Advancing Variant Detection for Gene Therapy Development

June 13, 2025
Genedata

AAV-based gene therapies are steadily entering the market to treat debilitating diseases.¹ However, manufacturing challenges still limit the efficient scaling of outputs while maintaining quality and reducing costs. These challenges include product heterogeneity and contamination which reduce quality, safety, and efficacy.² During recombinant AAV (rAAV) production, mutations can occur anywhere in the sequence leading to altered protein levels, packaging failures, or a non-functional protein that fails to induce the desired therapeutic effect.³ As these mutations can occur at a very low frequency, it is crucial to use a sensitive assay for sequence identity and integrity testing of the transfer plasmid and packaged rAAV DNA. This can be achieved through variant detection using the powerful multi-attribute method (MAM), next-generation sequencing (NGS), which is unbiased and highly sensitive. Analyzing NGS data requires a robust bioinformatics pipeline that aligns sequencing reads to a reference sequence and accurately detects mutations using a variant calling algorithm.

Successful variant analysis during gene therapy development requires all stakeholders within an analytical/process development team to make the right go/no-go decisions from NGS data. They need access to the appropriate sample-associated product- and process-related data, along with the resulting insights, to efficiently collaborate on quality controlling gene therapy products. While outsourcing NGS-based assays and data analysis has been an option, relying on service providers such as CROs generates high costs, leads to project delays, and limits control. With regulatory guidelines such as ICH Q5A(R2) encouraging the use of NGS as an alternative to in vitro and in vivo animal assays⁴, it is now even more important for companies to implement compliant NGS-based assays in-house. Genedata Selector^®, the advanced solution for in-house NGS data management and analysis, equips key stakeholders of analytical/process development to conduct NGS-based assays compliantly for accelerated gene therapy development.

Address Key Stakeholder Gene Therapy Development Needs

The key to success is equipping interdisciplinary teams to implement a fit-for-purpose solution that addresses the diverse needs of different stakeholders. Successful implementation requires domain expertise, resources, and seamless communication to implement a timely solution. The interdisciplinary team of Genedata ensures a smooth implementation process by complementing internal resources, expertise, and the leadership team.

Executive Leaders

In the final stage of gene therapy development, executive leaders are responsible for verifying that all operations are compliant, key milestones are met, and the product is free of defects. Genedata Selector empowers leadership to achieve these milestones, reducing dependency on service providers. With capabilities for ensuring GMP compliance, Genedata Selector supports leaders by introducing innovative technologies and automating result generation so they can replace traditional assays and equip their teams to perform NGS assays in-house and in a cGMP-validated environment. They benefit from multiple readouts with a single assay, achieve shorter turnaround times, and generate results that are immediately suitable for regulatory submissions. Analytical strategy and process development leaders seeking to advance CMC process development and advanced modality characterization use Genedata Selector to leverage NGS for method development and as a MAM to simultaneously assess several quality attributes.

Analytical Scientists

As operators of NGS-based assays, analytical scientists require clear, actionable insights to determine which samples are likely to be successful in production. After running experiments, they analyze the resulting data to identify which samples should progress in the development and manufacturing process. This involves inspecting for any impurities that could impact vector efficacy, safety, and quality, for example:

• Does the vector contain the transgene of interest?
• Are there any vector variants or truncations?
• Does the capsid contain any impurities (e.g., nucleic acids)
• Are there any adventitious agents present?

Often, progress can be hindered as they may require support from bioinformaticians to generate insights from NGS data. With Genedata Selector, analytical scientists can independently leverage workflows pre-built and approved by their bioinformatician team members. These easy-to-use, point-and-click Playbooks automate complex NGS analyses for efficient result generation. Results are displayed using a unique traffic light system that indicates which samples have met or missed pre-defined thresholds. If a sample fails during variant detection, scientists can investigate the result in detail using the genome viewer of Genedata Selector. This allows a deep dive into the genome and sequence read level to identify the location and type of mutations detected. This simplifies and accelerates results interpretation and troubleshooting. All experiments run in the Playbook are tracked in timestamped reports which contain the full chain of custody documenting all user data-related activity. The results are also linked to samples and any associated metadata. This makes it easier to report insights to the analytical lead or the quality assurance team for submission to regulatory authorities.

Analytical Leads

Throughout gene therapy development, the analytical lead requires a global overview of the different samples being analyzed and the results generated from them. They need to be able to quickly gain insights regarding critical quality attributes as well as understand and assess the various processes undertaken. Monitoring processes enable them to identify whether a methodological change or a different technological solution is required for process optimization. By leveraging Genedata Selector’s comprehensive overview of results, analytical leads remain informed of sample progress throughout the development cycle, without spending excessive hours navigating cryptic reports.

