ABSTRACT

The original goal of gene therapy was to substitute a healthy gene for a defective one or to repair a faulty gene, thereby eliminating symptoms of disease. But researchers have moved beyond inherited genetic disorders to treat other kinds of diseases. Today, almost 75 percent of all clinical trials involving gene therapy are aimed at treatments for cancer and acquired immunodeficiency syndrome. Cancer begins in genes and can be caused by an inherited defect or a mutation that causes a cell to malfunction, while AIDS is caused by a virus that disrupts the genetic material of immune cells. Other new gene therapy projects are targeted at conditions such as heart disease, diabetes mellitus, arthritis and Alzheimer’s disease, all of which involve genetic susceptibility to illness. Gene therapists hope to reduce or eliminate that susceptibility.

KEYWORDSGene Therapy, Gene , Science of Gene , Science of Gene Therapy

Introduction

The field of human genetics has been energized in recent years by the Human Genome Project, an international, collaborative research program whose goal was the complete mapping and understanding of all the genes of human beings — the human genome. Scientists working on the project have developed detailed maps that identify the chromosomal locations of the estimated 20,000 to 25,000 human genes.
Consequently, we now have an extraordinary resource of detailed information about the structure, organization and function of the complete set of human genes. That information can be thought of as the basic set of inheritable “instructions” for the development and function of a human being, and it might provide new strategies to diagnose, treat, cure and prevent human diseases.

Basic Parts
Genes
Genes are bits of biochemical instructions found inside the cells of every organism. Offspring receive a mixture of genetic information from both parents, a process that contributes to the great variation of traits that we see in nature, such as the color of a flower’s petals, the markings on a butterfly’s wings or a person’s blue eyes or curly hair. Geneticists seek to understand how the information encoded in genes is used and controlled by cells and how it is transmitted from one generation to the next. Geneticists also study how tiny variations in genes can disrupt an organism’s development or cause disease. Genes direct the synthesis of proteins, the molecular laborers that carry out all life-supporting activities in the cell. Although all humans share the same set of genes, individuals can inherit different forms of a given gene, making each person genetically unique.

Chromosomes
Genetic information is encoded and transmitted from generation to generation in DNA, which is a coiled molecule organized into structures within cells called chromosomes. Segments along the length of a DNA molecule form genes. Every chromosome in a cell contains many genes, and each gene is located at a particular site, or locus, on the chromosome. Chromosomes vary in size and shape and usually occur in matched pairs called homologues. The number of homologous chromosomes in a cell depends upon the organism .

Cells
The site where genes work is the cell. Some organisms are made up of a single cell, while other organisms are made up of many kinds of cells, each with a different function. Each cell’s function within an organism is determined by the genetic information encoded in DNA.
Within all organisms, cells divide to produce new cells, each of which requires the genetic information found in DNA. Yet, simply splitting the DNA of a dividing cell between two new cells would lead to disaster. The two new cells would have different instructions and each subsequent generation of cells would have less and less genetic information to work with. Organisms use two types of cell division to ensure DNA is passed down from cell to cell during reproduction. Simple one-celled organisms and other organisms that reproduce asexually — without the joining of cells from two different organisms — reproduce by a process called mitosis. During mitosis, a cell doubles its DNA before dividing into two cells and distributing the DNA evenly to each resulting cell. Organisms that reproduce sexually use a different type of cell division, one in which they produce special cells called gametes, or egg and sperm.
In the cell division known as meiosis, the chromosomes in a gamete cell are reduced by half. During sexual reproduction, an egg and sperm unite to form a zygote, in which the full number of chromosomes is restored.

Methods for Transporting Genes
In gene therapy, one or more genes are inserted into a cell to produce a missing protein or enzyme. Researchers have developed several methods for transporting genes into cells. The most common technique is to attach healthy genes to genetically modified viruses. These infectious agents, known as vectors, carry the genes into a cell’s nucleus and incorporate them into the genetic material of the infected cell. Another gene delivery method, one that still is under development, is chimeraplasty, in which segments of DNA are inserted into a cell’s nucleus. The DNA segment binds with a defective gene in a way that helps the cell’s repair mechanisms identify and fix the defective gene. About 15 years ago, scientists demonstrated that viruses could be used as vectors for the delivery of healthy genes. Scientists use four types of viruses, known as virus vectors, in gene therapy experiments: retroviruses, adenoviruses, adeno-associated viruses and herpes simplex viruses.

