Navigating Quality, Safety, and Analytical Testing Frameworks for Viral Vector–Based Gene Editing Therapies

Abstract

Viral vectors have become the backbone of delivery strategies in modern gene therapy, offering biologically robust means of transporting therapeutic transgenes and genome-editing machinery, most notably CRISPR-Cas nuclease systems, into target cells. Moving these intricate macromolecular assemblies from bench-scale research into human clinical application, however, is far from straightforward: it raises substantial analytical, manufacturing, and safety questions that cannot be resolved through conventional characterization alone. Meeting international regulatory expectations demands the establishment of harmonized, multi-layered quality control systems spanning every stage of vector production. This review takes a critical look at the analytical approaches currently used to characterize viral delivery platforms, with particular attention to recombinant adeno-associated viruses (rAAV), lentiviruses, and adenoviruses. We examine the core quality attributes that define these products—genome titer determination, ratios of physical to infectious particles, discrimination between empty and full capsids, and quantification of residual host-cell proteins and host-derived nucleic acids—and consider how well existing assays actually capture these properties in practice. Beyond product characterization, we turn to nuclease-associated safety concerns, reviewing the methods available for detecting unintended double-strand breaks at off-target sites, structural rearrangements within the genome, and the emergence of replication-competent viral variants. By weighing what current technologies can reliably achieve against their practical limitations, this article aims to provide a working framework for verifying the potency, genomic integrity, and clinical safety of vector-based gene therapies—one intended to be useful both to laboratories developing these products and to those responsible for regulating them.