Genetically Modified Tissue Implanted in Brain for Alzheimer's disease

The first surgical implantation of genetically modified tissue into the brain of an Alzheimer's patient took place on April 5, 2001 at UCSD's John and Sally B. Thornton Hospital in La Jolla.

In a groundbreaking procedure, physicians at the University of California, San Diego (UCSD) School of Medicine have surgically implanted genetically modified tissue into the brain of an Alzheimer's patient. This launches the first phase of an experimental gene therapy protocol for Alzheimer's disease.

The 11-hour procedure was performed April 5 at UCSD's John M. and Sally B. Thornton Hospital in La Jolla on a 60-year-old Caucasian woman in the early stages of Alzheimer's disease. The patient is recovering well, and was discharged from the hospital Saturday, April 7.

The study, led by UCSD neurologist Mark H. Tuszynski, M.D., Ph.D., is the first attempt to use human gene therapy to treat a disease of the nervous system. The researchers will attempt to prevent cell loss in Alzheimer's disease using gene therapy to deliver a natural brain-survival molecule called nerve growth factor (NGF) to the dying cells in the brain. The surgical procedure was led by UCSD neurosurgeon Hoi Sang U, M.D., who implanted the tissue in the patient's brain using specially designed surgical tools.

This Phase I clinical trial, also called a "safety/toxicity" study, is designed to determine whether the gene-transplantation procedure is safe. The patient is a former teacher from Oregon, diagnosed with Alzheimer's disease three years ago. She and her family have requested anonymity.

"We have four children and one grandchild. If there is a genetic trail associated with this disease, we are concerned about their future," said the patient's husband. "Our main motivation is to see if we can contribute to patient care in the future by participating in this study. If there are benefits for my wife, that will be a plus."

According to Tuszynski, "NGF gene therapy is not expected to cure Alzheimer's disease, but we hope that it might protect and even restore certain brain cells and alleviate some symptoms, such as short-term memory loss, for a period that could last a few years."

This procedure targets a class of cells located deep within the brain in an area called the cholinergic system, important for supporting memory and cognitive function. The cholinergic system profoundly degenerates in the course of Alzheimer's disease. These cells have been shown to respond to NGF in primate studies, and researchers hope that preventing extensive loss of these cells may slow intellectual decline seen in Alzheimer's patients.

If the protocol is successful, implanted cells could begin to affect brain function in a month or two, but Tuszynski cautions that "it may take several years to test the procedure in a large enough number of patients to determine whether it will be useful therapy."

A second patient has been accepted for the UCSD clinical trial and is expected to receive the gene implant in about three months. Six additional persons with a confirmed diagnosis of early Alzheimer's disease, who are otherwise healthy, are being sought for this phase of the clinical trial.

THE PROCEDURE

The process leading to this first surgery began several months ago. A small sample of the patient's own skin cells was collected in a biopsy procedure, and NGF genes isolated from nervous system tissue were inserted. Over a three-month period the genetically engineered cells were grown in culture in a commercial-grade GMP (Good Manufacturing Practices) facility. As they divided and increased in number, they began producing large quantities of NGF.

Before implantation, the scientific team verified that the genetically engineered cells produced the appropriate amount of NGF, and that no harmful contaminants were present.

In the surgical procedure on April 5, the patient received five implants of modified cells, targeting a region located at the base of the frontal lobe called the nucleus basalis of Meynert. This area contains cholinergic cells, and undergoes profound degeneration in Alzheimer's disease, which is thought to contribute to the decline of cognitive function.

First, the precise coordinates of the targets in the brain were determined with the use of a device called a stereotaxic head frame, which identifies the three-dimensional location for the implantation of the tissue. Surgical instruments designed by U and Peter Amis were used to insure that the cells would be injected in exactly the right location. Magnetic resonance scanning further verified the targeted area.

U then made a small hole on the right side of the patient's skull, exposing approximately one inch of the brain's surface. A fine needle was inserted and the genetically modified cells were inserted through the needle.

The patient underwent memory and neuropsychological testing before the surgery. Over the next several weeks, she will be monitored for any adverse events, and physicians will continue measurements of cognitive function. The size and location of the NGF implants will be monitored by MRI scans. She will be closely monitored for a year and then evaluated annually for an indefinite period.