Retroviruses
Retroviruses were the first viruses used as vectors in gene therapy experiments. They are unusual because instead of using DNA to carry their genetic information to the cell’s protein-making machinery, retroviruses use a related material called ribonucleic acid (RNA) as their primary carrier of genetic information. When retroviruses invade a cell, they use an enzyme called reverse transcriptase to make a DNA copy of their genes. Other enzymes then incorporate this DNA copy into the infected cell’s DNA. Although retroviruses have been used in most gene therapy experiments so far, they present problems. Retroviruses can invade only cells that are actively dividing, limiting potential targets for therapy to blood cells, skin cells, stem cells and other fast-growing tissues. In addition, the viruses have no specific targets in the infected cells chromosomes. As a result, the genes they carry are inserted in a haphazard manner.
Ideally, retroviruses insert genes into the middle of a strand of DNA that does not contain other genes. The genes might, however, be inserted in the middle of a crucial gene, rendering it defective and blocking key cellular functions — and causing more damage than repair. Retroviruses also could integrate new genes into a stretch of DNA where they could cause cancer. Despite the presence of promoters, the added genes typically do not produce sufficient amounts of proteins to treat disease effectively. In addition, the patient’s body generally recognizes retroviruses as foreign invaders, provoking adverse immune responses. Researchers approached the use of retroviruses with caution because of concerns they might attack inappropriate cells. To avoid the problem, researchers initially removed blood or other target cells from the patient’s body before treatment with the retrovirus. They then monitored the cells to ensure the therapy was working properly before returning the cells to the patient’s body. Researchers hope this treatment one day will help people with muscular dystrophy.

Adenoviruses
To avoid the problem of inserting genes at the wrong sites, some researchers have turned to other types of viruses, such as the adenoviruses, which cause the common cold. Stripped of their disease-causing genes, adenoviruses take healthy genes into the nucleus of cells, where the DNA is located but do not usually integrate them into a cell’s DNA. Researchers thus trade safety for impermanence, because the genes persist in the cell’s DNA only for days to weeks. Adenoviruses also can infect a broader variety of cells than retroviruses, including cells that divide more slowly, such as lung cells. However, adenoviruses also are more likely to be attacked by the patient’s immune system, and the high levels of virus required for treatment often provoke an undesirable inflammatory response. Despite these drawbacks, adenoviruses have been used in attempts to treat cancers of the liver and ovaries.

Adeno-Associated Viruses
One of the most promising potential gene-delivery systems, or vectors, is a recently discovered virus called the adeno-associated virus, which infects a broad range of cells, including both dividing and nondividing cells. Researchers believe most humans carry adeno-associated viruses, which do not cause disease and do not provoke an immune response. Scientists have demonstrated that the adeno-associated virus can be used to correct genetic defects in animals. It now is being used in preliminary studies to treat hemophilia, a hereditary blood disease, in humans.
The chief drawback of the adeno-associated virus is that it is small, carrying only two genes in its natural state. Its payload therefore is relatively limited. It can produce unintended genetic damage because the adeno-associated virus inserts its genes directly into the host cell’s DNA. Researchers have also had difficulties manufacturing large quantities of the altered virus.

Herpes Simplex Viruses
Scientists have found that the herpes simplex virus, the cause of the common cold sore, has a large genome compared to other virus vectors. This large genome enables scientists to insert more than one therapeutic gene into a single virus, paving the way for the treatment of disorders caused by more than one gene defect. The virus makes an ideal vector because it can infect a wide variety of tissues, including muscle, tumor, liver, pancreas, nerve and lung cells.
One problem with using herpes simplex virus is that the virus is cytopathic that is, it kills the cells that it infects. In addition, the virus can cause encephalitis (inflammation of the brain) if it replicates freely in the brain. Scientists are developing a form of herpes simplex virus in which the genes that direct the virus’s replication and cell-killing abilities have been removed.

