Adenovirus library and methods
Described herein is a method that generally includes infecting a host cell with a rescue adenovirus, wherein the rescue adenovirus genome comprises a loxP site and encodes at least one marker, and wherein the host cell comprises a library of polynucleotides that complement the adenovirus genome marker and encode a detectable polypeptide; incubating the infected host cell under conditions effective to permit recombination between the adenovirus genome and one or more of the library polynucleotides and the production of recombinant adenovirus particles comprising at least on detectable polypeptide; and detecting the at least one detectable polypeptide. Also described are adenovirus libraries constructed using such a method.
1. A method comprising:
infecting fiber transcomplementing mammalian host cells with an Ad5/3 pseudotyped, genetically fiberless rescue adenovirus, wherein the rescue adenovirus genome comprises a single loxP site, the host cells modified to express CRE recombinase;
transfecting the host cells with a library of plasmids, each plasmid:
comprising a loxP site; and
encoding at least one detectable polypeptide comprising a fiber polypeptide comprising an AB-loop comprising a targeting ligand, wherein the library of plasmids comprises a plurality of detectable polypeptides;
incubating the infected and transfected host cell under conditions effective to permit:
recombination between the adenovirus genome and the plasmid, and
production of recombinant adenovirus particles comprising at least one detectable polypeptide; and
detecting the at least one detectable polypeptide.
2. The method of claim 1 wherein the loxP site is located in place of a deletion within E3 of the rescue adenovirus genome.
3. The method of claim 1 further comprising collecting crude viral lysate and re-infecting the host cell with adenovirus in the crude viral lysate.
4. The method of claim 3 further comprising one or more additional rounds of collecting crude viral lysate and re-infecting the host cell with adenovirus in the crude viral lysate.
5. The method of claim 1 completed in no more than about 48 hours after plasmid transfection.
6. The method of claim 1 wherein infecting the host cell comprises adding adenovirus to a culture of host cells at a multiplicity of infection of about 10,000 vp/cell.
7. The method of claim 1 wherein the fiber polypeptide comprises an AB-loop that comprises an amino acid sequence that specifically binds to mesothelin.
8. The method of claim 7 wherein the amino acid sequence comprises VTINRSA (SEQ ID NO:12).
9. The method of claim 1 , wherein incubating the infected and transfected host cell produces an adenovirus library that has at least 10 10 recombinant adenovirus particles comprising at least one detectable polypeptide.