Gene therapy is an expanding area of medicine whereby specific medical conditions are treated at the DNA level through direct manipulation of the faulty gene. This has proven a viable approach through the use of viral vectors, serving as DNA delivery systems, that themselves have no negative pathogenicity to humans, whilst still being infectious and able to deliver the appropriate DNA. Conditions for which these gene therapy products are currently approved include spinal muscular atrophy and inherited retinal dystrophy with many, many more products under development.
Regulatory guidelines have quite appropriately placed strong emphasis on the necessity of confirming the correctness of the DNA to be delivered and that the particles are correctly packaged and the product is safe for use. However, it is also a requirement that gene therapy vectors are analyzed, since this constitutes a component of the drug (1). These vectors are, after all, multicomponent systems where all the individual components have to be correctly formed for maximal activity. The gene therapy guidelines do state that the structure of the drug substance must be elucidated including “…primary, secondary, or higher order structure; post -translational modifications…”. Thus, there is also a need for an investigation into the protein structures within the viral vector. This requirement to assess the structure of the viral proteins is strengthened in light of the fact that modifications to the protein coat, namely deamidation of asparagine residues, has been associated with loss of transduction activity for adeno-associated virus vectors (2). In other words, if there is an issue with the structure of the delivery vehicle then either the DNA may not get to where it needs to be or may do so with a lower efficiency.
One of the most, if not the most, common vector used in gene therapy is the Adeno Associated Virus (AAV) vector, because of its lack of pathogenicity. Recombinant adeno associated viruses are small viruses that small virus package the DNA within an icosahedral protein coat composed of 3 related proteins identified as VP1, VP2 and VP3. The nature of the capsid assembly process means that these proteins are present in non-stoichiometric but precise ratios. These three proteins are products of the same AAV DNA region of the vector genome, but are the result of alternative splicing of that DNA. All three proteins are what could be considered as mid-sized, having masses in the region of 60 to 90kDa. The structure of these proteins indicates the presence of various potential N-glycosylation sites as well as numerous Cysteine residues capable of forming disulfide bridges. Furthermore, since they are related proteins, varying in their N-terminal region as a result of the AAV ssDNA alternative splicing and transcription, identification of the N- and C-termini of the three proteins is important.
When we consider the various structural aspects of the VP proteins that need to be investigated – disulfide bridges and the associated higher order structure, N and C-termini, glycosylation and post translational modifications (PTMs) – it is clear that such an assessment falls within the ICH Q6B guidelines, which is the accepted guidance document for characterization of protein biotherapeutics. The use of mass spectrometric techniques in conjunction with various on- and off-line liquid chromatographic techniques produces a detailed structural understanding of the VP coat proteins and their PTMs and is applicable to different AAV serotypes. Furthermore, the application of these techniques, or at least a subset of them, during production of the gene therapeutic can highlight if any deleterious events have occurred to the proteins resulting in, for example, increased deamidation which could give cause for concern with regards to efficacy of the product. Of course, since therapies derived from adeno associated viral vectors require that the protein and nucleic come together to produce correctly packaged and active particles, it is necessary to analyze the final, packaged product to assess for the degree of capsid filling. This can be performed by analytical ultracentrifugation, which can also be used as a mechanism of identifying fragments and aggregates in the sample alongside the technique of differential light scattering.
It is worth bearing in mind that ICH Q6B also covers process impurity analysis such as process residuals and Host Cell Proteins (HCPs). The gene therapy guidelines from the FDA recommends that these types of components are screened for in an impurity analysis (1).
1. Chemistry, manufacturing and control (CMC) information for human gene therapy investigational new drug applications (INDs) Guidance for industry. FDA 2020.
2.Giles, A.R., et al. (2018). Deamidation of amino acids on the surface of Adeno-Associated Virus capsids leads to charge heterogeneity and altered vector function. Molecular Therapy, 26, 2848-2862.
