The second act of genetic healing / Ricky Lewis

A decade and a half after a series of tragic failures led to a critical reappraisal of gene therapy, scientists say it can be used again

The field of gene therapy (gene therapy) can finally realize the hopes placed on it: an experimental use of this process, in which healthy genes are inserted anywhere in the body where they are needed, has restored sight in the last six years to 40 people who suffered from a certain type of hereditary blindness. Doctors received unprecedented results in their treatment of more than 120 patients with various types of blood cancer and some of them remained cancer-free three years after treatment. Researchers have also used gene therapy to allow some patients with hemophilia, a potentially fatal excessive bleeding disease, to extend the periods of time in which dangerous incidents do not occur and without the need for high doses of blood-clotting drugs.

The positive results are even more impressive considering the shutdown of the gene therapy field 15 years ago following the untimely death of Jesse Gelsinger, a teenager who suffered from a rare digestive disease. Gelsinger's immune system responded to the gene therapy with an acute and surprising counterattack, which caused the boy's death. So it became clear that the early successes of gene therapy in the 90s had raised unrealistically high expectations among doctors and researchers to the point of overconfidence.

This case and other obstacles forced the scientists to rethink some of their approaches, and to be more realistic about the applications of gene therapy in the treatment of humans in various conditions. The researchers curbed their hopes and returned to basic research. They looked at potentially fatal side effects, like those seen in Gelsinger, and learned how to prevent them. And they paid more attention to explaining the risks and benefits to volunteers and their families.

For many, the turning point was six years later, when doctors treated Corey Haas, an eight-year-old boy who suffered from an inherited degenerative eye disorder that causes blindness. The gene therapy they used allowed the damaged retina in Haas's left eye to produce a protein that his body could not produce in any other way. Four days after the treatment Cory visited the zoo and was amazed when he saw a hot air balloon and the sun for the first time. Three years later he was treated in the same way in his right eye. Today Cory's eyesight has improved to the point that he joins his grandfather on turkey hunts.

Today gene therapy is still not available in hospitals and clinics, but this situation is going to change in the next decade. In Europe, in 2012, a gene therapy was approved for a rare disorder known as "familial deficiency of the lipoprotein-lipase enzyme" which causes severe pain. In late 2013, the US Institutes of Health (NIH) removed some of the regulatory restraints that the agency thought were no longer necessary. The industry predicts that the US will approve the commercial use of gene therapy in 2016. Gene therapy is finally beginning, after its lost decade, to fulfill its purpose as a revolutionary medical treatment.

heartbreak

The early failures of gene therapy made it clear how difficult it is to establish a safe and effective way to deliver genes to a target tissue. All too often the safest methods of gene transfer are ineffective, and some of the most effective methods have turned out to be unsafe enough and have caused an overwhelming reaction of the immune system, as in Gelsinger's case, or the development of leukemia.

To understand what triggers these side effects or to learn how to reduce the risk of them happening, the scientists focused on the most common gene transfer system for gene therapy: an engineered virus that would act as a microscopic injection gun.

At first, the researchers remove some of the genes of the virus itself to make room for the healthy genes they want to transfer to the patient. (This step is also intended to prevent the virus from replicating itself inside the body, thus increasing the risk of an immune reaction.) Then the engineered virus is injected into the person, and the virus inserts the genes into certain cells, according to its type.

When Gelsinger volunteered for a clinical trial, the common gene delivery system was adenovirus, a virus that in its natural state can cause mild upper respiratory tract infections in humans. Scientists at the University of Pennsylvania then determined that injecting the virus into the liver, where the cells that normally produce the enzyme missing in Gelsinger are located, had the highest chance of success. They inserted an active copy of the gene encoding the enzyme into an adenovirus that had some of its own genes removed and injected a trillion viruses, each carrying the intended payload, directly into the liver.

But some of the viruses made a tragic detour in Gelsinger's body. They entered the liver as planned, but also infected a huge number of macrophages, large, migratory cells that serve as sentinels of the immune system, and dendritic cells, which signal invasion. The immune system responded by destroying all the infected cells, a violent process that eventually destroyed Gelsinger's body from the inside out.

The wild reaction of the immune system surprised the researchers. None of the 17 volunteers who received such treatment for the same disorder had such serious side effects. Researchers knew that an adenovirus could provoke an immune response, but they did not imagine how explosive it could be, since until then they had not predicted such a response, except for the death of a monkey in one of the studies that used slightly differently engineered viruses. "Humans are much more versatile than animal colonies," says James Wilson of the University of Pennsylvania, who developed the gene transfer system used by the researchers in the clinical trial in which Gelsinger participated. "What we saw in the experiment was that in one individual out of 18 there was an exaggerated reaction of a host." In retrospect, it seems that it would have been correct to inject fewer viruses carrying the gene into his body, billions instead of a trillion. Another criticism leveled at the researchers was that they did not inform Gelsinger and his family of the monkey's death so that they could decide for themselves whether it was an event unrelated to the experiment.

