Capsid Shuffling

Limitations in tropism and transduction efficiency and widespread immunization of human targets are the drawbacks of current gene-therapeutic approaches based on wildtype adeno-associated viruses (AAVs). Bearing in mind that the efficiency of AAVs is determined by their capsids [http://2010.igem.org/Team:Heidelberg/Project/Capsid_Shuffling#References (Grimm et al,2008)],we sat out to develope improved capsids that are more specific and demonstrate better functionality. We were able to achieve this goal by using two different methods to shuffle capsid genes and produce synthetic viruses that can be evolved to infect tissues of choice specifically. Homology based shuffling by DNaseI digestion and self-primed PCR were used to produce a library of randomized novel viruses. Additionally, we introduce ViroBytes, a random assembly protocol based on rationally designed capsid parts.

Introduction

Adeno-associated viruses (AAVs) are a class of single stranded DNA viruses that are not able to replicate without a helper virus. This makes them a perfect tool for the iGEM community, as no special safety requirements have to be fulfilled to work with a non-replication virus, because it is non-pathogenic. Because of their wide range of tropism they are used for transgene delivery in a variety of gene therapeutic approaches.

In the class of AAVs, there are several serotypes that have been isolated from humans or non-human primates, the first and most well-known of them being AAV2. AAV serotypes are defined as naturally evolved variants of AAV that do not react to the same antibodies. All serotypes show different tissue specificites when injected into mouse or humans, and this tissue tropism is thought to be mainly due to interactions between the virus capsid and receptors on the cell surface.

Most AAVs exhibit a rather broad tropism, AAV2 and AAV9 for example have been shown to transduce liver, muscle, lung and nervous system. Other serotypes, for example AAV1 and AAV7, can infect very rapidly or more efficiently than others [http://2010.igem.org/Team:Heidelberg/Project/Capsid_Shuffling#References (reviewed in Wu et al., 2006)]. Although diverse, not one of the AAVs would make a good gene delivery shuttle by itself. Various approaches have been undertaken to change or combine AAVs in order to alter their tropism or transduction efficiency. These approaches mostly target the capsid genes by rationally creating AAV hybrids with certain properties or fusing targeting ligands to the proteins [http://2010.igem.org/Team:Heidelberg/Project/Capsid_Shuffling#References (reviewed in Gao et al., 2005)].

Another fundamental drawback of wild type AAVs for applicability in gene therapy is their high abundance in nature. It has been estimated that up to 80% of humans are immune against AAV2, which has a potentially fatal impact in clinical studies using AAV2 [http://2010.igem.org/Team:Heidelberg/Project/Capsid_Shuffling#References (Moskalenko et al., 2000)]. This can be circumvented by introducing functionally relevant sequences from AAV serotypes that have been isolated from non-human species while engineering virus hybrids.

The Heidelberg iGEM team 2010 uses two independent approaches to engineer synthetic AAVs based on capsid shuffling. In addition to miRNA-mediated On- and Off- Targeting of specific cell types, we created viruses with more specific tropism for delivery of our transgenes. Even more importantly, we developed a new method for a simplified and efficient production of shuffled capsid AAV libraries that we call ViroBytes.

Homology Based Capsid Shuffling

ViroBytes

References

Dirk Grimm, Joyce S. Lee, Lora Wang, Tushar Desai, Bassel Akache, Theresa A. Storm, and Mark A. Kay.In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses.J Virol. 2008; 82(12): 5887–5911. Wu Z, Asokan A, Samulski RJ. Adeno-associated virus serotypes: vector toolkit for human gene therapy. Mol Ther 2006;14(3):316–27
Gao, G., Vandenberghe, L. H., and Wilson, J. M. (2005). New recombinant serotypes of AAV vectors. Curr. Gene Ther. 5: 285 – 297.
Moskalenko, M., Chen, L., van Roey, M., Donahue, B.A., Snyder, R.O., McArthur, J.G., and Patel, S.D. (2000) Epitope mapping of human antiadeno-associated virus type 2 neutralizing antibodies: implications for gene therapy and virus structure. J. Virol. 74: 1761-6.