By David N. Leff
Do the following words remind you of anything?
"The act of bringing these two together in very close proximity allows them to see each other, and then to interact with each other and stimulate phosphorylation of themselves, and then a large number of downstream molecules."
They remind research hematologist C. Anthony Blau, who told them to BioWorld Today, of a wedding ceremony.
"Proximity is the way that molecules inside cells signal," he explained. "So by bringing the two copies of the fusion protein together, they interact with each other to stimulate phosphorylation. In fact," he observed, "I call it a shotgun marriage between two copies of the fusion protein. It really forced them together."
Blau, an assistant professor of hematology at the University of Washington, in Seattle, is senior author of a paper in today's Proceedings of the National Academy of Sciences (PNAS), dated July 7, 1998. Its title is "Targeted expansion of genetically modified bone marrow cells."
Bone marrow populates the blood with swarms of cells that operate the immune system, and erythrocytes — the red blood cells that oxygenate the body, not to mention platelets. Each clone of these diverse cells is the progeny of one rare ancestral stem cell. These have the totipotent gift of being able to differentiate into whatever blood cell line the body needs at a given moment.
In the bone marrow of mice, stem cells number one in every 100,000. In humans, their versatility makes stem cells a tempting target of gene therapists.
Gene Therapy Still Inefficient At Cellular Level
"Stem cell gene therapy is envisioned as a treatment for any number of inherited or acquired blood diseases," Blau observed. "The problem with the field has been that nobody figured out a good way to put genes into stem cells. Right now, using the best available technology, people feel great when they can get a gene into 1 percent of stem cells — far too low a frequency to expect any sort of therapeutic benefit.
"The motivation of our research," he continued, "was to try to circumvent or prevent what is arguably the single most important obstacle to stem cell gene therapy. That is, the process of introducing DNA into stem cells is extraordinarily inefficient.
"What we are doing is to envision a time when you'll have your therapeutic gene — the gene that corrects the cell — actually linked to a second gene that gives that cell, into which the gene's been transferred, a selective advantage. That is, when we put our gene into the stem cell, it already has a therapeutic gene on board."
Blau and his co-authors based their approach on a small, orally available molecule supplied to them by Ariad Pharmaceuticals Inc., in Cambridge, Mass. Its job is to join together two like proteins in a dimeric molecule.
Such dimerization incites the stream of signaling from a cell's outer surface to its nucleus. In Blau's case, the message is to divide and proliferate.
"You somehow have to give this cell a selective advantage. First you put in the selectable gene, which renders the cell competent to proliferate in the presence of a drug. Then you give it a secret decoder ring. The cells that have your gene decode its message and in response to your drug, divide. The other cells don't.
"Now, if you stop giving the drug, the cells stop dividing. It's like a cell-growth switch."
Blau imagines — and he stresses that word imagine — "this might be a very useful thing in innocuous types of treatment to allow very low levels of gene delivery into rare stem cells to suffice. Then, if you could specifically expand only those cells that had your gene, this could be potentially useful for stem cell gene therapy."
His experiment reported in PNAS used that approach to expand the blood's output of platelets, which fall dangerously low in much of cancer chemotherapy.
"We're also obviously interested in using it for other organs," he went on. "If you wanted to stimulate the expansion of liver cells, for example, that could be a clinical application. Or for nerve regeneration, as in a traumatized spinal cord. Or diabetes, to stimulate the expansion of pancreatic islet cells. It's all imagination so far," he allowed, "but could potentially be applicable to many diseases where cell growth would have a therapeutic effect."
Blau and his co-authors began with standard retroviral vector transfer of a recombinant complementary DNA gene into murine bone marrow cells.
"Essentially," he recounted, "our gene had two components.
"A receptor for mpl, a molecule involved in generating megakaryocytes, which are precursors of blood platelets. We used only the intracellular portion of mpl, because all we needed was the guts of the thing — the signaling portion of the molecule.
"We fused that to a protein called FK binding protein [FKBP]. The drug that this binds to is called FK1012.
"Then," he went on, "the trick was to make a dimeric version of two FK506s fused together. This drug is called FK1012, the arithmetic sum of two 506s. When you add FK1012, you reversibly dimerize, bring into apposition, two copies of this fusion protein. And its dimerization is what stimulates signal transduction — telling the cells to divide.
"We put this gene into the cell. It expressed a fusion protein. We'd targeted it so it goes to the intracellular surface of the cell membrane, to which it attached."
Cell Expansion Could Be Primary Target
Then the co-authors added Ariad's proprietary FK-based dimerizing drug, trademarked ARGENT. Being membrane-permeable, the drug diffused through the membrane into the cell, and bound that fusion protein.
"The FK1012," Blau pointed out, "one part of it, the 506 half, bound one of the two fusion proteins. The other FK506 portion bound the second, and brought the two close together.
"And that," he recalled, "stimulated an unbelievable expansion in culture, without any other growth factors. This is unheard of — just using this drug that sends this message to the cell to divide, to get this sort of amazing expansion of cells. We had 2.5 million or more cells at the end of 42 days in culture than we had at the beginning.
"Then, when we stopped the drug, the cells stopped dividing.
"This," Blau observed, "raises the question as to whether or not that might also have therapeutic applications —just the ex vivo or in vivo expansion of cells." *