Gene Therapy
The gene therapy involves inserting a functional copy of a normal defective or absent gene in the genome a individual in cells of the tissues of the individual with the aim of restoring normal tissue function and eliminate the symptoms of disease in general and hereditary diseases in particular, and to make a marking. Although the technique still is in development (which is why its implementation is carried out in controlled clinical trials), has been used with some success. Although at first it was a technique referred exclusively to treat genetic diseases, the fact is that currently being proposed for almost any disease, being one of the most promising mechanism.
In the 80's, advances in molecular biology had allowed human genes were located in the genome and cloned . Scientists looking for a simple method of producing proteins - such as insulin , deficit of the molecule in type 1 diabetes - introducing human genes into the bacterial DNA. The modified bacteria then produce the corresponding protein, can be cultivated and injected into patients who could not produce it naturally.
On 14 September 1990 , researchers National Institutes of Health the U.S.. UU. performed the first approved gene therapy procedure on a patient for four years, Ashanthi DeSilva, who had a rare genetic disease called severe combined immunodeficiency (SCID), characterized by the absence a competent immune system, so it was vulnerable to infection. Children with this disease usually develop severe infections and rarely reach adulthood, so that common childhood diseases like chickenpox are dangerous to your survival. Ashanthi had to be insulated, as should avoid contact with people outside their family, maintain a sterile environment in your home, and fight infections with antibiotics many .
Applications
- : The marking is not intended to cure genetic the patient but is used to improve the treatment of a particular disease. An example would be the development of vectors for clinical trials.
- monogenic disease therapy: Used on metabolic diseases in which there may or may not be efficient administration of a protein deficit. It provides the gene defective or absent.
- Therapy acquired diseases, such diseases among the most prominent is the cancer. Using different strategies such as the insertion of certain genes in the cell suicide tumor or the insertion of tumor antigens to enhance immune response .
Procedure
The procedure Ashanthi gene therapy, doctors removed white blood the child's body, let the cells grow in the laboratory by inserting the missing gene in cells, and then introduced genetically modified white blood cells within the patient's bloodstream. Laboratory tests have shown that the therapy strengthened Ashanthi immune system, since not reappeared recurrent infections, can attend school, leading a normal life and even be vaccinated against whooping cough . This procedure was not curative, as the genetically treated WBCs are effective only for a few months, after which the process should be repeated (VII, Thompson [first] 1993).
Although this simplified explanation of gene therapy procedure was a success, involved no more than an optimistic first chapter in a long history, the way to the first procedure approved gene therapy was complicated and fraught with controversy.
introduce genes directly into human cells, focusing on diseases caused by single gene defects such as cystic fibrosis , hemophilia, muscular dystrophy and sickle cell anemia . However, this process has been much more complicated than the one conducted in modified bacteria, mainly due to problems arising from the passage of large sections of DNA in the human genome a comparatively larger.
In most gene therapy studies, a gene "normal" is inserted into the genome to replace an "abnormal." A carrier called a vector is used to deliver the therapeutic gene to target cells of the patient. Currently, the most common vector are virus that have been genetically altered to carry normal human DNA. Viruses have evolved ways of encapsulating and delivering their genes to human cells in a pathogen. Scientists have tried to replicate this ability by manipulating the viral genome to remove disease-causing genes and insert therapeutic ones.
target cells such as liver cells or lung of the patient is infected with the vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product encoded by the therapeutic gene restores the target cell to a normal state.
Achievements
is theoretically possible transfected somatic cells (most cells of the body) or germ cells (such as gametes, and their precursors). All human gene therapy has so far focused on somatic cells while germ cell use in humans remains highly controversial just a perspective. So that the introduced gene is normally transmitted the offspring, must not only be inserted into the cell, but also be incorporated into the chromosomes by genetic recombination .
somatic gene therapy can be divided into two categories: ex vivo (where cells are modified outside the body and then transplanted again) and in vivo (where genes are modified in cells directly the body).
There are a variety of different methods to replace or repair the altered genes in gene therapy.
