O, Brave New World That Has Such People In It ...'

Gene Therapy Stirs A 'Tempest' In Some Circles, But IU Researchers Prudently Advance It With Basic Science, Safety And Collaboration.

It's a field that began with an explosion of publicity a decade ago, later was criticized by leading scientists who said it lacked a research foundation, and was shaken by the death of a young patient last year on the east coast.

Meanwhile, a group of physicians and scientists at Indiana University School of Medicine has been carefully crafting a nationally respected gene therapy program built on a decade of basic science research.

• Techniques developed at IU for inserting genes into cells are being adopted as standard procedure in gene therapy human trials around the world, including a French experiment that is believed to be the first to actually cure a human disease.

• IUSM now hosts the sole federally funded facility for manufacturing genetically engineered, virus-based vectors used to insert genes into cells.

• Three Phase I human trials have been successfully conducted at IU, including one that resulted in the highest levels of gene transfers into patients reported so far.

• Several more trials are planned, including experiments targeting prostate cancer and - eventually - heart disease as well as other cancers and genetic diseases of the blood.

IUSM's program is different than some because the School did not simply decide to create a gene therapy program, says David A. Williams, MD, Freida and Albrecht Kipp Professor of Pediatrics and a Howard Hughes Medical Institute researcher at the Herman B Wells Center for Pediatric Research.

"Here it occurred by basic scientists beginning to work together collaboratively," says Dr. Williams. "As the science progressed, basic scientists sought input from clinical researchers and clinicians in various fields to translate our findings into rational studies involving human subjects."

Dr. Williams adds, "This led to the development of the highly interactive group of scientists, physicians and research nurses called the Gene Therapy Working Group (GTWG), which became recognized and funded by the National Institutes of Health and continues to develop and implement clinical trials in gene therapy at IU."

False Start

Ten years ago, when Dr. W. French Anderson and colleagues at the National Institutes of Health (NIH) first injected modified blood cells into two young girls suffering from Severe Combined Immune Deficiency, the excitement - and the hype - was enormous.

Yet, just five years later, an NIH-appointed committee of scientists concluded there was no definitive evidence that gene therapy had helped any patient. Moreover, the committee judged that not enough basic research had been done on either the diseases being treated or the therapeutic tools being used.

But the field took its biggest jolt last year after the death of a young patient participating in a gene therapy trial in Pennsylvania. Soon after, the federal government announced several initiatives to stiffen reporting requirements and ensure that scientists understand and follow requirements for protecting human research subjects.

In the aftermath of this most recent controversy, IU researchers audited the records of all patients who had participated in gene therapy trials, says James M. Croop, MD, PhD, associate professor of pediatrics and leader of the GTWG. "We felt very comfortable and were very pleased with the results of our internal audit," says Dr. Croop.

Franklin O. Smith, MD, professor of pediatrics, medicine, microbiology and immunology, and director of the stem cell transplantation program at Riley Hospital for Children, whose duties include coordinating the regulatory aspects of the GTWG, says the level of federal oversight could easily reach the point where it's "cumbersome and inhibitory."

"The investigator has got to take responsibility," notes Dr. Smith. "Bad clinical research places patients at risk whether it is a gene therapy trial or any other type of clinical investigation. We've got good rules. It's a matter of investigators knowing them and following them." The investigator has got to take responsibility... We've got good rules. It's a matter of investigators knowing them and following them."

In The Beginning...

IUSM's gene therapy programs developed from a decade of research by IU scientists and physicians studying the fundamental biology of the blood system and cancer - researchers in pediatric and adult hematology and oncology, and at institutes such as the Wells Center for Pediatric Research and the Walther Oncology Center. The programs came of age in 1994 when the School received a $4.7 million NIH grant to establish a center of excellence in molecular hematology. It received competitive renewal funding in 1999.

The grant cemented the establishment of specialty operations, or "cores," around which the gene therapy efforts were built: specialty operations in stem cells, gene therapy vector production, molecular biology and research mice. Members of the Gene Therapy Working Group meet on a near-weekly basis to collaborate on projects. Face-to-face communication is vital as gene therapy trials become more complicated, interdisciplinary and expensive.

"It's really not very likely that any one given person is going to have the broad expanse of knowledge and the ability to put one of these complex protocols together," says Dr. Williams.

Like all Phase I trials, the gene therapy trials at IUSM to date have been mainly concerned with safety. So far, no ill effects from gene therapy have appeared. But the researchers must follow the patients for the rest of their lives, keeping an eye out for several potential, if unlikely, problems.

The virus-based vectors used to insert genes into cells are engineered to ensure that the viruses cannot replicate themselves once in the body. But we all carry bits of old retroviral DNA in our genome, and theoretically, it could link up with the vector to create a new "replication-competent" virus, explains Dr. Croop. The insertion of the new genes might also cause the creation of new cancers or immune system reactions to any of the proteins or other components used in the gene transfer process.

While safety is the main focus of these early experiments, scientists also want to know whether they're successfully introducing the new genes into their patients. In each of the initial trials, the researchers have been able to detect the presence of functioning versions of the genes in the patients who received them.

In fact, Rafat Abonour, MD, associate professor of medicine and medical director of the Stem Cell Laboratory, and his colleagues reported in the June 2000 issue of Nature Medicine that they had found evidence of the gene they inserted into patients more than a year later, at higher levels than have previously been reported by any other researchers.

"At each step we were very excited," says Dr. Abonour. "We were elated when we saw the amount of positive cells at one month; we were very excited to still see them at six months and we were more excited to see them at twelve months."

Promise Of Discovery

The excitement is understandable, because getting genes into bone marrow cells to work their magic long term turns out to be just plain hard to do. Whether to protect bone marrow cells from chemotherapy drugs or to fix an immune disorder, physicians would like to insert their genes into the DNA of stem cells, the relative handful of "mother" cells that are responsible for the billions of blood system cells the body produces every day.

To get the genes there, most IU researchers have recruited the retrovirus, which when properly engineered can carry a gene into a cell and insert it permanently into the cell's DNA. However, it can only do so when the cell is dividing, and stem cells spend most of their time not dividing. Moreover, human stem cells have relatively few surface receptors to which the retroviral vectors can attach.

To circumvent these problems, physicians remove blood from the patient, then separate the stem cells and other early-generation cells called progenitor cells (current technology cannot separate stem cells alone). The cells then are mixed with growth-stimulating proteins called cytokines to cause them to begin proliferating. Next, the gene-toting retroviral vectors are mixed with the patient's cells in hopes that a good number will be "transduced" with the vectors. Then the cells are given back to the patient, in hope that the stem and progenitor cells are able to find their way back to the bone marrow.

IU researchers have helped boost the percentage of stems cells transduced and returned to the patient's bone marrow by four-fold in recent years, thanks to a variety of improvements, such as painstakingly finding the optimum cytokine mixture used to stimulate the bone marrow cells.

Most notably, however, researchers now mix the cells and vectors in combination with fragments of a genetically engineered human protein called fibronectin. Dr. Williams and his research team discovered in the mid-1990s that fibronectin worked like Velcro, snagging the cells and vectors and forcing them to snuggle together.

"Fibronectin wasn't discovered because we were looking for something for gene therapy. It was discovered because Dave's (Williams) lab was very interested in learning how bone marrow cells normally grow," says Mary Dinauer, MD, PhD, Nora Letzer Professor of Pediatrics and director of the Herman B Wells Center for Pediatric Research.

Today the process, which was patented by IU, has become a standard part of the transduction process. It is a perfect example of how discoveries in the lab can serendipitously advance clinical care, and of the great rewards made possible through a strong, collaborative scientific environment.