BACKGROUND

This clinical trial is based upon a large body of experiments performed first in rats, then in monkeys, over the last 12 years by Tuszynski, U and Fred Gage Ph.D., who was on the faculty at UCSD before joining The Salk Institute for Biological Studies in 1995 (Gage is President-Elect of the Society for Neuroscience).

Early studies in rats demonstrated the feasibility of NGF therapy. For these studies, the researchers infused NGF via pumps directly into fluid-filled areas of animal brains. Although damaged brain cells were regenerated, the NGF also caused a proliferation of cell growth where it was not intended. To more precisely target NGF to specific brain regions, Tuszynski and Gage utilized a gene therapy method for inserting NGF into cells.

Tuszynski, in collaboration with Gage and Jeffrey Roberts, D.V.M., of the UC Davis Regional Primate Center, continued to demonstrate the feasibility of this gene therapy procedure in primates over the next several years. Skin biopsies from monkeys were modified to produce and secrete NGF. Then, the modified cells were surgically grafted directly into the brain tissue of aged monkeys.

In a study published in the Sept. 14, 1999 issue of Proceedings of the National Academy of Sciences (PNAS), the Tuszynski team reported that 40 percent of cholinergic neuron cell bodies had shrunk and atrophied in normal monkey aging, but were returned to nearly normal size and quantity following the surgical implant of cells genetically altered to produce NGF.

In February 2001, the researchers reported in PNAS that essential cellular connections called axons were also restored in primate brains using genetically modified tissue implants. The axons, which are essential for transmitting messages to and from neurons within the brain, had shriveled up and disappeared in aged monkeys. However, in monkeys that received genetically engineered NGF cells, the axons were restored to normal levels, and sometimes exceeded those levels.

In 1999, the initiation of human trials was approved by the Food and Drug Administration and the protocol was reviewed by the National Institutes of Health Recombinant DNA Advisory Committee (RAC).

UCSD ALZHEIMER'S DISEASE RESEARCH CENTER

The current patient clinical trial is taking place through the UCSD Alzheimer's Disease Research Center (ADRC), established in 1984 as one of the five original Alzheimer's Disease Centers supported by the National Institute on Aging of the National Institutes of Health. Currently there are 30 ADRCs in the U.S.

Under the leadership of Leon J. Thal, M.D., chair of neurosciences at UCSD, the ADRC provides patient evaluation, community outreach and education, clinical trials and basic research. Thal is also a co-investigator in the current clinical trial with Tuszynski and U.

According to the Alzheimer's Association, one in 10 persons over 65 and nearly half of those over 85 have Alzheimer's disease. This neurodegenerative disorder is characterized by build-up of protein plaque and tangles, leading to loss of function and death of brain cells. Alzheimer's patients suffer progressive loss of mental functions such as memory and learning.

THE SURGICAL TEAM

The team involved in this first surgery included U and UCSD neurosurgeon John F. Alksne, M.D.; Tuszynski; UCSD neuroradiology fellow Timothy Duncan, M.D.; neurosurgery resident Soren Singel, M.D; anesthesiologists Piyush Patel, M.D., and Christine Yeun, M.D.; nurses Robin Adduano, R.N., and Kathy Rajner, R.N., and from Tuszysnki's laboratory, Armin Blesch, Ph.D., project scientist and Lee Vahlsing, M.S., research specialist.

Consulting on the surgery were Roy Bakay, M.D., neurosurgeon from Rush- Presbyterian Hospital in Chicago, and Phil Starr, M.D., Ph.D., neurosurgeon from UCSF. Also in attendance were Peter Amis, who developed the surgical instruments with U, and Fred Gage, Ph.D., from the Salk Institute.

Some of the technology being utilized in this procedure has been licensed for commercial development to a company in which the University of California and some of the investigators have a financial interest.

ALZHEIMER'S DISEASE

Alzheimer's disease is a progressive, neurodegenerative disease characterized by loss of function and death of nerve cells in several areas of the brain, leading to loss of mental functions such as memory and learning. One of the characteristic structural abnormalities found in the brains of individuals with Alzheimer's are amyloid plaques, clusters of dead and dying nerve cells, other brain cells and amyloid protein fragments. Upon autopsy, the presence of amyloid plaques and neurofibrillary tangles is used to positively diagnose Alzheimer's.

