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Design and construction of targeted AAVP vectors for mammalian cell transduction

Abstract

Bacteriophage (phage) evolved as bacterial viruses, but can be adapted to transduce mammalian cells through ligand-directed targeting to a specific receptor. We have recently reported a new generation of hybrid prokaryotic–eukaryotic vectors, which are chimeras of genetic cis-elements of recombinant adeno-associated virus and phage (termed AAVP). This protocol describes the design and construction of ligand-directed AAVP vectors, production of AAVP particles and the methodology to transduce mammalian cells in vitro and to target tissues in vivo after systemic administration. Targeted AAVP particles are made in a two-step process. First, a ligand peptide of choice is displayed on the coat protein to generate a targeted backbone phage vector. Then, a recombinant AAV carrying a mammalian transgene cassette is inserted into an intergenomic region. High-titer suspensions (1010–1011 transducing units per μl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.

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Figure 1: Targeted AAVP vectors.
Figure 2: In vitro mammalian cell transduction with targeted AAVP particles.
Figure 3: In vivo transduction and molecular-genetic imaging of tumors in mice after systemic delivery of targeted AAVP.

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Acknowledgements

We thank Marco Arap, David Bier, Carlotta Cavazos, Carol M. Johnston, Erkki Koivunen, Darwin Lee, Frank C. Marini, Bradley H. Restel, Karen Schmidt, Yan Sun and Claudia Zompetta for advice and assistance. This work was funded by grants from the NIH (including the SPORE) and DOD (including the IMPACT) and by awards from the Gillson-Longenbaugh, the Keck Foundation and the Prostate Cancer Foundation (to R.P. and W.A.). A.H. received a Léon Fredericq award.

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Correspondence to Renata Pasqualini or Wadih Arap.

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Hajitou, A., Rangel, R., Trepel, M. et al. Design and construction of targeted AAVP vectors for mammalian cell transduction. Nat Protoc 2, 523–531 (2007). https://doi.org/10.1038/nprot.2007.51

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