Promise and Problems Loom for Stem Cell Gene Therapy

The Scientist 13[15]:0, Jul. 19, 1999 News Promise and Problems Loom for Stem Cell Gene Therapy By Paul Smaglik Photo courtesy of Harry Malech A network of gas-permeable plastic bags connected by tubing helps make ex vivo stem cell gene therapy more feasible. The insides of the bags are coated with recombinant human fibronectin fragments. That protein helps more retroviruses carrying therapeutic genes to enter the targeted stem cells, which are also grown in bags. The tubing allows growth factors to be pumped easily into and out to the network. The bags also maximize the interaction between stem cell, virus, and fibronectin. Stem cell transplantation and stem cell gene therapy share the same promise--and many of the same problems. The promise looms so large that gene therapy researchers and transplant scientists alike often refer to stem cells as the "ultimate target."1 In the transplantation field, such progenitor cells purified from either a donor or the patient could theoretically improve the efficacy of the procedure and reduce the risk of graft versus host disease (GVHD). 2 And in gene therapy, transducing a progenitor cell should give rise to an unlimited number of similarly "fixed" cells, potentially curing single-gene disorders. The problems, though, remain formidable, notes James Wilson, director of the Institute for Human Gene Therapy at the University of Pennsylvania, and the president of the American Society for Gene Therapy (ASGT). Wilson has been warily watching the progress of stem cell research in both fields, but he has yet to take the plunge in his own lab. "One of the problems with using cell transplantation to repopulate an organ is the cells that exist there have to get out of the way," he notes. That means stem cell treatments would theoretically involve eliminating muscle to cure Duchene's muscular dystrophy or creating massive liver damage to heal hemophilia. Bone marrow transplants, which require such cytoablation, have a 10 percent mortality rate, he notes. And even if the high doses of chemotherapy and radiation necessary to kill the existing bone marrow don't also kill the patient, they greatly weaken him or her, limiting the utility of such transplant procedures. Wilson notes that cytoablation causes "the same problems" in stem cell gene therapy. Repopulation is "going to be a major issue," he predicts. Curiously, Wilson sounds confident about overcoming one obstacle in stem cell gene therapy: getting a retroviral vector to enter cells that seldom divide. "We could get the genes into those cells. There's no doubt in my mind. But I think it's premature to get too excited about genetically modifying a human stem cell until we can figure out how to control its proliferation and differentiation." Fortunately for both fields, problems facing both may also share the same solutions--solutions that are growing tantalizingly closer to resolution, according to Curt I. Civin, Johns Hopkins University oncologist, and Harry L. Malech, deputy chief of the laboratory of host defenses at the National Institute of Allergy and Infectious Diseases. Civin is primarily interested in stem cell-based transplants to treat cancer, and Malech is in the midst of a stem cell gene therapy trial to treat a single-gene disorder. "They each inform the other," Malech notes of the two fields. Indeed, on June 30 Civin received an "Inventor of the Year" award from the Intellectual Property Owners Association, for developments that help both. In 1984 Civin developed the CD34 monoclonal antibody that binds to antigens only present in hematopoietic stem cells. Those cells give rise to blood, marrow, and immune cells. More recently, he developed a process to purify hematopoietic stem cells on a larger scale. Those two discoveries have helped solve a problem key to both fields--isolating the target cells, which make up about 1 percent of bone marrow cells. That step represents the foundation on which both stem cell gene therapy and transplantation can be built. The next step, making stem cells differentiate into specific cell types, is close to being solved, Civin claims. "If you gave me a pot of CD34 positive cells, I could make it so that, by giving them the right growth factors and conditions, most of the cells become either white cells, or red cells, or platelets." However, for stem cells to be more useful in either procedure, researchers must find a way to make stem cells beget stem cells. "We don't quite yet have the self-renewal problem licked," Civin explains. "We're getting there." The success of stem cell gene therapy depends on that problem's resolution for two reasons. First, even the best viral vector will likely only enter a small fraction of stem cells, so growing more would result in a bigger pool. And second, because viral vectors can only enter cells while they are dividing, making them grow would unlock the door to transduction. Still, Civin suspects that problem, too, is on the threshold of resolution. Researchers, including Malech, are getting some of the cells to divide at least once, allowing retroviral vector entry. "The trick is to use optimal combinations of cloned recombinant human growth factors and stem cells in special media." Using that approach--and a painstakingly complex protocol (see related story, below), Malech achieved subtherapeutic levels of transdution in a single-gene disorder. He transduced about 0.5 percent of targeted cells in two of four patients with the rare immune deficiency chronic granulomatous disease (CGD), Malech reported at last month's ASGT meeting in Washington, D.C. "We're at the borderline of most peoples' definition of a permanently engrafting stem cell." He estimates that they need a minimum long-term level of 5 percent correction to lessen the disease's impact. "We have to boost it a lot more," Malech says of the existing transduction level. Many animal models have already yielded higher levels of expression, but researchers destroyed existing bone marrow to achieve those results. 