Alzheimer's disease is the most common cause of dementia, a term used to describe the loss of cognitive or intellectual function. First described by Dr. Alois Alzheimer in 1906, Alzheimer's disease usually begins gradually, causing a person to forget recent events or familiar tasks. How rapidly it advances varies from person to person, but the brain disease eventually causes confusion, personality and behavior changes, and impaired judgment. Communication becomes difficult as the affected person struggles to find words, finish thoughts, or follow directions. Eventually, most people with Alzheimer's disease become unable to care for themselves.

According to the Alzheimer's Association, one in 10 persons over 65 and nearly half of those over 85 have Alzheimer's disease. Today, four million Americans have Alzheimer's disease. Unless a cure or prevention is found, that number will jump to 14 million by the year 2050. Worldwide, it is estimated that 22 million individuals will develop Alzheimer's disease by the year 2025. Caregivers are affected by this disease, too. In a national survey, 19 million Americans said they have a family member with Alzheimer's disease, and 37 million said they knew someone with the disease.

CHOLINERGIC SYSTEM

The cholinergic system of the brain includes neurons that produce neurotransmitters, the chemical signals used by the brain to process information and function normally. Nerve cells in this system atrophy and stop producing neurotransmitters in the brains of Alzheimer's patients.

NERVE GROWTH FACTOR (NGF)

Nerve Growth Factor (NGF) is one of several naturally occurring proteins found in the brains of all vertebrate animals. NGF promotes nerve cell growth and survival.

RESEARCH TIMELINE

• 1950s - NGFs were first discovered by developmental biologist Rita Levi-Montalcini, who won a Nobel Prize in 1986 for the finding.

• Mid 1986-88 - Studies by three separate groups of scientists - headed by Franz Hefti (1986), Fred Gage and Silvio Varon at UCSD (1986), and Lawrence Kromer (1987), - demonstrated NGF's potential to protect or repair the adult brain. They showed that pumping NGF into the adult brain could completely prevent injury-induced cell death in the rat. In addition, in 1987, Gage and his team demonstrated functional anatomical and behavioral recovery in aged rats. It was later found that pumping NGF into the brain caused side effects, and a better method of delivering growth factor to just the degenerating cells was needed.

• 1988 - To better target NGF to specific brain regions, UCSD researchers Gage and Theodore Friedmann, M.D., devised a method to genetically modify cells that produce and deliver the NGF. In culture dishes, skin biopsies from monkeys were modified to produce and secrete NGF. The genetically modified cells were then surgically grafted directly into the brain tissue.

• 1990 - Studies by Mark Tuszynski, Gage, Hoi Sang U, and colleagues at UCSD and The Salk Institute demonstrated that pumping NGF into the primate brain also prevented cell death in the adult primate brain. However, once again side effects occurred due to the pumping. Thus, an attempt began to deliver NGF to specific areas of the primate brain using gene therapy.

• 1994 - Studies by Tuszynski, Gage and colleagues demonstrated that NGF could successfully be introduced into the primate brain by gene therapy.

• 1996 - Studies by Tuszynski, Gage and colleagues demonstrated that NGF gene therapy would prevent cell death in the young monkey brain.

• September 14, 1999 - The first studies of NGF gene therapy in aged monkeys were reported in the Proceedings of the National Academy of Science. Tuszynski's team reported that 40 percent of cholinergic neuron cell bodies had atrophied in normal monkey aging, but were returned to nearly normal size and quantity following the surgical implant of cells genetically altered to produce NGF.

• October 1999 - The researchers received federal regulatory approval (U.S. Food and Drug Administration) to conduct human clinical trials of the gene implant procedure for people suffering from early Alzheimer's disease.

• December 1999 - The gene therapy protocol presented by Tuszynski and Hoi Sang U and Leon Thal was reviewed by the Recombinant DNA Advisory Committee (RAC) of the National Institutes of Health. As an advisory group, the RAC does not take action, but its review is considered an important step in the process of launching a patient trial involving gene transfer.

• February 2001 - In research that built upon previous work with atrophied brain cells in aging monkeys, Tuszynski's team found that essential brain fibers called axons were restored to normal levels in primate brain cells, with infusion of tissue that had been genetically altered to produce NGF. This research was reported in the Feb. 12, 2001 issue of Proceedings of the National Academy of Science.

• April 5, 2001 - UCSD team performs first implant of genetically modified tissue into the brain of an Alzheimer's patient.

This project is supported by donations from the Shiley family and the Institute for the Study of Aging in New York.