It’s an exciting time in gene therapy, with the recent approvals of lifesaving products where few (or no) treatments used to exist. Like the rapid biologics boom in the 1980s, significant improvements are being made in manufacturing and testing.
Currently, recombinant adeno-associated virus (rAAV) is the most common viral vector used for the in vivo administration of gene therapies. Despite the demonstrated success of rAAV products, several instances of treatment-emergent serious adverse events (TESAEs) — some even leading to patient deaths — have occurred following intravenous administration at high doses. The field is in critical need of a more detailed product understanding in order to increase product safety without sacrificing efficacy.
rAAV are complex biologics composed of ~60 assembled viral proteins which form the protein capsid ‘shell’ along with an internal DNA sequence carrying the necessary elements for therapeutic effect. An innate level of heterogeneity (diversity, variation) in product and product impurities exists in rAAV within both the protein and the DNA sequence. Heterogeneity can impact safety, efficacy and product quality. Broadly, heterogeneity in rAAV can be classified into the following categories:
- Heterogeneity in encapsidated rAAV DNA sequence lengths
- Heterogeneity in rAAV DNA sequence identity and composition
- Heterogeneity in rAAV capsid protein integrity, stoichiometry and post-translational modifications
Understanding these product attributes and their role in product safety, efficacy and quality is critical, particularly because many rAAV products target rare indications with aggressive disease phenotypes. After a number of TESAEs occurred following treatment with high dose, systemic rAAV products, an FDA advisory committee met in September 2021 to discuss toxicity risks. While a singular root cause of the TESAEs was not identified, a total capsid dose correlation emerged.
The total capsid dose is typically calculated from both the overall dose (the number of ‘full’ capsids, measured in vg/kg) and the percentage of empty capsids (viral particles that don’t contain any DNA). While empty capsids are considered an impurity in rAAV products and likely play a role in immunogenicity, there are additional impurities and variants that can contribute to the quality profile, such as capsids that do not contain the correct length of DNA (aka ‘partial’ or ‘overpackaged’ capsids) or those that don’t contain the correct DNA sequence. Some of these are not routinely characterized in rAAV products and require greater analytical standardization across products.
Historical rAAV analytical methods have not been sensitive enough to quantify both a product’s heterogeneity and its product-related impurities. Methodologies for quantifying this heterogeneity are advancing rapidly, enabling a more comprehensive understanding of characteristics than has been historically possible. These advances provide the possibility of characterizing rAAV to a higher standard but have yet to be widely adopted. As the rAAV field advances and matures, sponsors may see increased standards aligning with harmonized guidelines, such as those described in ICH Q6B.
A subset of product heterogeneity currently receiving increased focus is capsids containing a shorter-than-intended DNA sequence. Termed ‘partial capsids,’ the identity of the included partial sequence can determine whether it contributes to the therapeutic effect. Advancements in Next-Generation Sequencing (NGS) methods paint a picture of sequence identity, size distributions and relative abundance within individual capsids. Additionally, NGS provides a window into product-related heterogeneity that can impact efficacy, efficiency and safety of rAAV products. While NGS and other analytical methods have provided drastic advancements in rAAV heterogeneity characterization, there are still significant limitations for implementation to be addressed.
While rAAV characterization has improved substantially, there is still a long way to go. In a recent white paper, Dark Horse Consulting sought to classify heterogeneity and impurities in rAAV products, discuss emerging technologies for characterization, and introduce how these attributes can improve product understanding and further define manufacturing strategy. With these advancements, there is an opportunity to gain greater understanding of rAAV product heterogeneity and related impurities, which will allow for better product quality understanding across the gene therapy industry — paving the way for the development of safer and more efficacious rAAV products.