3,4 The transplantation field again points to a gentler alternative. Malech discussed an experiment applying that approach to people during a recent Lake Tahoe, Nev., stem cell conference. Physicians isolated stem cells from CGD patients' siblings with matching tissue type, then purified them. Meanwhile, they gave the patient mild marrow suppression and immune suppression, rather than complete bone marrow ablatement. The patients then received the purified stem cells. Two patients have been completely engrafted after seven months, Malech notes. "There's a side benefit to the gene therapy program from this work," he adds. "I believe some kind of conditioning in the gene therapy setting is going to be necessary." That kinder, gentler conditioning could allow higher levels of transduction without requiring that existing cells be destroyed--one of Wilson's biggest qualms about stem cell gene therapy. That strategy shows signs of working in animal models. In a paper recently accepted for autumn publication in the journal Blood, Malech and colleagues show "reasonable" stem cell gene transfer in monkeys. They used small doses of full-body radiation to achieve it--a tactic Malech admits might be too harsh for humans. "But the principle is there." Humans could instead receive mild chemotherapy, he thinks. Still, stem cell gene therapy faces other issues. Some researchers fear that forcing retroviral vectors into stem cells could cause an immune response. "Maybe we need to go on to other kinds of vectors," Malech notes. Unfortunately, few acceptable alternatives exist. Adenoassociated viruses have problems getting into stem cells and nonviral vectors still require refinement, he explains. "Many people are pinning some hopes on these lentiviral vectors. We might be able to get into a lot more of the stem cells with lenti." Malech remains optimistic about the approach, despite the considerable puzzles that remain to be solved. "I'm not one of those people waving the flag saying, 'Hang on. Gene therapy's just around the corner and we're going to cure everybody,'" he explains. "I think the days of that kind of flag-waving hopefully are gone. I think many people in the field raised a lot of hopes that couldn't be matched in a short time frame. But I see light at the end of the tunnel." P. Smaglik, "Stem cell scientists caution: clinical applications remain years away," The Scientist, 12 [23]:1, Nov. 23, 1998. S. Latta, "Advances in bone marrow transplantation improve safety," The Scientist, 11[24]:11, Dec. 8. 1997. H.P. Kiem et al., "Improved gene transfer into baboon marrow repopulating cells using recombinant human fibronectin fragment CH-296 in combination with interleukin-6, stem cell factor, FLT-3 ligand, and megakaryocyte growth and development factor," Blood, 92 :1878-86,1998. J.F. Tisdale et al., "Ex vivo expansion of genetically marked rhesus peripheral blood progenitor cells results in diminished long-term repopulating ability," Blood, 92:1131-41,1998. Building a platform for stem cell gene therapy required timing, technology--and a bit of luck, notes Harry L. Malech, deputy chief, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases. The treatment begins by mobilizing patients' hematopoietic stem cells from their bone marrow into their blood. In his trial for chronic granulomatous disease (CGD), Malech used granulocyte macrophage-colony stimulating factor (GM-CSF) and Flt-3 ligand, a novel experimental bone marrow-stimulating growth factor, both provided by Immunex of Seattle. "It greatly raises the number of stem cells in the blood," Malech says of the experimental combination. After seven or eight days of treatment with the cocktail, the patient's level of stem cells rises to a harvestable amount. Then a machine plucks the mononuclear cells from the patient but returns the plasma, platelets, and red blood cells. After this apheresis, the blood monocytes, now in plastic bags, are further processed by an Isolex 300i machine provided by Nexell Therapeutics of Irvine, Calif. That machine uses immunomagnetic beads together with antibodies against CD34 and spinning chambers, to separate the stem cells from the rest of the blood product. "After two or three hours, you end up with the waste cells in one bag and purified CD34 cells in another bag," Malech notes. Takara Shuzo www.takara.co.jp/english/bio_e/index.htm Nexell Therapeutics http://www.nexellinc.com/main.htm Immunex www.immunex.com Those purified CD34 cells are immediately pumped into a new set of special gas-permeable plastic bags, provided by Nexell. Similar bags have been used in earlier gene therapy experiments, but only to grow lymphocytes. The new Nexell bags differ in chemical composition and charge, and therefore are conducive to growing stem cells. Those bags, Malech notes, make the protocol feasible. The bags have insides coated with recombinant human fibronectin fragments (RetroNectin) CH-296 from Takara Shuzo of Otsu, Japan. The product boosts levels of retrovirus-mediated gene transfer. When Malech heard of the product's transduction-enhancement properties several years ago, he was worried that he would have to abandon his system of bags, which makes handling the cells much easier. "I purchased some of that stuff and it really worked well in my system. I then demonstrated that it worked in the bags the same way it worked in a tissue culture flask. You get a molecular adherent layer that sticks to the inside of the bag." Other growth factors, including the Flt-3 ligand, as well as a purified human serum component, are pumped into the bags. The RetroNectin and the growth factors essentially help force the retroviral vector into the stem cell. "Retroviruses will not go into anything except dividing cells," he notes. "We want to get them [dividing] just long enough to get genes into them, but not hold them in culture too long before they go back into the patient." After a day of growing, lab workers drain the old media and pump in a new mixture, this time containing the retrovirus, which holds a corrective gene for CGD. The bags again play a key role here, because they allow for a maximum amount of surface area for the vector and the cells to interact--essential for optimal transduction. Researchers then repeat that process for several days, hoping to bump up the level of transduction each time. After four days, transduced cells can be detected on the bag's surface with a microscope. The insides of the bags are vigorously rubbed together, since some of the transduced cells cling tenaciously to their inside surface. Finally, the transduced cells are spun down and washed several times in a salinelike solution fit for infusion. About 30 to 50 milliliters are infused into the patient in 15-20 minutes--one of the quickest parts of the protocol. Without the bags, the protocol would be even more difficult to perform--and likely less dependable. "You can imagine if you're doing this all in flasks that there would be a lot of pipetting in and out; that now is replaced by running fluid through tubing." He expects that other gene therapy researchers, including Indiana University pediatric hematologist David Williams, who pioneered the use of RetroNectin in Indianapolis, will use similar systems. --Paul Smaglik

Promise and Problems Loom for Stem Cell Gene Therapy

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