GENE THERAPY

INTRODUCTION:

DEFINITION: Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development. The most common form of gene therapy involves the insertion of functional genes into an unspecified genomic location in order to replace a mutated gene, but other forms involve directly correcting the mutation or modifying normal gene that enables a viral infection.

Types of gene therapy:

Gene therapy may be classified into the two following types:

1.      Germ line gene therapy

         In the case of germ line gene therapy, germ cells, i.e., sperm or eggs are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. This new approach, theoretically, should be highly effective in counteracting genetic disorders and hereditary diseases. However, many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.

2.      Somatic gene therapy

         In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations.

Approach:

Scientists have taken the logical step of trying to introduce genes directly into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, haemophilia, muscular dystrophy and sickle cell anemia. However, this has proven more difficult than modifying bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering them to the correct site on the gene. Today, most gene therapy studies are aimed at cancer and hereditary diseases linked to a genetic defect. Antisense therapy is not strictly a form of gene therapy, but is a related, genetically-mediated therapy.
The most common form of genetic engineering involves the insertion of a functional gene at an unspecified location in the host genome. This is accomplished by isolating and copying the gene of interest, generating a construct containing all the genetic elements for correct expression, and then inserting this construct into a random location in the host organism. Other forms of genetic engineering include gene targeting and knocking out specific genes via engineered nucleases such as zinc finger nucleases, engineered I-CreI homing endonucleases, or nucleases generated from TAL effectors. An example of gene-knockout mediated gene therapy is the knockout of the human CCR5 gene in T-cells in order to control HIV infection. This approach is currently being used in several human clinical trials.
The biology of human gene therapy remains complex and many techniques need further development. Many diseases and their strict genetic link need to be understood more fully before gene therapy can be used appropriately. The public policy debate surrounding the possible use of genetically engineered material in human subjects has been equally complex. Major participants in the debate have come from the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different views to the discussion.

Procedure:

Gene therapy is designed to introduce genetic material into cells to compensate for abnormal genes or to make a beneficial protein. If a mutated gene causes a necessary protein to be faulty or missing, gene therapy may be able to introduce a normal copy of the gene to restore the function of the protein. A gene that is inserted directly into a cell usually does not function. Instead, a carrier called a vector is genetically engineered to deliver the gene. Certain viruses are often used as vectors because they can deliver the new gene by infecting the cell. The viruses are modified so they can’t cause disease when used in people. Some types of virus, such as retroviruses, integrate their genetic material (including the new gene) into a chromosome in the human cell. Other viruses, such as

Adenoviruses, introduce their DNA into the nucleus of the cell, but the DNA is not integrated into a chromosome. The vector can be injected or given intravenously (by IV) directly into a specific tissue in the body, where it is taken up by individual cells. Alternately, a sample of the patient’s cells can be removed and exposed to the vector in a laboratory setting. The cells containing the vector are then returned to the patient. If the treatment is successful, the new gene delivered by the vector will make a functioning protein. Researchers must overcome many technical challenges before gene therapy will be a practical approach to treating disease. For example, scientists must find better ways to deliver genes and target them to particular cells. They must also ensure that new genes are precisely controlled by the body.

Conventional Vectors in gene therapy

Viruses

Ø  Viral vector:

                     All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. There are two main types of virus infection: lytic and lysogenic. Shortly after inserting its DNA, viruses of the lytic cycle quickly produce more viruses, burst from the cell and infect more cells. Lysogenic viruses integrate their DNA into the DNA of the host cell and may live in the body for many years before responding to a trigger

Ø  Retroviruses:

             The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is termed reverse transcription. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase.

Ø  Replication-Competent Vectors:

                            A replication-competent vector called ONYX-015 is used in replicating tumor cells. It was found that in the absence of the E1B-55Kd viral protein, adenovirus caused very rapid apoptosis of infected, p53(+) cells, and this results in dramatically reduced virus progeny and no subsequent spread. Apoptosis was mainly the result of the ability of EIA to inactivate p300. In p53(-) cells, deletion of E1B 55kd has no consequence in terms of apoptosis, and viral replication is similar to that of wild-type virus, resulting in massive killing of cells.

Ø  Herpes Simplex Virus:

                            The Herpes simplex virus is a human neurotropic virus. This is mostly examined for gene transfer in the nervous system. The wild type HSV-1 virus is able to infect neurons. Infected neurons are not rejected by the immune system. Though the latent virus is not transcriptionally apparent, it does possess neuron specific promoters that can continue to function normally^. Antibodies to HSV-1 are common in humans; however complications due to herpes infection are somewhat rare.

Non-viral methods:

                              Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses.

Injection of Naked DNA:

                          This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success. Cellular uptake of naked DNA is generally inefficient. Research efforts focusing on improving the efficiency of naked DNA uptake have yielded several novel methods, such as electroporation, sonoporation, and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.

Physical Methods to Enhance Delivery

·         Electroporation

              Electroporation is a method that uses short pulses of high voltage to carry DNA across the cell membrane. This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecules to pass through. Electroporation is generally efficient and works across a broad range of cell types. However, a high rate of cell death following electroporation has limited its use, including clinical applications.

·         Gene Gun

                The use of particle bombardment, or the gene gun, is another physical method of DNA transfection. In this technique, DNA is coated with gold particles and loaded into a device which generates a force to achieve penetration of DNA/gold into the cells.

Chemical Methods to Enhance Delivery

Oligonucleotides:

The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene. Another uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression. Additionally, single stranded DNA oligonucleotides have been used to direct a single base change within a mutant gene. The oligonucleotide is designed to anneal with complementarity to the target gene with the exception of a central base, the target base, which serves as the template base for repair. This technique is referred to as oligonucleotide mediated gene repair, targeted gene repair, or targeted nucleotide alteration.

Read About Recent advance in Gene Therapy

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 - PARIDHI PITALIYA

BITS Pilani