Instead of using viruses to insert normal genes into the body of patients, one can use proteins that are equipped with an "entry permit" into the cell nucleus
Uri Nitzan, Haaretz
Doctoral student Adi Mesika and Dr. Ziv Reich. Potential for selective killing of Hodgkin's lymphoma cells. Photo: Ariel Shalit
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Gene therapy began more than twenty years ago, but despite the high expectations attached to it, very few successes have been recorded in the field and it is still impossible to find drugs based on gene therapy in pharmacies. Despite this, many resources are being poured into research laboratories in the hope that a breakthrough is imminent.
The initial logic behind gene therapy was simple: in hereditary diseases such as cystic fibrosis (CF), the defect is in a specific gene in the patients' hereditary material. The defective gene has been identified, its sequence is known, and with the existing technology it is possible to produce it
the normal gene and try to insert it into the diseased cells. The integration of the normal gene into the DNA of a diseased cell means the recovery of the cell and its return to normal function. The working assumption was that even if the gene penetrated only some of the damaged cells, the patients would benefit from the treatment.
Over the years, attempts to treat acquired diseases were added to the genetic treatment of hereditary diseases. Cancer is an example of an acquired disease, the compatibility of which with genetic therapies is controversial. Theoretically, if genes were inserted into the malignant cells that would cause them to die, it would be possible to reduce the severity of the disease and possibly even cure the patients.
Researchers trying to implement gene therapy technologies encounter a variety of obstacles. The normal gene is at their disposal, but in order to inject it into the patient's cell they have to break through a set of fortifications that consists of two walls. The first wall is the lipid envelope membrane of the cell, which separates the intracellular environment from the outside world. The hereditary material is housed in the nucleus, so the second wall facing the researchers is the nuclear membrane of the cell. Between these two walls lies the intracellular space, the cytoplasm.
One of the ways to break through the cell's fortifications is based on viruses. "At first glance, it seemed that viruses are the most appropriate tools for this task," explains Dr. Ziv Reich from the Department of Biological Chemistry at the Weizmann Institute, "because this is exactly what they have been doing for millions of years: they inject their genes into living cells, incorporating them into the genetic material of the host cell and force it to replicate more and more viruses. In order to enjoy the 'honey' of the viruses and not be harmed by their 'sting', the harmful genes are removed from them and replaced with the normal genes that you want to inject into the patient's cell."
The use of viruses seemed promising at first, but over time its shortcomings were discovered. The immune system may react sharply to the presence of the virus in the body, and significant damage may be caused to the patient. In addition, the inserted gene may integrate into essential places in the hereditary material and cause further genetic damage. For example, the integration of the inserted gene into the hereditary material may damage the function of healthy genes, including genes that are responsible for suppressing the development of cancerous tumors. Damage to such a gene can cause the cell to become cancerous.
An alternative method for introducing genes into the cell nucleus is based on packaging the DNA segment containing the gene in a liposome (fatty envelope). The liposome penetrates the cell and the DNA inside it is released into the cytoplasm. The main obstacle facing the success of liposome treatment is the nuclear membrane. "The ability to break through the second wall and insert the genes into the nucleus is limited," explains Reich, "and most of the DNA that is inserted into the cell remains in the cytoplasm and is broken down. To ensure the passage of the normal gene into the nucleus, we make use of the cell's natural mechanisms and recruit a 'Trojan horse' to our aid ".
In the life routine of the cell there is a ceaseless passage of molecules from the cytoplasm to the nucleus and in the opposite direction. The transition takes place through channels in the membrane of the nucleus. Small molecules pass freely through the channels, and the passage of molecules such as DNA, whose diameter is greater than nine nanometers (one millionth of a millimeter), is conditioned by the fact that a "recognition sequence" is attached to the molecule. The recognition sequence is used by various molecules as confirmation of entry into the nucleus through the channels, but the DNA molecules do not carry it naturally.
Dr. Reich and the members of his research group have developed a new tactic for breaking through the cell's fortifications. "Our set of experiments recruits the recognition sequence to the service of gene therapy. The basic premise of the research is that linking the DNA molecules to a recognition sequence will ensure their entry into the nucleus. We achieved the connection to the recognition sequence through the mediation of intracellular proteins called transcription proteins (TF)," explains Reich. "The TFs are already equipped with permission to enter the nucleus, because they have a role in controlling the replication of the hereditary material in the nucleus. In every healthy cell, signals from the environment activate the transcription protein, then it enters the nucleus through the recognition sequence and binds to the cellular DNA."
Dr. Reich developed a method that allows the gene that is being inserted to bind to the transcription protein. "We engineer the inserted DNA so that immediately after its release from the liposome in the cytoplasm, it will bind to TF. In this way, it reaches the nucleus as a hitchhiker on the back of the transcription protein."
There are many types of transcription proteins, and it is possible to ensure binding of a specific TF to the DNA fragment inserted into the cell. "If in a certain type of cancer there is an overproduction of a certain TF, we will try to engineer DNA segments that will bind specifically to it," says Dr. Reich. "This way we can ensure selective entry of the normal gene into the nuclei of diseased cells."
In blood cancers, for example, and especially in Hodgkin's lymphoma, there is an overactivity of a transcription protein known as NFkB. In the diseased cells, there is an accelerated and uncontrolled "running" of these transcription proteins into the nucleus, and as a result the malignant cell divides without control. According to Adi Mesika, a doctoral student in Reich's laboratory, "it is possible to utilize the properties of NFkB to selectively kill malignant cells. To carry out the task, a gene is used that causes the cell to 'suicide'. We will introduce the lethal gene into the cell using a liposome, and engineer it so that the DNA segment that carries it will bind to the transcription protein "NFkB." The proposed method could be used for the selective destruction of lymphoma cells, because the lethal genes will not penetrate into the nucleus of the healthy cells. The technology is currently being tested in the laboratory, and the research work is being done by Mesika in collaboration with Dr. Mural Zohar.
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