Standardisation in the Analytical Characterization of Adeno-Associated Virus (AAV) Vectors.

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

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.

Leveraging host cell modulators of adeno-associated vectors transduction to tailor viral biodistribution.

Clinical successes in gene therapy using adeno-associated vectors (AAV) are offering a hopeful path toward the correction of several monogenic disorders. At its core, AAV gene delivery relies on multiple interactions between the capsid or vector genome and numerous host cell factors, a complex process that remains incompletely understood. Whether different serotypes similarly hijack the intracellular machinery in a cell, and whether these processes are necessarily conserved across species, are not known. To identify host factors enabling or preventing transduction across species or serotypes, CRISPR/Cas9 whole-genome knockdown screens were conducted in mouse (AML12) and human (HuH-7) cell lines before transduction with AAV2 or AAV9. Key common entry factors (AAVR, GPR108) were confirmed, with many other serotype- and/or species-specific host cell modulators identified. Interestingly, while serotype-specific differences were observed for successful transduction of a specific cell line, species-specific divergences were even more striking when comparing results across murine and human cells for the same serotype. Leveraging these data, we developed a novel approach to detarget liver transduction by transiently downregulating the expression of key entry factors in this tissue using GalNac-siRNAs prior to AAV9 administration. Such manipulation not only blunted hepatic transduction but also redirected the vector to other transduction-permissive tissues.

A novel adeno-associated viral vector derived from human spleen isolate AAV.hu.S17

Adeno-associated viruses (AAVs) are widely used vectors for gene therapy owing to their nonpathogenic nature, low immunogenicity, and ability to support long-term transgene expression. However, the tissue tropism and immune response of AAVs are strongly dependent on their capsid serotype. Although hundreds of naturally occurring AAV variants have been isolated, many remain unvectorised and poorly characterized. Therefore, the discovery and development of previously uncharacterised native serotypes retain valuable. Here, we generated a novel recombinant AAV vector rAAV.hu.S17, derived from the human spleen isolate AAV.hu.S17, and systematically evaluated its capsid features, in vitro transduction, and in vivo tissue tropism. Sequence analysis showed that AAV.hu.S17 is closely related to AAV2 and AAV3, and shares 90.9% amino acid identity to AAV2 across the capsid proteins. High-titer vector production was achieved using the triple-plasmid system, with rAAV.hu.S17 yielding approximately 2.68-fold more vector than that of rAAV2. Although rAAV.hu.S17 showed ∼20-fold lower transduction in HEK293T cells than rAAV2 in vitro, it mediated higher neuronal transduction in the mouse primary visual cortex (V1) in vivo. Following intravitreal injection into C57BL/6 mice, rAAV.hu.S17 preferentially transduced photoreceptors in the outer nuclear layer (ONL), with minimal transduction of the inner nuclear layer (INL) and the ganglion cell layer (GCL). Collectively, these results demonstrate that rAAV.hu.S17 exhibits transduction characteristics distinct from those of rAAV2 in both the brain and retina. This study provides the first vectorization and biological characterization of the previously uncharacterized human AAV isolate AAV.hu.S17 and expands the repertoire of naturally occurring AAV capsids available for future biological investigation and vector engineering.

Combined Plasmid Redesign and Transfection Optimization Significantly Increases Upstream AAV Titers While Maintaining Vector Quality and In Vivo Potency

A high manufacturing cost of goods (CoG) remains a critical barrier to the broad clinical adoption of gene therapies and is driven in part by limited productivity in adeno-associated virus (AAV) manufacturing. Here, we report an optimized AAV production process developed to markedly increase upstream titers while preserving vector quality and potency. The process combines a redesigned plasmid system, an optimized plasmid ratio, and a novel synthetic transfection reagent and was benchmarked against a conventional triple-plasmid/PEI MAX workflow. Across multiple AAV capsids and independent production runs, the optimized process reproducibly increased crude harvest titers by approximately 10- to 33-fold relative to the standard process, while maintaining key vector quality attributes. Notably, within the detection limits of the assay, rcAAV was undetectable at 1 × 1010 vg input with the optimized process, whereas the conventional triple-plasmid (with native Rep-Cap sequence)/PEI MAX workflow remained rcAAV-positive under identical conditions. Importantly, the in vivo potency was comparable to that of vectors produced by the conventional process. These results position our optimized AAV production process as a promising strategy to materially reduce AAV manufacturing CoG per patient.

The Characterization of AAV Capsid Individual VP-Specific Charge Heterogeneity Using a High-Throughput Reduced Denatured iCIEF–Western Method

In recent years, advancements in gene therapy have highlighted the important role of adeno-associated viruses (AAVs) due to their favorable characteristics, such as low immunogenicity compared to other viral vectors, e.g., lentivirus and HSV, and the ability to maintain gene expression in a variety of tissues. However, the production of recombinant AAVs in biological systems can lead to variability in the biophysical properties of viral capsid proteins, primarily due to post-translational modifications (PTMs) and cleavage events during downstream processing and storage. A critical quality attribute of AAV capsids is charge variant heterogeneity, which is significantly influenced by PTMs like deamidation, phosphorylation, acetylation and glycosylation. These modifications can impact the safety and efficacy of the viral vectors. The traditional imaged capillary isoelectric focusing (iCIEF) method, which uses absorbance or fluorescence detection, has been the primary choice for characterizing charge variants. However, it often lacks the sensitivity and resolution needed for AAVs’ charge variants. We introduce an optimized, highly sensitive capillary-based Western method to measure the apparent isoelectric point (pI) and detect charge heterogeneity at the individual VP protein level under reduced denatured conditions. This approach involves generating and purifying polyclonal antibodies to detect charge variants specific to the VP1, VP2, and VP3 proteins across different AAV serotypes, including AAV1, AAV8, and AAV5. This method is a valuable tool for the characterization of AAV capsids and can be utilized for the stability assessment and analysis of in-process and purified samples during process optimization from a charge heterogeneity perspective.

Site-Specific Chemical Conjugation of Recombinant Proteins onto the AAV Capsid.