality, purity, and strength, including potency, and NGS can support several of these areas when appropriately developed and validated.

NGS for AAV Genome Identity and Integrity

One of the strongest applications of NGS in AAV quality control is confirmation of vector genome identity and integrity. Recombinant AAV vectors should contain the intended expression cassette, including the promoter, transgene, regulatory elements, and polyadenylation signal, in the correct sequence and configuration. NGS can help confirm whether the packaged genome matches the designed construct and whether unexpected mutations, deletions, insertions, rearrangements, or truncations are present.

This is particularly important because AAV preparations may contain a mixture of full-length genomes, partial genomes, fragmented genomes, and unintended nucleic acid species. Genome integrity is increasingly recognized as an important CQA for AAV products because it can affect both therapeutic efficacy and safety.

Detection of Sequence Variants and Product Consistency

NGS can detect low-frequency sequence variants that may not be visible using conventional sequencing methods. These may include single-nucleotide variants, small insertions or deletions, recombination-derived sequences, or unexpected junctions. Detecting such variants helps confirm sequence consistency across production batches and supports comparability studies during process development, scale-up, or manufacturing site transfer.

However, NGS results must be interpreted carefully. Low-frequency variants can arise from true product heterogeneity, PCR amplification bias, sequencing errors, sample preparation artifacts, or bioinformatics pipeline settings. Therefore, NGS-based variant analysis should include appropriate controls, validated workflows, and clearly defined acceptance criteria.

Analysis of Packaged DNA Impurities

Another important use of NGS is the identification of unintended DNA packaged within AAV particles. These impurities may include host-cell DNA, plasmid backbone sequences, helper plasmid fragments, rep/cap-related sequences, or other process-derived nucleic acids. Long-read sequencing and short-read sequencing can both contribute to impurity profiling, although they provide different types of information.

Recent studies using long-read sequencing have shown that residual non-transgene DNA can be detected in recombinant AAV preparations, including host-cell and plasmid-derived DNA species. Such analyses can support a more data-based assessment of co-packaged DNA impurities and their potential safety relevance.

It is important to clarify that NGS is not used to directly measure protein impurities. Host-cell proteins, residual nucleases, and other protein contaminants are typically assessed by methods such as ELISA, HPLC/UPLC, capillary electrophoresis, SDS-PAGE, mass spectrometry, or other protein analytics. NGS is most useful for nucleic-acid-related characterization.

Relationship Between NGS and Titer Testing

NGS can provide detailed sequence composition data, but it is not usually the primary method for routine AAV titer determination. Vector genome titer is typically measured by qPCR or digital PCR, while capsid titer may be measured by ELISA, HPLC-based methods, mass photometry, or other biophysical assays. NGS can complement these assays by showing what DNA species are present and whether the intended genome is intact, but dose calculation generally still relies on validated quantitative titer methods.

In this way, NGS should be viewed as a complementary characterization tool rather than a standalone replacement for established titer assays.

NGS and Functional Assessment

NGS can support functional interpretation, but it does not directly replace infectivity or potency assays. AAV infectivity and potency are usually evaluated using cell-based assays that measure transduction, transgene expression, enzymatic activity, genome editing activity, or another mechanism-specific functional readout.

In some advanced applications, NGS may be used in cell-based studies to quantify delivered vector genomes, barcode distributions, or cell-type tropism in complex systems. However, functional activity still requires biological confirmation. Therefore, NGS data should be integrated with infectivity, potency, biodistribution, and expression data rather than interpreted alone.

Future Outlook and Challenges

NGS-based AAV quality control is expected to become increasingly important as AAV products become more complex. Engineered capsids, dual-vector systems, self-complementary AAV, large or difficult-to-package genomes, and high-dose systemic delivery all create a greater need for detailed molecular characterization.

At the same time, NGS presents several challenges. Sample preparation, library construction, sequencing depth, read length, amplification bias, data analysis pipelines, and reference database quality can all influence results. Standardization, automation, validated bioinformatics workflows, and appropriate reference materials will be essential for broader adoption in regulated AAV development.

NGS-based quality control represents an important advancement in AAV vector characterization. By providing high-resolution insight into genome identity, sequence integrity, variant profiles, and packaged DNA impurities, NGS helps developers better understand AAV product quality and manufacturing consistency.

However, NGS should be used as part of an integrated analytical strategy. When combined with qPCR or ddPCR, capsid titer assays, purity testing, empty/full capsid analysis, genome integrity assays, and cell-based potency testing, NGS can significantly strengthen the overall quality control package for AAV gene therapy products.