One promising potential treatment for diseases caused by genetic mutations is to harvest cells from the patient, correct the DNA mutation that causes the disease, and then return the cells to the patient. The key to this approach is finding a way to efficiently correct the mutation without major side effects. Using zinc finger nucleases (ZFNs) to edit the genome at specific, predetermined sites is one possible strategy that Steffen Meiler (Georgia Regents University), William Dynan (Emory University), and colleagues have been working on for eight years now.
When introduced to cells via cDNAs, ZFNs create a double-stranded break at the mutation site, allowing the cell to replace the mutated DNA with healthy DNA also provided to the cell on a plasmid. But ZFNs often cut areas of the genome in addition to the intended site, an undesirable effect that has been the Achilles heel of this technique.
Now, a paper by Meiler and Dynan in Nucleic Acids Research presents a new method for delivering ZFNs to cells. “We thought, wouldn’t it be nice if we could produce a ZFN as a protein by attaching it with a disulfide linker to the transferrin ligand,” said Mieler, explaining that this approach should enhance endocytosis of the ZFN. The idea was that the reducing environment inside the cell would split the disulfide bond in the linker, releasing the ZFN from the transferrin ligand.
“The big question we had was, would it, once it was in the endosome, be able to escape?” For reasons Meiler and colleagues don’t quite understand, the ZFN protein had no difficulty moving from the endosome into the cytosol; they suspect this effect may be due to the ZFN’s high positive charge. From there, the ZFN targeted the nucleus and cleaved a specific sequence of DNA.
“The gene correction activity we saw was almost as high as in cells that had been treated with zinc finger cDNA,” said Meiler. But this method of delivery was highly efficient, providing better temporal control and control of the ZFN quantity delivered while maintaining roughly the same cutting efficiency as a ZFN expressed from cDNA. “What is really novel about this study is that we were able, in very low nanomolar concentrations, to deliver a nuclease as a protein therapeutic,” explained Meiler.
The next step, Meiler said, is to look at whether the transferrin system is in fact the ligand-specific pathway to use. The team is looking at other receptor-mediated pathways that are more specific to hematopoietic stem cells. Meiler and his colleagues are also experimenting to see whether this method can be used for other nuclease systems such as TALENs, which may have some competitive advantages due to a greater cutting efficiency and less off-target activity.
Chen Z, Jaafar L, Agyekum DG, Xiao H, Wade MF, Kumaran RI, Spector DL, Bao G, Porteus MH, Dynan WS, Meiler SE. Receptor-mediated delivery of engineered nucleases for genome modification. Nucl. Acids Res. (2013) 41 (19): e182.