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Washington University Researcher Envisions RNAi Cure for Severe Congenital Neutropenia


Severe congenital neutropenia is a rare inherited disease characterized by low levels of neutrophils, a type of white blood cell responsible for fighting bacterial infections, and a predisposition to the development of acute leukemia. While treatments for the condition exist, these therapies have certain risks and limitations.

As a result, there is a pressing need to better treatments for SCN, and Daniel Link, a physician and associate professor of medicine and pathology at the Washington University School of Medicine, thinks RNA interference may be the key to developing them.

“For severe congenital neutropenia, there are really two treatment options,” Link told RNAi News this week. “One is G-CSF, which is a growth hormone that will improve the major phenotype of this disease — low neutrophil counts.” According to Link, G-CSF therapy is effective enough to allow children affected with SCN, who without treatment are likely to die from infection before the age of 10, to live well into their 20s.

But G-CSF is not perfect; it does not prevent the development of acute leukemia associated with SCN. “These kids are getting fatal leukemia at an extremely high rate despite this growth factor,” Link said. “The most recent study would suggest that as you go out, potentially as many as one out of three patients are getting leukemia.” He added that he is concerned this rate could become much higher as SCN patients age, although there is no solid evidence to support this belief.

The other option for SCN patients is an allogeneic bone marrow transplant. “I think everyone knows that [this] is a very morbid procedure, with a mortality of a few percent in kids,” Link said. “Most of these kids have lifelong graft-versus-host disease, which is … something you can live with, but it needs its own treatment.” Additionally, in many cases appropriate donors are not on hand, so the transplant is not even an option, he noted.

The majority of SCN cases are due to mutations in a gene called ELA2, which encodes the protease neutrophil elastase. “These, we know, are heterozygous mutations,” Link said. “So we think they act in a dominant fashion to cause neutropenia, but what we don’t know yet is the pathophysiology — what is the molecular mechanism by which these mutations cause neutropenia?”

However, evidence exists that suggests this molecular mechanism is not due to the protease activity of neutrophil elastase, Link said, “so we don’t think that using a protease inhibitor is likely to have much affect on this disease.” RNAi, however, appears to be a very promising way of treating SCN, he said.

“We know that complete loss of neutrophil elastase, complete suppression of this gene by knockouts, has no affect on hematopoiesis,” Link explained. “So we don’t think that knocking down the hematopoiesis gene [using RNAi] will be associated with significant hematopoietic problems.

“Our prediction is that if you were able to get RNAi effectively into hematopoietic stem cells and knock down expression of neutrophil elastase, you would revert the phenotype [of neutropenia associated with SCN], potentially get a good hematopoietic response, and maybe even cure these patients,” he said.

So far in his effort to develop this kind of RNAi-based treatment for SCN, Link and his colleagues have developed a lentiviral vector that expresses an siRNA targeting human ELA2. “We have shown in [human] cell lines we can suppress neutrophil elastase activity significantly,” although the exact magnitude of suppression is unknown, he said.

“This lentivirus, we know, will efficiently infect human … hematapoietic stem cells,” Link said. “Our … long-term goal would be to take [stem cells from] patients who have SCN, infect [the cells] with this lentivirus, get stable silencing through this vector, and transplant those back into patients.”

Link concedes that significant hurdles face this effort, such as getting the siRNA-transfected stem cell to expand and take over the marrow, and that these are issues that are “beyond the stage we’re at right now.” However, he remains optimistic that these issues can be resolved, and will be by others working in the gene therapy field.

Currently, Link is working on demonstrating that his lentiviral vector is able to suppress wild-type neutrophil elastase in [normal] primary human stem cells, and that it has no effect on hematopoietic development. “We’re looking for toxicity here,” he said.

“The next step would be to take a patient who has SCN, take his stem cells, and infect them with this lentivirus to see if we can suppress the mutant neutrophil elastase and whether that corrects the in vitro defect in maturation,” Link said. “These cells do not mature normally in vitro. We’re hoping that if we express this siRNA in these progenitor stem cells, that they will have normal maturation. That would be pretty darned good evidence that we’re getting what we want.”

Should these experiments be successful, Link said that he plans on transplanting these human stem cells into non-obese diabetic/severe combined immunodeficiency (NOD-SCID) mice. “We’d look for engraftment and hematopoiesis over weeks and months after transplant, and ask, ‘Is the block in myeloid maturation correct?’ If it is, then I think we would then start really moving into the translational realm and think about how to potentially do this in patients,” he said.

As far as how long all this might take, Link said that the work testing the siRNAs in normal human stem cells should wrap up within a year. “Showing that [the cells] can revert the SCN phenotype in vitro and possibly in a NOD-SCID [mouse could be done] in about two to three years,” he said. “Then, planning for clinical trials, [that would be] year three and onwards — that’s a big hurdle.”

While this work is going on, Link and his colleagues at Washington University are also trying to develop a transgenic mouse model of SCN in which mutant neutrophil elastase is expressed under the regulatory control of the endogenous ELA2 locus.

“We’ve already made one mouse model and it didn’t work,” he said. “We have two more mouse models that are in progress, and we should have data on [them] in the next six months. But they are either going to work or they ain’t — I can’t honestly tell you if they’ll work or not.

“If they do, we’ll have it within six months,” he added, “and that will be nice because it will help our siRNA studies tremendously.”

— DM

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