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Home | Applications | Imaging of AAV2 Virus Particle

Imaging of AAV2 Virus Particles

AAV

Fig 1. Reconstruction of AAV2 virus particle. nanoparticles.

AAV

Fig 2. Schematic of TEM sample preparation.

Adeno-associated virus type 2 (AAV) is a non-pathogenic DNA virus which has been utilized as a eukaryotic gene transfer vector in vitro and in vivo. The advantages of AAV-2 vectors as gene therapy vectors include the nonpathogenic nature of the wild-type (wt) virus, the ability to infect dividing and nondividing cells, and the establishment of long-term expression of heterologous genes by recombinant AAV [1-4]. AAV vectors have been used to transduce a wide range of cell types in vitro including respiratory epithelial cells as well as bone marrow and lymphocyte-derived cells. Experimental AAV vectors have been designed to combat a variety of diseases such as AIDS, high blood pressure, cystic fibrosis, and Parkinson's disease[5]. However, to improve the specificity of the gene vectors for target cells, an improved understanding of the basic biology of AAV-2 including structural information regarding host-cell interactions is required. Adeno-associated viruses (AAVs) are small, icosahedral viruses of the Parvoviridae family with a capsid of 20 to 25 nm in diameter.

Electron Microscopy is a powerful tool that can be used to probe this structural information. In a recent publication, O’Donnell, et.al. reported on the structure of AAV2 and its primary receptor via cryoEM. The current study builds upon this work to introduce a new sample preparation approach that will facilitate a deeper understanding of the structural interactions of antigenic regions of the AAV2 with monoclonal antibodies.

The general protocol used in the preparation of sample is shown in the schematic on the right. After each successive step, the grids were thoroughly rinsed in DI H2O. Conditions were optimized to block non-specific absorption of either the primary or secondary antibody. For our initial samples, the grids were stained with 0.2% uranyl acetate.

References

  • 1) Flotte, TR, Carter, B.J. , Adeno-associated virus vectors for gene therapy. Gene Ther. 1995 Aug;2(6):357-62.
  • 2) Samulski R J, Sally M, Muzyczka N. Adeno-associated viral vectors. In: Friedman T, editor. The development of human gene therapy. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1999. pp. 132–172.
  • 3). Kotin R M. Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum Gene Ther. 1994;5:793–801.
  • 4) Muzyczka N. Use of adeno-associated virus as a general transduction vector for mammalian cells. Curr Top Microbiol Immunol. 1992;158:97–129.
  • 5)O’Donnell, J, Taylor, KA, Chapman, MS, Adeno-associated virus-2 and its primary cellular receptor—Cryo-EM structure of a heparin complex. Virology 385 (2009) 434–443.