A normal gene can be inserted into a nonspecific location within genome to replace a nonfunctional gene. This approach is the most common. An abnormal gene could be exchanged for a normal gene with homologous recombination. The abnormal gene could be repaired with selective reverse mutation, which returns the gene to normal function. The regulation (the degree to which a gene is active or inactive) ...
Standards to receive gene therapy
The rules for receiving gene therapy are well established and include several requirements:
- must be an effective means of gene transfer . For now, many trials using retroviral vectors , but also used other methods such as adenoviral vectors and physical and chemical techniques.
- The target tissue must be accessible for gene transfer. The first generation of gene therapy processes used white blood cells or their precursors as the target tissue.
- There should be no any form of effective therapy available , and gene therapy should not harm the patient.
Diseases treated by gene therapy
Although gene therapy was originally developed to treat diseases caused by a single gene (monogenic), this technique was quickly adapted to treat acquired diseases such as cancer and infectious diseases like AIDS . Are currently also trying several hereditary diseases such as severe combined immunodeficiency (SCDI, English Severe Combined Inmunodeficiency ), the Familial hypercholesterolemia the cystic fibrosis and muscular dystrophy, among others.
Cystic Fibrosis
In cystic fibrosis (CF, cystic fibrosis English ) called Δ508 deletion (mutation) is found in 70 percent of all copies of the mutant gen. The CF is a disease autosomal recessive associated with a defect in a protein called conductance regulator Cystic fibrosis transmembrane regulator (CFTR), which regulates the transport of ions in the plasma membrane . CF affects approximately 1 in 2000 persons of northern European descent. One of the most vectors have been used in the fight against fibrosis has been the retroviral or retroviral. A cDNA for conductance regulator gene for cystic fibrosis transmembrane regulator (CFTR) was constructed by the overlap of three cDNA clones, and a complete cDNA was cloned into a retroviral vector. CF cells were infected with the vector , carrying a CFTR gene, and cell "virus", expressing the CFTR gene.
experiments showed that the flow of chlorine (one of the ions that pass through the membrane) were detected only in cells CF CFTR. Significantly, the response of CF cells with CFTR is comparable with normal human tracheal responses and encourages the hope that gene therapy is effective in cystic fibrosis , using retroviral vectors directly on the lung epithelium through aerosols.
Hypercholesterolemia
is an inherited disease caused by mutations in receptor low density lipoprotein (LDLR), and those patients who are homozygous for mutation of LDLR gene generally die by a heart attack before age 20. In this experiment, retroviral vectors carrying the gene for human LDLR used to infect cultures of hepatocytes (liver tissue cells). Cell lines were obtained and were able to degrade low density lipoproteins at a level or less normal. So far this experiment has only been used in cell culture, although one of the safest to try individuals.
Severe Combined Immunodeficiency (SCID)
gene therapy began in 1990 with the treatment of a girl named Ashanti De Silva who had the disease.
Affected individuals do not have a functional immune system and usually die of infections. Ashanti had an autosomal SCID, and is due to a defect in the gene encoding the enzyme adenosine deaminase (ADA). Its gene therapy began with the isolation of a type of white blood cells called T. These cells, which were part of the immune system, mixed with retroviral vector carrying a normal ADA gene copy inserted. The virus infected thousands of T cells and a normal copy of the ADA gene was inserted into the genome some of them. After mixing these cells with the vector and the occurrence of the infection, T cells were cultured in the laboratory and analyzed to ensure that the transferred ADA gene was expressed. The last step of this gene therapy was the injection of billions of these cells T, GM, in your bloodstream. Some of these T cells migrated to the bone marrow Ashanti, and began to divide and produce ADA. 25% to 30% of T cells expressing the Ashanti protein ADA, which is sufficient to allow a normal life. Another example is that of David, "bubble boy" who had to live in sterile chamber to avoid any risk of infection because your immune system produces defenses having the mutation of the gene coding for the enzyme adenosine deaminase.
Duchenne Muscular Dystrophy
The Duchenne Muscular Dystrophy (DMD) is a genetic disease that begins in early childhood, causing progressive loss of muscle strength and volume. Appears when a gene on the X chromosome stops making essential muscle protein, dystrophin the . This gene is one of the largest in the human body (it takes almost a 1% of the X chromosome).
