Genetic Modification of AAV Vector
It is well known that adeno-associated virus (AAV) as a gene delivery vector has been widely tested in human clinical trials. Creative Biolabs leverages the current scientific knowledge of AAV to perform reasonable capsid engineering design of AAV, such as point mutation of amino acid and insertion of peptide domain, to further regulate, enhance and optimize the performance of AAV vector, so that it can be better applied to the field of gene therapy.
Genetic Modification of AAV Vector
AAV is a small, single-stranded, DNA-containing, non-pathogenic parvovirus whose protein capsid is not encapsulated. AAV is an efficient and safe gene transfer vector. However, one obstacle inhibiting the development of AAV vectors is the natural tropism of the virus. For example, the primary attachment receptor for AAV type 2 (AAV-2) is heparan sulfate proteoglycan. This receptor is widely expressed in many cell types and can cause problems when transmitting genes that promote cell death, such as in anti-cancer gene therapy strategies. With the further development of gene therapy, it will be necessary to modify the AAV vector to improve cell transduction and evade the host immune response, thereby fully exploiting the potential of the vector system. To date, genetic capsid modification remains a viable alternative to enhance the transfer of AAV genes to target cells by replacing cellular receptors.
Figure 1. AAV 2 vector genome conformation. (Büning, 2019)
Methods of Genetic Capsid Modification for AAV Vector
The capsids of all AAV serotypes are icosahedron, consisting of 60 viral proteins (VPs) monomers. There are about 50 VP3 copies, 5 VP2 copies and 5 VP1 copies. Variable regions (VRs) between serotypes are different, leading to a change in serotype specificity of antibody-receptor binding. Due to their exposed position and their function in receptor binding, the protuberant loops formed by VRs are an ideal location for capsid modification, intended to redirect or extend AAV tropism. There are two common methods of genetic capsid modification.
By inserting peptide ligands into the AAV capsid at receptor binding sites, AAV-derived vectors can be re-target to other cell types. Typically, receptor-directed targeting of AAV has been achieved by inserting the peptide into AAV-2, the capsid region surrounding amino acid R588 (VP numbering) being the most commonly used insertion site. Compared with wild-type AAV-2, such targeting peptides are 60 times more prominent at the top of protruding capsid domains within the threefold spike region, which helps to interact with potential cell surface structures. In addition, we have successfully inserted peptides into the capsid of AAV-8 and -9, which can significantly change and improve their efficiency and specificity, making these insertion sites attractive to the production of novel targeted vectors in these serotypes.
Specific capsid amino acid residues play an important role in AAV's function. Specifically, the use of point mutations in the capsid of the virus can improve the efficiency and specificity of AAV gene transmission. For example, mutation of various serine, threonine and lysine residues in AAV-2 capsid to alanine or arginine can solve the degradation of capsid caused by undesirable post-translational modification, thereby improving cell transduction efficiency. In addition, we found that only two residues, H527Y and R533S, are changed in the simple modification of parent AAV-9, resulting in a new variant with specificity in the central nervous system.
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Reference
- Büning, H.; Srivastava, A. (2019). Capsid modifications for targeting and improving the efficacy of AAV vectors. Molecular therapy. Methods & clinical development. 12: 248.
