By David N. Leff

When gene therapy delivers a sequence of interest into a patient's body, that foreign DNA often encounters the silent treatment. In fact, "Irreversible human stem cell gene silencing remains one of the main obstacles to the gene therapy of hematological disorders."

So said molecular biologist Philippe Leboulch, of Harvard Medical School in Boston and the Massachusetts Institute of Technology in Cambridge. He also is co-founder and chief scientific officer of Genetix Pharmaceuticals Inc., of Cambridge.

"This transcriptional silencing," Leboulch told BioWorld Today, "poses one of the most significant challenges to the success of gene therapy. It's not at all like an autoimmune counterattack by the body's immune system," he pointed out. "We believe gene silencing is a mechanism that cells - particularly stem cells - are prone to have as a means of defense against invasion by viruses and transposons. These can insert their DNA at random into the chromosomes of cells, or for example propagate multidrug resistance genes.

"What's happening in gene therapy," he went on, "is that when genes have been transferred into cells, especially stem cells, very often they don't express much - not only per cell, but also the large proportion of cells that have the new gene permanently integrated don't express at all. And that's what we call the phenomenon of gene silencing."

Leboulch is senior author of a paper in the current Proceedings of the National Academy of Sciences (PNAS), dated May 9, 2000. Its title: "Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human b-globin in engrafted mice."

Saving Globin Genes From Silencing

In this project, he and his co-authors set out to transfect human beta-globin genes into the bloodstreams of mice. Their strategy was to ensure the expression of these genes by preselecting their stem cells, while preventing these from silencing those globin genes. Globin is the protein payload of red blood cells (RBCs), which fuel the body's tissues with oxygen and nutrients.

Different mutant globin genes cause sickle cell disease (SCD) and thalassemia. "Between them," Leboulch observed "they are the No. 1 causes of morbidity and mortality by genetic disease worldwide. SCS," he pointed out, "is most prevalent in Africa, South India and among African-Americans. Beta-thalassemia is widespread in the countries around the Mediterranean basin, and among Americans of Italian or Greek ancestry."

The co-authors, he recounted, "took bone marrow stem cells from mice and exposed them to a retroviral vector that contained the beta-globin gene of interest. Cells that received the vector without our intervention would not have had an advantage.

"However," Leboulch continued, "we linked in the same vector a second gene, which encodes the green fluorescent protein [GFP] of jellyfish. This gave rise to fluorescence in cells we exposed to wavelengths of light that excite the GFP. Then - without the need for any antibody - we got autofluoresence of the cells, so the machine automatically sorted by fluorescence-activated cell sorter only those stem cells that expressed GFP, and left behind those that did not.

"So when we took that pure population of fluorescent cells," he went on, "and injected them intravenously into the recipient mice, which had received total-body irradiation to remove as much as possible of the non-transduced marrow, there was complete engraftment.

"By doing so," he recounted, "we were pleasantly surprised to find that we avoided the phenomenon of gene silencing because we selected stem cells that did not silence the gene originally, but expressed it in the GFP. Moreover, they also impacted on the linked globin gene, which meant that it, too, was expressed. In other words, we had eliminated a subset of stem cells in which the gene was silenced permanently."

Leboulch made the point that "in conventional gene transfer experiments, one would also expect to have a subset of cells in which the gene is not silenced, but a few weeks after bone marrow transplantation, expression goes down to very low levels. It's sort of a secondary silencing. We found we had avoided that outcome as well. Of the seven mice we treated, 100 percent expressed both the globin gene and GFP gene permanently for up to 9.5 months - the longest time period we assayed.

"In humans," Leboulch presumed, "we would use the patient's own bone marrow to have an autograft, but in mice we have strains that are syngeneic [matching immune type], so we can use donor and recipient animals without any problem. We don't know whether the GFP will be the way to go with humans," Leboulch observed, adding, "I would say, 'Why not?' because we didn't detect any toxicity. The mice remain perfectly fine for many months, and they don't care whatsoever at having GFP at low levels in their blood cells. But we are pointing in the direction of imposing the selection for gene expression on transduced stem cells ex vivo, which doesn't require more than a day in order to eliminate silenced stem cells. That would do a lot of good by ensuring long-term expression at significant levels in host cells' blood."

Testing Animals With Diseases For Real

Genetix Pharmaceuticals holds an exclusive license from MIT to the elements of that globin gene expression technology, shared with Cobra Therapeutics Ltd., of Keele, UK. "These elements," Leboulch mentioned, "are strong enhancers of human beta-globin in RBCs. So we have incorporated them to boost expression per cell. Now not only do we have a high proportion of red blood cells that are modified, which we have reported in this week's PNAS, but in addition to that, our next step is to test our approach in animal models of sickle cell disease and thalassemia.

"We have in our possession," he noted, "spontaneous beta-thalassemic mice, and also a transgenic animal model of sickle cell disease, which symptomatically expresses human sickled beta-globin. So we are about to begin trying to cure those animals of their diseases. If that works," he concluded, "it could be an important step toward human trials."