Design and construction of targeted AAVP vectors for mammalian cell transduction.
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 (approximately 10(10)-10(11) transducing units per microl) can be produced within 3 days after vector construction. Transgene expression by targeted AAVP usually reaches maximum levels within 1 week.
Hajitou
Amin
A
Department of Genitourinary Medical Oncology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, TX 77030, USA.
Rangel
Roberto
R
Trepel
Martin
M
Soghomonyan
Suren
S
Gelovani
Juri G
JG
Alauddin
Mian M
MM
Pasqualini
Renata
R
Arap
Wadih
W
D000818:Animals; D001435:Bacteriophages / Q000235:genetics*; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000138:chemical synthesis* / Q000737:chemistry; D008156:Luciferases; D051379:Mice; D014161:Transduction, Genetic / Q000379:methods*; D019076:Transgenes / Q000235:genetics
D017931:DNA Primers; D008156:Luciferases