Immune Deficiencies : New Genetic Experiments Spark Hope

On first glance, Laura Cay Boren looks like any normal 3-year-old. But all her life she has been severely ill, suffering with a rare immune deficiency disease that leaves her vulnerable to many life-threatening infections.

Because of this inherited condition, Laura Cay cannot go to school or even play with other children. She learns the numbers and the alphabet from her mother. She leaves her Shelbyville, Ky., home only for visits to the doctor or the hospital. And she requires six to eight medicines a day, intravenous feedings 12 hours a day and oxygen around the clock.

Two transplants from her father’s bone marrow have failed to cure her body’s inability to fight infections.

‘Bubble Boy’ Disease

Laura Cay has “ADA deficiency,” the same disease that afflicted “David,” the boy who lived in a bubble in a Houston hospital for 12 years before he died in 1984.

But for all her problems, Laura Cay has reason for hope. Having lived longer than many children with severe ADA deficiency, she is now on the precipice of a fast-moving medical frontier, commonly called gene therapy.

Traditionally, doctors have used medicines or surgery to treat illnesses. Never before have they attempted to cure disease by adding a laboratory-engineered gene to a patient’s own cells.

But now, as a result of a crescendo of advances over the last decade in recombinant DNA technology, scientists, mostly in the United States, have learned to isolate and copy human genes, package them in genetically engineered viruses and--perhaps soon--insert these genes into the bone marrow cells of children like Laura Cay.

The hope is that such a replacement piece of the genetic code will trigger her bone marrow cells to produce large quantities of the missing ADA enzyme, whose absence causes the accumulation of toxic molecules that poison her immune system.

Adenosine deaminase deficiency is so rare that physicians know of fewer than 100 cases. But because this single-gene defect is simple compared to other diseases and is seemingly well understood, it is considered a prime candidate for gene therapy.

‘Only Ultimate Hope’

Laura Cay’s physicians say outright that gene therapy is probably her “only ultimate hope.”

“If gene therapy doesn’t work for ADA deficiency, it probably won’t work for anything,” said Dr. W. French Anderson of the National Institutes of Health, who leads one of the research teams that intends to submit one of the first proposals to perform human gene therapy. His group’s work has focused on ADA deficiency.

If gene therapy works in patients like Laura Cay, the potential ramifications could be far-reaching. The most immediate beneficiaries of this unprecedented technology would likely be children--who may have the most to gain.

In all, more than 3,000 genetic diseases have been discovered, as have additional genetic factors that contribute to heart disease, some cancers and diabetes.

Most genetic diseases are either poorly understood or too complex for gene therapy. But scientists hope that the more common single-gene diseases--such as cystic fibrosis, hemophilia or sickle cell anemia--eventually also can be candidates for gene therapy.

The procedure is also being contemplated for other single-gene defects where the gene has been cloned, including another immunodeficiency disease called PNP and the Lesch-Nyhan syndrome, a disease that causes mental retardation and self-mutilation.

Such single-gene defects may be rare individually, but together they affect 1% to 2% of all newborns and are responsible for 6% of hospitalizations of children.

Federal approval for such experimental treatments is likely within the next several years.

Debate on Ethics, Safety

Because of the revolutionary nature of such experiments, however, their safety, effectiveness and ethics have been subjected to one of the most extensive debates and scrutiny in the history of medical science.

The ongoing review is sparked by the specter of misguided attempts to create “more perfect” humans. Parents might ask physicians, for example, to place an additional growth hormone gene into cells of a child who is likely to be short.

In gene therapy, DNA, the molecule that contains hereditary information, is removed through genetic engineering from a type of virus called a retrovirus, which can be found in both humans and animals. The DNA is then replaced with the normal gene for the missing human enzyme.

In the laboratory, the retrovirus infects the bone marrow cells and inserts the gene randomly into the chromosomes. The blood cells that develop from the bone marrow cells are supposed to manufacture the deficient protein.

The critical challenge for researchers is getting these added genes to work.

“It is possible at any point we could hit an absolute dead block,” said the NIH’s Anderson.