Quality Assurance Experts

Quality and GxP validation are central to the development of gene therapies and the main priorities of Quality Assurance (QA) Experts. To ensure the end product meets the required standards, QA experts oversee the entire process to verify compliance with regulatory requirements. From sample registration and wet lab experimentation to sequencing, data processing, analysis, and reporting, they develop standard operating procedures (SOPs) and aggregate documentation regarding the entire assay. QA experts also ensure their teams follow protocols that meet the requirements of a GxP-validated environment. This involves having the necessary documentation, which contains the full chain of custody and evidence that all computerized systems are suitable and performant for the use cases involved in the development process.

Genedata Selector helps quality assurance experts ensure a GxP-compliant environment by:

• Governing user activities through assigning specific roles and permissions with user access controls, enabling only authorized users to access certain data or modify information relevant to regulatory submissions
• Standardizing NGS data processing and analysis while ensuring reliability and reproducibility using automatic workflows
• Delivering easily customizable automatically generated reports on the platform and actions performed that can be exported for archiving in compliance with regulatory requirements
• Tracking all actions performed in a transparent and complete audit trail

To further simplify the process of GxP validation, Genedata provides standard off-the-shelf documentation to help customers meet regulatory requirements. This includes IQ protocols, end-user and system administration documentation, configuration specifications, testing reports, and maintenance release notes.

Bioinformaticians

Bioinformaticians can play a key role in NGS-based quality control of gene therapies. Based on an organization’s available resources, it can opt to utilize its internal bioinformatics team, the scientific services of Genedata, or a collaborative approach that combines both. Bioinformaticians develop methods for data processing and analysis, so results can be interpreted by analytical scientists to detect deviations in quality, safety, and efficacy. Typically, this involves building NGS data processing and analysis pipelines that their team members can use. They also need to ensure that everything they do is tracked and the data lineage is maintained so that the entire process is transparent. Genedata Selector gives full control to bioinformaticians, equipping them to easily build workflows to standardize data processing and analysis. Designed with built-in logic for automated NGS analyses, Genedata Selector also provides out-of-the-box workflows that users can use to quickly get started — these are configurable to user experiments, data types, and samples. Once satisfied with the performance and outputs of their designed workflows, bioinformaticians can “lock” and convert them into guided user interfaces called “Playbooks”. Team members can run these by simply selecting the data source, genomic references, and the viral database. They guide users through a variety of analyses, such as mutation profiling (for variant analysis), adventitious agent detection, and plasmid contamination checks. Rather than inefficiently using different tools, bioinformaticians make use of a single unified interface in the Genedata Selector platform to intuitively keep track of all workflows and efficiently collaborate with team members.

Unlock Return on Investment

Leveraging Genedata Selector for in-house NGS-based assays can yield significant time and cost savings. It eliminates reliance on service providers, granting full control and increased sample throughput — from starting material and in-process control to lot release testing. For example, when operating NGS on a small scale, rather than waiting 10 weeks for results from CROs, results can be obtained in just 1.5 weeks. When operating in a scale-up environment, where there can be multiple product lines and, e.g. up to 20 various reports produced for submission to regulatory authorities, rather than waiting 12 weeks for results, in-house NGS can deliver results in just 1 week. In this same environment, costs can be reduced by a factor of 5.5, saving pharmaceutical companies an impressive 80% annually compared to outsourcing. With Genedata Selector, the bottleneck of which sample to send is also solved. NGS-based analyses can be done efficiently in-house at any time, for any project and any modality.

Gene therapy development is an intricate process due to the complex biological nature of the various viral-vector-based therapy components. Unwanted product or process alterations may hinder the therapeutic from producing the intended outcome or could result in adverse effects. This makes it essential to perform variant detection and define critical quality attributes up front for monitoring quality throughout the development cycle. With the regulatory push for NGS adoption, it is even more important that companies have a solution that enables them to reap the benefits of NGS while complying with regulatory guidelines. Genedata Selector not only provides this solution, but Genedata’s scientific consultants offer support for assay definition, building workflows, bioinformatics expertise, and GMP validation. Genedata’s dedicated IT team ensures seamless integration of the software into IT infrastructure and with any internal legacy systems. 

To learn more about Genedata Selector and its support for variant detection during gene therapy development, request a demo.

^{Marie-Ange Kouassi, Ph.D., Scientific Marketing Specialist, Genedata AG
Qiu Ruan, Ph.D., Scientific Consultant, Genedata Inc.
Devon Ryan, Ph.D., Head of Scientific Consulting, Genedata AG
Marcell Veidner, Business Development Manager, Genedata  AG}

References
 

1. Liu et al. Adeno-associated virus therapies: pioneering solutions for human genetic diseases. Cytokine and Growth Factor Reviews (2024).
2. Fuentes C et al. Beyond empty and full: understanding heterogeneity in rAAV products and impurities. Dark Horse Consulting Group Whitepaper (2023).
3. Tam Tran N et al. AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity. Mol Ther Methods Clin Dev (2020).
4. European Medicines Agency. ICH Q5A(R2). Guideline on viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (2023).