Gelsinger's death was not the only tragic event in the genetic healing. Treatment for another disorder, severe combined immunodeficiency (SCID-X1), soon after caused five of 20 children to develop leukemia, which even resulted in the death of one of them. Once again it turned out that the culprit was the gene transfer system. In this case the microscopic injection gun was a retrovirus, a type of virus that injects the genetic cargo directly into the cell's DNA. However, the place where the healing gene is inserted into the DNA is somewhat random, and sometimes it happens that the retrovirus inserts the payload into an oncogene, a gene that under certain circumstances can cause cancer.

rethinking

When the researchers knew about adenovirus's propensity to trigger deadly immune responses and retrovirus's propensity to trigger cancer, they began to see if other viruses had better results. They soon zeroed in on two suitable common candidates. The first of the new gene transfer systems is based on a virus related to the adenovirus (AAV) that does not cause disease in humans (although most of us are infected with it at least once). Since the virus is so common it is unlikely that it provokes extreme immune responses. This virus has another feature that helps reduce the side effects: it is available in several forms (serotypes) and each of them favors certain types of cells or tissues. For example, AAV2 works well in the eye, AAV8 prefers the liver, and AAV9- gets into heart and brain tissues. Researchers can select the best AAV for a particular organ in the body, thereby reducing the number of viruses injected and reducing the risk of an acute immune response or other undesirable outcomes. On top of that, AAV unloads the genetic cargo outside the chromosomes, so it cannot randomly cause cancer through oncogene disruption.

The first clinical trial of AAV was done in 1996 in the treatment of cystic fibrosis. Since then, 11 serotypes of the virus have been identified, and parts of them have been mixed and matched to engineer hundreds of delivery vehicles that appear to be safe and selective. Research is now being conducted on gene therapy using AAV for several diseases that affect the brain, including Parkinson's and Alzheimer's, as well as for hemophilia, muscular dystrophy, heart failure and blindness.

The second gene transfer system is surprisingly based on a version of HIV, the virus that causes AIDS, with some of its genes removed. If the killer image of HIV is ignored, its benefits in gene healing are becoming more and more apparent. HIV belongs to a particular family of retroviruses, called lentiviruses, which are able to evade the immune system, a necessary property for retroviruses, which normally do not bother oncogenes.

After removing the genes that make HIV deadly, what remains is a viral envelope "with a large capacity," says Stuart Naylor, former senior chief scientist at Oxford Biomedica in England, which specializes in gene-based drugs for eye diseases. Unlike AAV, which is a small virus, HIV is "large enough to carry many genes or one large gene," he says. "It is non-toxic and does not create an immune counter-reaction." Engineered lentiviruses are now being used as tools in several clinical trials, including the treatment of adreno-leukodystrophy (ALD), the fatal disease depicted in the 1992 film Lorenzo's Oil. Some of the children who received the treatment have already recovered enough to return to school.

Although the use of AAV and HIV in clinical trials is expanding, researchers are also re-engineering and modifying older gene delivery systems so that they can be used in certain circumstances. For example, scientists are developing non-HIV retroviruses that will inactivate themselves before causing leukemia.

Even adenovirus, the virus that caused Gelsinger's death, is still being used in clinical trials for gene therapy. The researchers limited its use to areas of the body that are not expected to provoke an immune response. One of the promising uses is the treatment of "dry mouth" in patients undergoing radiation therapy for head or neck cancer. The radiation damages the salivary glands, which are located just below the surface of the inner part of the cheek.

Researchers at NIH are conducting a small clinical trial that involves inserting into the glands a gene that encodes a protein that serves as a channel for water to enter. Because the glands are small and circumscribed, and the experiment uses 1,000 times less virus than the number of viruses used in Gelsinger's treatment, the chance of an exaggerated immune response occurring is considerably less. In addition, viruses that do not penetrate the target cells will reach the saliva of the patients, from where they will be eliminated by swallowing or spitting, therefore there is little chance of "upsetting" the immune system. Six out of 11 people who received the treatment since 2006 produce much more saliva. Dentist and biochemist Bruce Baum, who led the research before retiring, calls the results "quite encouraging."

New goals

Encouraged by the successes, medical researchers moved from treating hereditary diseases to attempts to repair genetic damage that occurs naturally over time. Scientists at the University of Pennsylvania, for example, are using gene therapy to treat a common childhood blood cancer: acute lymphoblastic leukemia (ALL).

About 80% of children with ALL respond to chemotherapy. Researchers are now trying to turn to gene therapy to encourage the children's immune system cells that do not respond to treatment, to find and destroy the rebellious cancer cells.

