By David N. Leff

After years of hiding away from isolation and experiment, bone-marrow stem cells came out of the closet in the late 1990s, and are now a hot property of research and development.

It looks as if the next such starring role is reserved for another biologic dark horse — bone-marrow stromal cells. They drew considerable attention last week at the Fourth Annual Cold Spring Harbor Laboratory Meeting on Gene Therapy, in New York.

One poster session, headed "Ex vivo gene therapy of hemophilia A and B using bone marrow stromal cells in a canine model," was presented by molecular biologist David R. Hurwitz, scientific director of the ALG Co., of Marlboro, Mass. A group of academic investigators from Belgium reported similar stromal cell research in mice.

"We work with 50- to 60-pound dogs," Hurwitz observed, "so there's a major difference in gene expression levels in animals that size.

"The bone marrow stromal cell system is starting to come into its own," he told BioWorld Today. "People are beginning to see the advantages of using this ex vivo system. The levels of gene expression we are getting with human factors VIII and IX — especially VIII — represent highly therapeutic results in our canine models.

"Stromal cells," Hurwitz explained, "are non-hematopoietic cells of the bone marrow microenvironment. They're not to be confused with hematopoietic stem cells. The stromal cells physically and physiologically support the differentiation and self-replication of the stem cells. They produce growth factors such as GM-CSF, [granulocyte macrophage colony-stimulating factor], stem-cell factor, and a number of others, which allow the hematopoietic stem cells to replicate, and to differentiate into blood-forming lineages. (See BioWorld Today, Sept. 28, 1998, p. 1.)

"There's also a lot of evidence," he added, "that bone marrow stromal cells can differentiate into other cells, notably osteoblasts, and be involved in the formation of bone."

Hurwitz and his associates at the five-year-old ALG Co. have dragooned these stromal cells into eventually correcting the genetic defect that causes hemophilia in humans, and certain breeds of dogs. The disorder results primarily from mutations in the X-linked genes for one of two proteins in the blood-clotting cascade, factor VIII or factor IX. These cause hemophilia A or B respectively, essentially in males only.

'Highly Therapeutic Results' — Then Immune Rejection

As he reported at Cold Spring Harbor, Hurwitz withdrew bone marrow from three anesthetized, mongrel, non-hemophiliac dogs, and separated out the stromal cells. These are far less frequent than already-rare stem cells, so he and his co-workers expanded them in culture.

They then transduced these cells with human factor VIII or factor IX cDNA genes, and injected the package intravenously back into their canine models via a slow 10-minute to 30-minute drip.

"We administered three graduated doses of expression capacity into the three dogs — 8, 12 and 51 micrograms over 24 hours," he recounted. "The factor VIII level rose over the first two days, and gave us peak plasma readings on day two. In the highest-dose dog, it reached 54.5 nanograms per milliliter [ng/ml].

"A normal human being has about 200 ng/ml of factor VIII in his or her blood, so that result represented 27.3 percent of normal human levels."

Hurwitz recalled there are "differing severities of hemophilia. In the severest, worst-case scenario of the disease, patients have less than 1 percent of normal clotting activity.

"A moderate case, from 1 to 5 percent, has a considerably better life. And the mildest form ranges from 5 to 50 percent. So to convert a patient from less than 1 percent severe to over 27 percent normal would restore him to being a mild, nearly normal person. And that would be highly therapeutic."

Hurwitz went on: "In our second dog, with the second dose, of 12 micrograms per 24 hours, we obtained a level of 21.7 ng/ml, equal to 10.4 percent of normal human levels, and that is also highly therapeutic."

A similar canine study with human factor IX genes also yielded respectable therapeutic results. In both cohorts, those high-expression numbers hung in for about five days, before the dogs' immune systems started rejecting the alien human protein. Therefore, the ALG group is now tooling up to repeat the experiment in hemophiliac canines, engineering their own clotting factors.

"We'll be looking at the actual efficacy in those dogs," Hurwitz said. "Whether it restores their clotting activity."

He made the point that current therapy for human hemophilia is transfusion of donor blood or anti-clotting factors in response to every bleeding episode throughout life. "Those treatments are reactive, not preventative, as ours will be," he predicted.

Human Trials Likely Next Year

ALG estimates the worldwide hemophilia population at 600,000, of whom 20,000 are in the U.S. "Hemophilia patients spend an estimated $100,000 annually for these reactive infusions of clotting factor, with prophylactic therapy costing three times as much," Hurwitz observed.

Hurwitz foresees initial clinical trials will follow in 1999. "In fact," he observed, "we are already starting some studies in obtaining bone marrow stromal cells from humans."

Beyond hemophilia lie other candidate disease entities for stromal cell gene therapy.

"We believe that it really has great potential in the treatment of other diseases," he said, "because of the fact that stromal cells home to the marrow. That's a beautiful source for production of product that needs to go directly into the bloodstream, as opposed to intramuscular expression.

"We've done some preliminary studies of human growth-factor deficiencies," he observed. "Also in terms of cancer treatment, particularly in using the anti-angiogenic factors, because one can engineer the stromal cells, put them back into the patient, and get continuous expression over potentially long periods of time against metastases, promoting remission."

Then there's rheumatoid arthritis.

"This is a systemic, as well as a specifically articular, disease," Hurwitz said. "So, the interleukin-1 receptor antagonist protein looks very promising in treating rheumatoid arthritis." *