AAV Gene Therapy Product Characterization using Next-Generation Sequencing (NGS)

Next-generation sequencing (NGS) technologies have transformed gene therapy research, development, and manufacturing. Not only has it helped identify genomic biomarkers of rare diseases and disease heterogeneity within a population, but it has also helped characterize adeno-associated virus(AAV)-based gene therapies in pre-clinical development and assess critical quality attributes (CQAs), such as capsid content and viral titer, of manufactured AAVs.¹ 

However, widely used methodologies used to characterize drug candidates or assess CQAs need to be improved, either in throughput or accuracy.  For gene therapies to reach their full potential, ensuring their quality, stability, consistency, and comparability is crucial. NGS is an essential technique for assessing capsid content and the presence of non-therapeutic capsids, which can impact the potency, purity, and safety of AAV gene therapy candidates and manufactured products. 

Below, we discuss the different types of NGS, how it’s enabling gene therapy development, and how the future of NGS is shaping the gene therapy regulatory approval landscape.

What is Next Generation Sequencing (NGS)?

NGS is a high-throughput, massively parallel sequencing technique capable of determining the sequence of DNA or RNA. Several different NGS platforms are widely used in the life science industry that have helped to birth the fields of genomics, the study of the structure, function, and mapping of genomes, and transcriptomics, the study of the complete set of RNA being expressed in cells and tissues.²‍

NGS-based genome analysis can be categorized into three primary approaches: Whole genome sequencing (WGS), whole exome sequencing (WES), and targeted sequencing.³ WGS provides comprehensive coverage of the entire genome, including coding, non-coding, and regulatory regions. In contrast, targeted sequencing focuses on specific genomic regions, with WES specifically targeting protein-coding exons. 

NGS-based transcriptome analysis is performed using RNA-seq, where cellular RNA is used to construct a complementary DNA (cDNA) library, which is then used for sequencing.³ Total RNA-seq analysis looks at most of the cellular RNA (ribosomal RNA has been depleted), whereas targeted RNA-seq looks at a panel of RNAs of interest. 

NGS techniques have recently been categorized into two distinct sequencing principles; Short-read sequencing, which generates reads up to 600 bp and long-read sequencing, which produces reads over 10 kb.  While more accessible due to cost and industry-wide adoption, short-read sequencing has limitations, such as amplification bias and the inability to recognize certain genomic features. 

‍Long-read sequencing addresses these limitations, offering improved accuracy and applicability, particularly in identifying genomic targets for gene therapy, characterizing gene therapy candidates, and assessing CQAs for manufacturing runs.⁴ 

Why is NGS Relevant for AAV Gene Therapy Characterization?

In early gene therapy development and later manufacturing stages, there is significant QC that needs to be performed on AAV gene therapies to ensure that drug products are safe and efficacious. Recombinant AAVs (rAAVs) go through a complex production and purification process to remove potentially harmful cellular and viral impurities before they can be administered to patients.

Among these hazardous impurities are residual cellular or non-therapeutic viral DNA, which can produce immunogenic proteins and RNAs or activate the innate immune system via Toll-like Receptor 9 (TLR9).^5,6 ‍

NGS can be used to characterize these DNA contaminants during early- or late-stage AAV production runs. However, widely used short-read sequencing platforms are insufficient at sequencing high GC regions and inverted terminal repeats (ITRs) in the rAAV vectors, providing an incomplete picture of the impurities produced during biomanufacturing runs.⁷ ITRs are prone to mutation, which can impact the packaging efficiency of rAAVs, making them essential to characterize as part of AAV-based gene therapy development.⁸ 

For that reason, long-read sequencing techniques are more suitable for rAAV characterization applications, including drug product lot release and early-stage biomanufacturing runs. Techniques such as AAV-genome population sequencing (AAV-GPseq) provide the throughput and accuracy needed for gene therapy development and can resolve non-therapeutic rAAV impurities, such as truncated genomes.⁹ 

How NGS is Shaping the Game in AAV Gene Therapy Regulatory Approvals

The FDA has long been gearing up for alterations in the regulatory procedures for gene therapies. They have been exploring various methods of data submission, ensuring the relative safety of AAVs, and verifying the production of full capsids rather than empty ones. 

At ASGCT 2024, there was a notable emphasis on a regulatory landscape as Peter Marks, director of the FDA's Center for Biologics Evaluation and Research, addressed current manufacturing and regulatory barriers to gene therapy products to market. 

The FDA recognizes the potential of NGS technologies to enhance drug quality, safety, and efficacy assessments demonstrating the purification process and effectively eliminating impurities.  This paradigm shift is evident in integrating NGS guidelines into preliminary regulatory frameworks, most notably by the International Council for Harmonisation (ICH) Q5A(R2) guideline and Bespoke Gene Therapy Consortium Regulatory Playbook. By incorporating NGS methods for AAV-based gene therapy characterization regulators aim to foster innovation while ensuring robust evaluation standards and accelerated regulatory approval processes.

The Future of AAV Gene Therapy Characterization with NGS

To reach the full potential of NGS data in AAV gene therapy development, AI and other in silico tools must be deployed. Form Bio’s newest product, in silico Assays^TM accelerates the process from gene-of-interest (GOI) to BLA, by exploring full AAV vector designs and identifying the best candidates before in vitro screening. 

Moreover, in collaboration with our industry and academic partners, we have recently developed and published a standardized nomenclature and reporting for PacBio HiFi sequencing and analysis for rAAV gene therapy vectors.¹⁰‍

As a proof-of-concept, we released our biological validation report on our AI predictions for 42 candidate constructs. You’ll find details on our AI-optimized designs, reducing the accumulation of truncation peaks and increasing full-length AAV production by 28%. 

AI Disclosure: The image was generated with AI image generator MidJourney.