Human Trials on Hold

Earlier in the year, the federal Recombinant DNA Advisory Committee had anticipated that it would have gene therapy proposals to review this year. But most researchers say additional technical problems still must be solved before human trials can begin.

“Don’t hold your breath,” said Dusty A. Miller of the Fred Hutchinson Cancer Research Center in Seattle.

In the last six months, NIH researchers in Anderson’s lab have been testing gene addition in monkeys. So far the animals have produced only minuscule amounts of the necessary ADA enzyme. To consider human gene therapy, “we need 100-fold more activity,” Anderson said.

Despite the unknown long-term consequences of altering a human being’s genetic code, parents of children with crippling genetic diseases usually view gene therapy as one more option for their loved ones.

“It doesn’t scare me,” said Laura Cay’s mother, Linda Boren, 28. “If she were the first, it wouldn’t bother me, if that is what the doctors say she needs.”

Worth a Try

Added Dr. Sheldon Horowitz, a pediatric immunologist at the University of Wisconsin Hospital who has cared for a number of patients with ADA deficiency: “Parents look at their kids dying, and anything reasonable they would be willing to try. They don’t think about it as man interfering with God’s will.”

There are actually two tactics in gene therapy.

The first is “somatic cell” therapy, in which replacement DNA is inserted into body cells, such as the bone marrow. It is the only type for which the government’s Recombinant DNA Advisory Committee will review proposals.

“If there is a crippling life-threatening disease and the only medical option available is (somatic cell) gene therapy, the decision is up to the doctor, the patient and federal authorities,” said Jeremy Rifkin, an activist who has repeatedly called for a go-slow approach in the field.

But it is the less severe cases that raise “larger ethical questions,” said Rifkin, president of the Foundation on Economic Trends in Washington. “We all have various forms of physical handicaps, from acne to myopia. Where do we draw the line?”

Far more controversial than “somatic cell” therapy is “germ line” gene therapy. It would allow recipients to pass on a new gene to their succeeding generations because replacement DNA would be inserted into reproductive tissue.

Enhancing Characteristics

Some find the notion of “germ line” therapy objectionable because it raises the possibility of enhancing human characteristics, such as height or appearance, and therefore might tempt unscrupulous scientists to perform unsupervised experiments to “improve” the human race--with potentially catastrophic consequences.

In the laboratories, “germ line” therapy has been performed in animals--to create, for example, giant mice.

The initial human gene therapy experiments will closely parallel the established techniques for transplanting human bone marrow, which produces the body’s red and white blood cells and platelets.

Since the first successful transplant in 1968, more than 5,000 patients have received transplants for ADA deficiency and other genetic diseases as well as for aplastic anemia and leukemia, with mixed but improving results.

Some leaders in the field, such as Dr. Robertson Parkman of Childrens Hospital of Los Angeles, believe that a genetic disease must first be shown amenable to successful bone marrow transplant treatments--at least in some patients--before gene therapy can be considered for others with the same disease who, like Laura Cay Boren, do not respond to bone marrow transplants.

Therefore, the Lesch-Nyhan syndrome that causes mental retardation and self-mutilation, for example, is a less likely candidate for gene therapy than ADA deficiency because bone marrow transplants have not worked for Lesch-Nyhan patients, according to researchers like Parkman.

Marrow Transplants

In Laura Cay’s two marrow transplants, both done at the Duke University Medical Center in Durham, N.C., normal bone marrow cells first were removed from her father. The cells likely to damage Laura Cay’s tissues and cause the transplant to fail then were separated and removed. Finally, the remaining cells were infused through a vein into the girl.

Her father’s cells migrated through her bloodstream to the bone marrow, where they multiplied and produced blood cells.

When a patient has a brother or sister whose bone marrow is perfectly “matched,” and thus is less likely to be rejected, diseases such as ADA deficiency can usually be cured because the donor’s bone marrow contains the missing ADA gene.

Difficulties arise when a patient, like Laura Cay, lacks a “matched” sibling, which happens about 70% of the time. “Mismatched” transplants--usually from a parent, the next best source--are more likely to fail because of incompatibilities between the bone marrow cells and the patient’s tissue. In severe cases, death or worsened disease may result.