Chimeraplasty
Some researchers believe that in the near future a process called chimeraplasty might make it possible to fix defective genes within a cell directly, making it unnecessary to insert new genes into cells. Researchers have developed short segments of DNA called oligomers, whose nucleotide sequences complement those of gene in which a defect occurs. When inserted into the cell’s nucleus, oligomers bind to the defective gene where the sequences are correct, but they do not bind properly at defective sites. The cell’s repair machinery sees this “bump” in the DNA and interprets it as a signal to repair the defective gene. Chimeraplasty has been tested successfully in animals, and investigators recently have begun to test it in humans.

Types of Genetic Disorders
Scientists have identified certain categories of genetic disorders, some of which have characteristic inheritance patterns. One category consists of single-gene disorders — disorders that involve an error in the DNA that makes up an individual gene. A second category of genetic disorders involves abnormalities of chromosomes in which too much or too little chromosomal material is present. Some genetic disorders are said to be multifactorial, because they are caused by the combined effects of multiple genes and environmental factors, such as diet and exposure to certain chemicals. Still other genetic disorders are caused by mutations in mitochondrial DNA.

Dominant and Recessive Gene Transmission

Single-gene disorders result from errors within an individual gene. Each gene contains information used by cells to manufacture a specific protein or a component of a protein. A tiny alteration, or mutation, in the DNA that makes up a gene could cause a person’s cells either to fail to produce sufficient quantities of a crucial protein or to synthesize a protein with an altered form. Such a protein cannot perform its normal role.
The impact of a single-gene disorder sometimes depends on whether a person has inherited a faulty version of a gene from only one parent or from both parents. The genes carried on each of the 22 pairs of autosomes always occur in pairs — one of which is inherited from the mother and the other from the father. In some instances, a faulty gene has a dominant effect, meaning that the person who inherits one faulty gene and one normally working gene eventually will develop a disorder. In other instances, the faulty gene is recessive — it will not cause a disorder unless a person inherits two copies of the faulty gene, one from the mother and one from the father.

Types of Gene Therapy
The two forms of gene therapy currently used involve somatic gene therapy, which involves introducing a “good” gene into targeted cells with the end result of treating the patient. This type of therapy does not treat the patient’s future children since these genes are not passed along to offspring. This is the more common form of gene therapy that is being performed throughout the world. The second type of gene therapy is germ line gene therapy, which involves modifying the genes in egg or sperm cells. This therapy is aimed at either correcting defective genes responsible for certain diseases or enhancing the genetic makeup of one’s offspring. Contrary to somatic cell gene therapy, which affects only he target cells of the individual being treated, germ line therapy has permanent hereditary consequences.
Although it has the potential for preventing inherited disease, germ line gene therapy for the purpose of enhancing future generations has stirred great controversy.

Conclusion
Eventually, gene therapy might help older people regain strength in withered muscles and increase pumping power in aging hearts. Some researchers predict that in the distant future gene therapy could be used to eliminate genetic defects from families or to produce “designer babies” with more muscle strength, higher intelligence, sweeter dispositions or whatever traits parents desired. Gene therapy carries the excitement of a promise of a type of medical treatment most of us never have imagined possible and the promise of a cure for a host of diseases. It also prompts considerable controversy. With its potential to eliminate and prevent hereditary diseases such as cystic fibrosis and hemophilia and its use as a possible cure for heart disease, AIDS and cancer, gene therapy is a potential medical phenomenon. However, despite the benefits derived from genetic advancements, some observers have voiced concerns that altering human genes could cause harm to people or the environment. Others fear that new genetic technologies may enable scientists to modify genes that affect characteristics other than those responsible for disease. They warn that determining who has undesirable genetic characteristics could lead to discriminatory practices. And those and other challenging issues place geneticists directly at the crossroads of science and social responsibility.

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Dr. Manjul Tiwari MDS ,Oral Pathology & Microbiology Senior Lecturer ,Hindustan Institute of Dental Sciences Plot No. 32-34 , Knowledge Park – III Greater Noida -201306 Uttar Pradesh