ABSTRACT
The field of gene therapy has evolved and improved so that today the treatment of thousands of genetic diseases is now possible. An integral aspect of the drug development process is generating analytical methods to be used throughout clinical and commercial manufacturing. Enumeration and identification assays using genetic testing are critical to ensure the safety, efficacy, and stability of many active pharmaceutical ingredients. While nucleic acid-based methods are already reliable and rapid, there are unique biological, technological, and regulatory aspects in gene therapies that must be considered. This review surveys aspects of method development and validation using nucleic acid-based testing of gene therapies by focusing on adeno-associated virus (AAV) vectors and their co-transfection factors. Key differences between quantitative PCR and droplet digital technologies are discussed to show how improvements can be made while still adhering to regulatory guidance. Example validation parameters for AAV genome titers are described to demonstrate the scope of analytical development. Finally, several areas for improving analytical testing are presented to inspire future innovation, including next-generation sequencing and artificial intelligence. Reviewing the broad characteristics of gene therapy assessment serves as an introduction for new researchers, while clarifying processes for professionals already involved in pharmaceutical manufacturing.