In recent years, physicians, including those at Duke, have developed experimental techniques to successfully treat an increasing number of children with incomplete immune systems through “mismatched” transplants. But the two such attempts did not work for Laura Cay.

Since March, the child also has been treated with another form of experimental therapy--a manufactured form of the missing ADA enzyme that is given as a shot into the muscle. It isn’t known yet whether this treatment will help Laura Cay or not, although her doctors are not overly optimistic.

Laura Cay became sick right after birth with a rare form of pneumonia called pneumocystis pneumonia, her mother recalled in an interview. The child also developed skin infections from her baby shots.

At age 3 months, her physicians in Kentucky, after reviewing blood test results, recognized that the child had a serious underlying illness.

They referred her to Dr. Rebecca Buckley, an expert in childhood immune deficiency diseases at Duke.

To pay for her subsequent bone marrow transplants, $125,000 was raised after newspaper and television publicity.

After each transplant, about a year apart, laboratory tests showed that Laura Cay’s immune function became normal--but only for several months, until her body rejected the father’s cells.

The rejection episodes, which occurred while she was still hospitalized, were subtle, causing the child to lose her appetite and stop gaining weight, Buckley explained.

“Gene therapy is probably going to be Laura Cay’s only ultimate hope,” she said. “But I’ve talked with her mother and told her we don’t know if that will be next year or in five to 10 years.”

Unlike bone marrow transplantation, in human gene therapy the patient’s own bone marrow is removed, treated and infused back into the body, thus eliminating the “matching” problem. The original aberrant gene is not affected.

Bone marrow cells are an excellent vehicle for gene addition, because they can be easily removed, manipulated in the laboratory and then returned to the body.

Additional Hurdle

But for Anderson and his NIH group, who are using monkeys to study ADA deficiency, there was an additional hurdle. Because monkeys--one of the closest animal models to people--do not develop ADA deficiency, the researchers first had to bombard them with radiation, thus destroying their bone marrow cells, to allow the transplants to take.

Initially, eight monkeys died when viral infection damaged their treated bone marrow cells, Anderson said. The next four monkeys survived, although there were complications. The next two did well from the start, he said.

Researchers are now trying to determine if the treated cells survive and multiply and make sufficient amounts of the deficient protein when returned to the monkeys. Scientists agree that this latter step of protein manufacture, called gene expression, is the major obstacle to be surmounted.

Researchers also have yet to learn how to control where the gene is inserted. Each human gene has a specific location on a chromosome. They are concerned that random placement of the additional DNA in the chromosomes by a virus may upset delicate cell functions--perhaps, for example, activating tumor-causing substances, called oncogenes, within cells.

To address safety concerns over human gene therapy, “self-inactivating” viruses are now being designed and will first be tested in several animal species. These modified viruses are capable of delivering the missing gene but are unable to reproduce or spread infection. If the viruses could reproduce, they might spread and cause an infectious disease in the patient or in family or medical personnel who have contact with the patient.

The ultimate question--whether gene therapy will make the immune system normal--can only be answered after patients like Laura Cay are treated.

Extensive Review

The initial proposals to perform human gene therapy will receive an extensive review that may take six months or more by various committees and officials at the National Institutes of Health and the Food and Drug Administration. The only comparable review of scientific investigations is the scrutiny given the initial recombinant DNA research in the 1970s, which led to many restrictions and safeguards for such experiments, many of which have been subsequently relaxed.

The federal review is only to determine whether human gene therapy trials, as a matter of practice, should be allowed to proceed. Individual parents and physicians still must choose between gene therapy or alternative treatments, like “mismatched” marrow transplants.

There is a small network of physicians at academic medical centers throughout the country who care for most of the patients likely to be eligible for the first trials, and they maintain close contact with the gene therapy research groups, including groups at NIH, Harvard and Baylor universities, the University of Washington and University of California at San Francisco and San Diego.

Federal officials are mindful of the controversial--and unauthorized--human gene therapy experiments conducted in 1980 by Dr. Martin Cline of UCLA Medical Center in Israel and Italy.

