Artificial Intelligence

Solving Gene Therapy Product Development Challenges Through AAV Analytical Method Development

Learn how to overcome product quality challenges when moving from pre-clinical to commercialization of AAV gene therapy.

Jill Roughan, PhD

Jill Roughan, PhD

October 17, 2023

Solving Gene Therapy Product Development Challenges Through AAV Analytical Method Development

So often in biomedicine, we celebrate the two opposite ends of the therapeutic pipeline: the discovery and the phase III clinical readout. But, what frequently goes underappreciated is what happens between the beginning and end of drug development.

In gene therapy product development, these steps – viral vector analytical development and standardization, production optimization, quality control, etc. – transform a candidate into an efficacious and safe medicine.   This thorough product characterization analysis also plays a pivotal role in providing a comprehensive and well-documented account of gene therapies. This meticulous approach not only facilitates regulatory compliance but also establishes a clear and traceable record of the therapeutic development journey that will help determine which patient will benefit the most.

In this blog, we focus on critical quality attributes (CQAs) for adeno-associated virus (AAV)-based therapies and how we’re helping gene therapy developers assess them with innovative and proven technologies.

Prioritizing AAV Analytical Methods Early in R&D

The first step in AAV-based gene therapy development is preclinical research and development, which focuses on target validation, vector design, and preliminary safety and efficacy testing. Early on, safety testing is particularly important to support moving to IND and first-in-human trials. From there comes first-in-human clinical testing to ensure preclinical efficacy and safety results translate into humans. With promising results on efficacy and safety in a phase III trial, the hope for drug developers is that regulatory bodies find their data convincing and they can move into the commercialization of the new gene therapy.

With AAV-based gene therapies, there has been an increased focus by regulatory bodies on ensuring product safety. Impurities generated during AAV production can increase safety risks, so regulatory bodies are working to solidify AAV analytical requirements with specific tests and assessments around critical quality attributes (CQAs).1 Because safety issues have derailed clinical trials before, gene therapy developers are prioritizing AAV analytical development and biomanufacturing in the preclinical stage to avoid costly rework down the road. 

Assessing Critical Quality Attributes for AAV Gene Therapies

The FDA hasn’t issued official regulatory guidance yet regarding gene therapy CQAs, but for AAV-based therapies, several have emerged as essential.2 Content ratio (or the number of full capsids to partially filled or empty capsids) is one the most important CQAs, as empty and partially filled capsids represent the most common product-related impurity in AAV production and can lead to adverse events in clinical trials.3 

A related CQA, viral titering, is focused on quantifying the number of total capsids (regardless of content) or endonuclease-resistant (and thus, encapsidated) genomes. While both are important, genome titering is used to determine potency and, therefore, is used to determine clinical dosing. 

Both CQAs discussed above require a validated assay for detecting encapsidated full-length or partial viral vector and non-vector DNA. Methods such as qPCR and ddPCR have been the gold standard, but recently, long-read sequencing has shown significant promise.3 It enables the characterization and quantification of complete genomes, partial genomes, and non-vector genomes within AAV capsids.4 In addition, reads can span the entire 5kb AAV genomes, thus reducing the need for complex bioinformatics pipelines used for processing and analyzing short-read sequencing data.

Shaping the Future of AAV Gene Therapy Product Development

Form Bio provides powerful gene therapy product characterization and analytic development capabilities. Using our PacBio’s HiFi long-read sequencing platforms, we can help assess the quality of bioreactor runs to gain a deeper understanding of product contents, quality, and potential risks early in pre-clinical development stage. HiFi reads provide base-level resolution with 99.9% single-molecule read accuracy. Read consists of a DNA sequence, which includes the entire ITR-to-ITR target vector sequence in either (+) or (-) polarity.

Once your research has demonstrated effectiveness in an animal or iPSC model, we collaborate with you to enhance the viral vector for biomanufacturing by developing a custom tailored viral construct comparison report.  By leveraging the data from your biological laboratory experiments, our in silico platform, FORMsightAI, has the capability to predict your gene therapy payload before investing any resources in bioreactor experiments. This analytical and product development approach empowers you to take proactive measures in overcoming obstacles as you embark on Investigation New Drug (IND) enabling studies.

AI Disclosure: Feature image was generated by an AI image tool MidJourney.

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  1. Toxicity Risks of Adeno-associated Virus (AAV) Vectors for Gene Therapy (GT). FDA website Accessed November 2, 2023. Published August 18, 2021.
  2. FDA Clinical Trial Guidance For AAV Gene Therapy | Form Bio. Accessed September 13, 2023. Published March 15, 2023.
  3. Gimpel AL, Katsikis G, Sha S, et al. Analytical methods for process and product characterization of recombinant adeno-associated virus-based gene therapies. Mol Ther - Methods Clin Dev. 2021;20:740-754. 
  4. Tran NT, Heiner C, Weber K, et al. AAV-Genome Population Sequencing of Vectors Packaging CRISPR Components Reveals Design-Influenced Heterogeneity. Mol Ther Methods Clin Dev. 2020;18:639-651. 

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