Prodrug converting enzyme gene delivery by L-monocytogenes

Background: Listeria monocytogenes is a highly versatile bacterial
carrier system for introducing protein, DNA and RNA into mammalian
cells. The delivery of tumor antigens with the help of this carrier
into tumor-bearing animals has been successfully carried out
previously and it was recently reported that L. monocytogenes is
able to colonize and replicate within solid tumors after local or
even systemic injection. Methods: Here we report on the delivery of
two prodrug converting enzymes, purinedeoxynucleoside phosphorylase
(PNP) and a fusion protein consisting of yeast cytosine deaminase
and uracil phosphoribosyl transferase (FCU1) into cancer cells in
culture by L. monocytogenes. Transfer of the prodrug converting
enzymes was achieved by bacterium mediated transfer of eukaryotic
expression plasmids or by secretion of the proteins directly into
the host cell cytosol by the infecting bacteria. Results: The
results indicate that conversion of appropriate prodrugs to toxic
drugs in the cancer cells occured after both procedures although L.
monocytogenes-mediated bactofection proved to be more efficient
than enzyme secretion 4T1, B16 and COS-1 tumor cells. Exchanging
the constitutively PCMV-promoter with the melanoma specific
P4xTETP-promoter resulted in melanoma cell-specific expression of
the prodrug converting enzymes but reduced the efficiencies.
Conclusion: These experiments open the way for bacterium mediated
tumor specific activation of prodrugs in live animals with tumors.