Mammalian cell transduction and internalization properties of lambda phages displaying the full-length adenoviral penton base or its central domain.
In recent years a strong effort has been devoted to the search for new, safe and efficient gene therapy vectors. Phage lambda is a promising backbone for the development of new vectors: its genome can host large inserts, DNA is protected from degradation by the capsid and the ligand-exposed D and V proteins can be extensively modified. Current phage-based vectors are inefficient and/or receptor-independent transducers. To produce new, receptor-selective and transduction-efficient vectors for mammalian cells we engineered lambda by inserting into its genome a GFP expression cassette, and by displaying the penton base (Pb) of adenovirus or its central region (amino acids 286-393). The Pb mediates attachment, entry and endosomal escape of adenovirus in mammalian cells, and its central region (amino acids 286-393) includes the principal receptor-binding motif ((340)RGD(342)). Both the phage chimerae lambda Pb and lambda Pb (286-393) were able to transduce cell lines and primary cultures of human fibroblasts. Competition experiments showed that the transduction pathway was receptor-dependent. We also describe the different trafficking properties of lambda Pb and lambda Pb (286-393). Bafilomycin, which blocks endosome maturation, influenced the intracellular distribution of lambda Pb (286-393), but not that of lambda Pb. The proteasome inhibitor MG-132 improved the efficiency of lambda Pb (286-393)-mediated transduction, but not that of lambda Pb. In summary, this work shows the feasibility of using lambda phage as an efficient vector for gene transfer into mammalian cells. We show that lambda Pb and lambda Pb (286-393) can both mediate receptor-dependent transduction; while only lambda Pb is able to promote endosomal escape and proteasome resistance of phage particles.
Piersanti
Stefania
S
Department of Genetics and Molecular Biology, University La Sapienza and Parco Scientifico Biomedico di Roma, S. Raffaele, Rome, Italy.
Cherubini
Gioia
G
Martina
Yuri
Y
Salone
Barbara
B
Avitabile
Daniele
D
Grosso
Fabiana
F
Cundari
Enrico
E
Di Zenzo
Giovanni
G
Saggio
Isabella
I
D010582:Bacteriophage lambda / Q000235:genetics* / Q000378:metabolism; D002213:Capsid; D036022:Capsid Proteins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism*; D015316:Genetic Therapy / Q000379:methods; D049452:Green Fluorescent Proteins; D006801:Humans; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011991:Receptors, Virus / Q000378:metabolism*; D014161:Transduction, Genetic / Q000379:methods*
D036022:Capsid Proteins; D011991:Receptors, Virus; C043322:penton protein, adenovirus; D049452:Green Fluorescent Proteins; D046988:Proteasome Endopeptidase Complex