Sickle cell anemics have one mutation that leads to two problems: too much mutated and not enough normal beta-globin, which transports oxygen in blood cells.
Sickle cell anemia is a good case for how harmful mutations remain in the population. The heme group, which is the business part of the oxygen-carrying molecule hemoglobin, consists of four chains.
Sickle cell anemics have a mutation in their beta chain that is prone to misfolding. One copy of the mutated gene will protect carriers against malaria, which is why it hasn't disappeared from the gene pool long ago; two copies will lead to sickle cell disease, which is "by far the most common monogenic disease on the planet," Michel Sadelain told BioWorld Today.
Sadelain is a member of New York's Memorial Sloan-Kettering Cancer Center's immunology group. He is also the senior author of a paper in the January 2006 issue of Nature Biotechnology, in which he and his colleagues from the University of Medicine and Dentistry of New Jersey, New Brunswick; the V. Cervello hospital in Palermo, Italy; and the National Institutes of Health in Bethesda, Md., describe how gene therapy might simultaneously correct both problems: by delivering a gene for a normal globin that has an interfering RNA for the mutant globin in one of its introns.
Sickle cell anemia can be cured by a bone marrow transplant, but most patients cannot find a suitable marrow donor, so that possibility remains theoretical far more often than not. Current therapies for sickle cell disease focus on pharmacologically inducing expression of gamma globin, the type of globin normally expressed during fetal development that will not sickle.
Sadelain and his colleagues also used the gene for gamma globin in their studies. Asked why they chose to add gamma rather than a normal beta-globin, Sadelain said that adding the beta chain is "certainly what we plan to do." Currently, however, the siRNA that is the other half of their therapy still knocks down some of the normal beta chain as well as the mutant chain, so at this point, "adding the beta chain would be shooting yourself in the foot."
In the paper, the scientist described constructing, in blood-forming stem cells, a transgene for gamma globin that contained a small interfering RNA for the mutant beta-globin in one of its introns. Sadelain pointed out that the use of that co-regulation approach is not limited to beta-globin.
"The technique could also be used to knock down an oncogene and deliver a tumor suppressor," or any number of other combinations, he said.
When the transgene was inserted into adult stem cells from sickle cell disease patients, both the gamma-globin and the interfering RNA were expressed in red blood cells descended from those stem cells; those cells produced normal gamma-globin, while the production of mutant beta-globin was suppressed by up to 95 percent.
Encoding the interfering RNA for the mutant beta-globin within the gamma-globin had two advantages. First, despite the fact that blood-forming stem cells make many different types of progenitor cells, interfering RNA was expressed only in red blood cells progenitors. Second, because strange RNAs most often signal a viral infection, the body will react by trying to destroy them. Sadelain and his colleagues managed to avoid that immune response by encoding the interfering RNA within an intron of the therapeutic gene. The scientists tried a number of different intron positions for the interfering RNA and found they were not all created equal; Sadelain said there were "definite winners," though at this time, his team can only speculate as to why some locations are better than others.
Somewhat unusual for a recessive disorder, sickle cell anemia is caused by a misfolded protein. Misfolding mutations often are dominant, because if even half of a cell's supply of a particular protein is misfolded, it can be sufficient to bring the pathway in question to a screeching halt. While recessive mutations, on a genetic level, often lead to the complete absence of a protein - in such a case, the cell will be able to limp along, or in some cases move along quite well, with only half the normal supply. It is only a complete absence of the protein in question that will bring the cell to that screeching halt.
Sadelain said that "the proof is in the patients" with sickle cell anemia, who rarely have clinical symptoms despite producing half of their beta-globin in a useless form, as long as they have one normal copy of the beta-globin gene. From his perspective, that also means that the expression of mutant beta-globin does not need to be completely silenced: "Knock down half the protein and you're in business" - a minimal requirement that the experiments described in Nature Biotechnology exceeded by a considerable margin.