Altering Adeno-Associated Virus (AAV) Tropism
Together, the various serotypes and variants of AAV that have been described can transduce a wide array of cells. Nevertheless, certain cells are refractory to transduction. In addition, for certain gene therapy applications, it is desirable to transduce only specific cell types. Over the past few years, several methods—biochemical as well as genetic—have been developed to expand and/or restrict AAV tropism. One of the most widely used techniques involves the insertion of a targeting ligand within the AAV capsid. Unfortunately, because of structural constraints, such insertions of ligands often result in reduction in particle and/or transducing titers. Consequently, the investigation of additional methods to alter AAV tropism is an active area of research.
Recently, the groups of Xiao and Samulski have described chimeric vectors between two different AAV serotypes. These vectors retain the tropism of both serotypes. We reasoned that the principle of mosaicism could be used to alleviate the detrimental effects that the insertion of a ligand into the AAV capsid has on viral titers.
To test our hypothesis, we used a system developed by Hallek and colleagues1. We inserted a small fragment of protein A into the AAV capsid. This fragment allowed the binding of antibodies to AAV that then could be used to transduce cells expressing the receptor against which the antibody is directed. Because of the disturbance of the capsid structure, however, the titers obtained were comparatively low.
Therefore, we decided to generate recombinant AAV with mosaic capsids, containing capsid proteins with and without the protein A insertion. To eliminate wild-type AAV2 tropism, we mutated two lysine residues involved in binding of the primary AAV2 receptor, heparan sulfate proteoglycan (HSPG). We produced mosaic viruses that contained between 25 percent and 75 percent of capsid proteins carrying the protein A insertion. We then tested if we could specifically and efficiently transduce Jurkat cells, a T-lymphoma-derived cell line that expresses the CD29 antigen (beta-1 integrin). The results are shown in the figure below.
Specific transduction of Jurkat cell using rAAV mosaics
All viruses contained the arginine to alanine mutations at positions 585 and 588, eliminating wild-type tropism. Jurkat cells were transduced with either wild-type R585/588A or mosaic-capsid virus in the presence or absence of targeting antibody against beta-1 integrin (CD29) and inhibitors as indicated. (In the diagram above, Hep = heparin, CD29 = antibody against CD29 (beta-1 integrin), IgG = rabbit IgG, Prot A = soluble protein A.)
As expected, AAV2 in which HSPG binding has been eliminated was unable to transduce Jurkat cells. Viruses that contained the protein A fragment, in contrast, were infectious, but only if the targeting antibody against CD29 was present. This transduction was inhibited by the competitive inhibitors IgG and protein A, but not by heparin, which inhibits transduction by wild-type AAV2. Confirming our hypothesis, mosaics containing 25 percent protein A were the most infectious, and higher amounts of protein A-containing capsid proteins resulted in a reduction of specific transduction. As a result of the detrimental effect of the insertion of the protein A fragment into the capsid, we were unable to produce sufficient virus to test the transduction efficiencies of viruses that are composed entirely of capsid proteins with the protein A fragment2.
We are currently extending this approach to insert other ligands that are otherwise difficult to insert into the viral capsid.
Note: The images on this page are not in the public domain. The images can be used only for educational purposes.
1 Ried MU, Girod A, Leike K, Buning H, Hallek M. Adeno-associated virus capsids displaying immunoglobulin-binding domains permit antibody-mediated vector retargeting to specific cell surface receptors. J Virol. May 2002;76(9):4559-4566.
2 Gigout L, Rebollo P, Clement N, et al. Altering AAV tropism with mosaic viral capsids. Mol Ther. Jun 2005;11(6):856-865.