The experimental approach is particularly complicated and is based on a technology that includes the hybridization or chimera of the CAR receptor antigen. Like the chimera in Greek mythology made of different animals, an antigen receptor chimera consists of two molecules of the immune system that are not normally connected. The chimera is expressed in the patient's T cells and allows them to recognize proteins that are found on leukemia cells in a greater quantity than normal cells. The engineered T cells, armed with a chimera, are returned to the patient's body and destroy the cancer cells. The first experimental patients were adults with chronic leukemia, and they responded well to the treatment. The next experiment was done on a girl, and its results exceeded the researchers' wildest dreams.

Emily Whitehead was five years old in May 2010, when she was diagnosed with leukemia. Two series of chemotherapy treatments did not help, and in the spring of 2012 she underwent a third series. "She received a dose of chemotherapy that would have killed an adult, but she still had metastases in her kidneys, liver and spleen," says Bruce Levin, one of the doctors who treated her. The girl was on the verge of death.

The doctors took a blood sample and isolated some T cells. The cells were injected with an antivirus that carried the appropriate genes. After a difficult start in which, fortunately, Whitehead responded to treatment, her condition improved rapidly. Three weeks after the treatment, a quarter of the T cells in her bone marrow carried the genetic correction. Her T cells homed in on the cancer cells, which soon disappeared. "In April she was bald," Levin recalled, "and in August she went to her first day of second grade."

Although the engineered cells will probably not remain forever, and when they are gone the doctors can repeat the process, the beautiful girl with wild brown hair has been cancer free for two years. And she is not alone. In late 2013, several groups of researchers reported that they had used the CAR technique to treat more than 120 patients who had leukemia of the type Whitehead had and three other types of blood cancer. In five adults and in 22 out of 29 children the disease regressed and they are now cancer free.

to the clinic

Now that they have safe viral gene transfer systems in hand, gene therapy experts are about to face the biggest challenge any new drug faces: getting approval from the US Food and Drug Administration (FDA). This daunting phase involves phase III clinical trials, which last one to five years (times vary widely) and are supposed to measure the effectiveness of the cure in larger groups of volunteer patients. At the end of 2013, about 5% of about 2,000 clinical trials in gene therapy had reached phase III. One of those that have come closest to the goal is the treatment of a hereditary degenerative eye disease (LCA)), the disease that robbed the child of Hass of his sight. So far, the corrective genes have been inserted into the eyes of several dozen patients, and all patients can now enjoy the world's best.

China was the first to approve, in 2004, gene therapy for head and neck cancer. In 2012, Europe approved the use of the drug Glybera for the treatment of gene therapy in familial deficiency of the lipoprotein lipase enzyme. Active copies of the mutant gene packaged in AAV are injected into the leg muscles. The Dutch company UniQure is conducting preliminary negotiations with the FDA regarding the approval of the drug in the United States. One possible obstacle in the way is the price of one cure dose: $1.6 million. But the price may decrease following the development of more efficient procedures.

As with many medical technologies, the path to achieving successful genetic healing, which has been going on for several decades, was and still is full of pitfalls and far from over. However, as success stories like those of Corey Haas and Emily Whitehead accumulate, gene therapy will be an acceptable treatment for some diseases and a promising treatment option for other diseases.

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About the author

Ricky Lewis (Lewis) is a science reporter with a PhD in genetics. She has written several non-fiction books, many articles in newspapers and the book: "Repairing the Worlds: Geni Healing and the Boy Who Saved Him" ​​(St. Martins Publishing, 2012)

in brief

Early excitement about gene therapy experiments in the 90s raised unrealistic expectations about the technology's potential for use in humans.

After several tragic failures, the researchers set aside several years to improve the understanding of basic biology and the techniques involved in the field.

New and safer treatments are about to enter the clinic. Europe approved the use of gene therapy in 2012. The United States will likely follow suit in 2016.

Two options for gene transfer

In addition to injecting viruses directly into patients, researchers can collect cells from the body, inject into them the viruses carrying the healing gene, and inject back the engineered cells. Since the corrected genetic information is contained in the DNA of the cells, the correction will be passed on to all the daughter cells that are created.

increasing safety

Researchers reduce the risk of causing cancer or a dangerous attack of the immune system by carefully choosing the type of viruses they use, limiting their number or limiting the tissues treated.

And more on the subject

Gene Therapy of Inherited Retinopathies: A Long and Successful Road from Viral Vectors to Patients. Pasqualina Colella and Alberto Auriccio in Human Gene Therapy, vol.8, No.23, pages 796-807; August 2012. http://www.ncbi.nlm.nih.gov/pubmed/22734691

The American Institutes of Health's gene therapy website:

Animation video about liver gene healing in the online version of the article on the Scientific America Israel website, www.sciam.co.il

The article was published on the Scientific American Israel website