Cline, a hematologist, mixed bone marrow cells from two patients suffering from the blood disease beta-thalassemia with DNA coding for normal hemoglobin and then returned the cells to the patients.

UCLA officials had refused to approve the research because they believed that more work in animals had to be done first. After Cline’s experiments became public, NIH imposed sanctions against his work and stripped him of some of his research funds.

No Change in Patients

The patients were neither better nor worse after the therapy, Cline said in a recent interview. Both are still living.

“Gene therapy, at least for certain diseases, is now widely accepted (by the public),” Cline said. “It is clear that we are not going to artificially create a series of monsters.”

To avoid a repetition of such unapproved human experiments, the federal Recombinant DNA Advisory Committee has prepared a detailed “points to consider” document, giving researchers a step-by-step outline of how to design and submit their plans for review.

In sharp contrast to the normal review of research behind closed doors, the proposals will be public documents from the time they are submitted and are to be debated in open meetings at NIH.

“We feel that the public and opinion leaders who speak on behalf of the public are satisfied that somatic cell gene therapy is being adequately planned and that there are appropriate safeguards,” said Prof. LeRoy Walters of the Kennedy Institute of Ethics at Georgetown University. Walters chairs the committee’s working group on human gene therapy.

“In my more optimistic moments, I hope that we have discovered a model to apply in the future when a radically new area of therapy begins to open up,” he said.

In Shelbyville, Ky., Linda Boren also is hopeful. “I hope medical science can give me a normal child, just like yours.”

ADVANCES IN GENE RESEARCH

Here are some of the key scientific advances that have made human gene therapy a possibility:

1865: Gregor Mendel postulates the existence of specific inherited factors, later called genes.

1953: Francis Crick and James Watson determine the structure of DNA, the substance of which genes are made.

1966: Scientists establish the complete genetic code, which is the information carried by the DNA molecule that determines physical traits and other human characteristics.

1970: Discovery of retroviruses, the best current vehicle for inserting genes into cells.

1972: Scientists create recombinant DNA molecules.

1973: Researchers learn to make large quantities of human genes, from which proteins such as insulin or growth hormone are manufactured.

1982: Researchers perform successful gene therapy in fruit flies, correcting an eye color defect. Experiments show that the normal gene can be transmitted to subsequent generations. In the same year, “supermice” are born and grow to twice their normal weight after researchers inject the cloned gene for rat growth hormone into fertilized mice eggs.

1985: Viruses are used to transfer genes into mice bone marrow cells, which in turn make substantial amounts of protein from the added gene.

1986: First gene therapy experiments in monkeys in progress at the National Institutes of Health.

HOW HUMAN GENE THERAPY WORKS In human gene therapy, the patient’s own bone marrow is removed. In the laboratory, a place of the genetic code, created with recombinant DNA technology, is added. Then the marrow cells are infused back into the body. Gene therapy will likely be used first for a severe childhood disease, such as ADA deficiency, which cripples the body’s ability to fight infections. THE PROCEDURE 1. The patient’s own bone marrow cells with defective immune system gene are obtained. 2. In the laboratory, through genetic engineering, DNA is removed from a virus--called a retrovirus, and replaced with the normal gene for the missing human enzyme. 3. The retrovirus with the normal human gene is mixed with the bone marrow cells, infecting them. 4. The retrovirus inserts the normal gene into the chromosomes of the bone marrow cells where it becomes part of the cell’s genetic makeup. 5. The repaired cells are returned to the body through a vein. They migrate through the blood stream to the bone marrow, where they multiply and produce the deficient protein, restoring immune function. THE CHALLENGES

Some researchers feel that successful treatment of a disease by bone marrow transplant between individuals is a prerequisite to human gene therapy, because bone marrow is the vehicle that will be used to introduce the new gene. Patients who lack suitable donors for marrow transplants may be candidates for gene therapy.

Researchers are developing “self-inactivating” viruses that are capable of delivering the missing gene but would not reproduce or spread infection.

The gene must be permanently inserted into a large number of bone marrow cells, without killing or otherwise altering the cells.

The greatest challenges are learning how to coax the bone marrow cells to make sufficient amounts of the deficient protein and seeing if the body’s immune system responds by becoming normal.