E. coli nitroreductase NfsA is a reporter gene for non-invasive PET imaging in cancer gene therapy applications.

The use of reporter genes to non-invasively image molecular processes inside cells has significant translational potential, particularly in the context of systemically administered gene therapy vectors and adoptively administered cells such as immune or stem cell based therapies. Bacterial nitroreductase enzymes possess ideal properties for reporter gene imaging applications, being of non-human origin and possessing the ability to metabolize a range of clinically relevant nitro(hetero)cyclic substrates. Methods: A library of eleven Escherichia coli nitroreductase candidates were screened for the ability to efficiently metabolize 2-nitroimidazole based positron emission tomography (PET) probes originally developed as radiotracers for hypoxic cell imaging. Several complementary methods were utilized to detect formation of cell-entrapped metabolites, including various in vitro and in vivo models to establish the capacity of the 2-nitroimidazole PET agent EF5 to quantify expression of a nitroreductase candidate. Proof-of-principle PET imaging studies were successfully conducted using 18F-HX4. Results: Recombinant enzyme kinetics, bacterial SOS reporter assays, anti-proliferative assays and flow cytometry approaches collectively identified the major oxygen-insensitive nitroreductase NfsA from E. coli (NfsA_Ec) as the most promising nitroreductase reporter gene. Cells expressing NfsA_Ec were demonstrably labelled with the imaging agent EF5 in a manner that was quantitatively superior to hypoxia, in monolayers (2D), multicellular layers (3D), and in human tumor xenograft models. EF5 retention correlated with NfsA_Ec positive cell density over a range of EF5 concentrations in 3D in vitro models and in xenografts in vivo and was predictive of in vivo anti-tumor activity of the cytotoxic prodrug PR-104. Following PET imaging with 18F-HX4, a significantly higher tumor-to-blood ratio was observed in two xenograft models for NfsA_Ec expressing tumors compared to the parental tumors thereof, providing verification of this reporter gene imaging approach. Conclusion: This study establishes that the bacterial nitroreductase NfsA_Ec can be utilized as an imaging capable reporter gene, with the ability to metabolize and trap 2-nitroimidazole PET imaging agents for non-invasive imaging of gene expression.

Stem cells: reparative medicine and nephrology.

Stem cells (SC) provide a way to repopulate damaged organs, to deliver genes or growth factors to privileged sites in the body after gene transfection and to reconstitute organs in vitro for transplantation. This reparative or regenerative medicine is currently used in the care of hematological and neoplastic diseases, but promising results have been obtained in the care of other diseases involving heart, arteries, liver and brain. SC have also been used in experimental models of mice with glomerulonephritis or with unilateral uretheral obstruction. SC may be involved in the care of damaged organs in two ways. Firstly, niche cells reside in privileged regions of an organ and are protected from environmental damage; their reproduction is asymmetric, some of them self-generate and others differentiate in the specific tissue for organ repair. Secondly, SC from bone marrow, in which multiple distinct types are present, repair the damaged organ. Data from experimental animal models of renal diseases have shown that infused bone marrow-derived SC may repair the injured glomeruli. However, we cannot exclude that niche adult SC may reside in the outer medulla of the kidney. Therefore, niche cells may be the El Dorado for promising care for renal diseases in the near future.