The March 28, 2006, at Children's Hospital in Columbus, Ohio, began a trial in six boys with DMD who received replacement genes for an essential muscle protein via injection into the biceps of an arm . This therapy was developed based on adeno-associated virus, specifically designed to carry the dystrophin gene into muscle cells.
Standard treatments for this disease have been the chemotherapy and radiotherapy . Gene therapy is presented as a possible weapon for action against this disease. Essentially, gene therapy will focus on the stimulation of natural immunity to tumor cells, remove or infer growth drugs, insert a tumor suppressor gene or slowing drug resistance. Virus used lytic cycle and is activated only in tumor cells, with the help of promoters are activated only in these cases. If infected normal cells, this promoter can not be activated and the introduced gene is not activated. But it may be another case and is to be carried out the necessary lytic cycle expression of certain tumor genes. In the second case, is placed under a promoter of the tumor cell, a gene that expresses a toxic enzyme. This enzyme will kill the cell that contains it and it will come out by removing neighboring cells, which are possibly also tumor.
vectors in gene therapy
The great diversity of situations in which gene therapy could be applied makes impossible the existence of only one type of vector . However, you can define characteristics for the "ideal vehicle" and then adapt specific situations.
- Reproducibe.
- Stable.
- for the insertion of genetic material regardless of size.
- allowing transduction in both dividing and quiescent cells.
- which enables integration of a therapeutic gene specific.
- To recognize and act on specific cells. That
- therapeutic gene expression can be regulated.
- that lack elements that induce an immune response.
- That may be fully characterized.
- Safe. To minimize potential side effects.
- Easy to produce and store.
- reasonable cost.
The system consists of "vector" itself, which is the means of transport, and the "cargo", the transported material. The latter in turn consists of two components: The "effector" which is the gene to be introduced, and the "medium", which are the elements that control the expression of this gene. The most important is the developer, but there may be others: enhancers and repressors sequences, isolation, integration, packaging , homologous recombination, immunostimulatory sequences, ...
can be separated into two types of vectors for gene therapy: viral vectors and nonviral vectors. Viral vectors have high transformation efficiency, but low specificity of tissue-infect all cells have receptors for the viruses used. By contrast, non-viral vectors are highly tissue-specific or protected inject naked DNA with various molecules directly into the tissue, but we want to infect your transformation efficiency is low.
Virus
All virus attach to their hosts and introduce their genetic material in the host cell as part of its replication cycle . This genetic material contains "instructions" core of how to produce more copies of these viruses, making the normal body becomes a machine that serves to meet the needs of the virus. The host cell will carry out these instructions and produce additional copies of the virus. This will lead to a greater number of infected cells. Some viruses insert their genes physically in the host genome (a typical feature of retrovirus , such as HIV , introducing transcriptase enzyme reverse the host and also uses its RNA to give "instructions"). This makes mixing the genes of the virus with host cell genes during the life of that cell.
has been shown that viruses of this type can be used as vectors to carry genes "benign" in a human cell. This will eliminate the virus genes that cause disease and then be replaced by the genes that encode the desired effect (eg, insulin production in the case of diabetics ). This procedure should be such that genes inserted the virus with the host genome intact.
There are many problems that prevent successful gene therapy using viral vectors, such as:
- Difficulty prevention effects.
- We must ensure that the virus infects cells of the body and the inserted gene does not disrupt any other critical gene in the genome.
Retrovirus
The genetic material of retroviruses is in the form of RNA molecules, while their host's DNA. When a retrovirus infects a cell host, introduces its RNA together with some enzymes mainly reverse transcriptase (RT) and integrase (IN). This RNA molecule should produce a DNA copy of of RNA molecule to be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is called reverse transcription. Is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is free in the host cell's nucleus, should be incorporated into the genome. This process is mediated by the integrase enzyme of the virus. The viral genome contains essentially three regions, gag, pol and env, which encode capsid proteins, viral proteins (RT, IN, and protease inhibitors) and proteins involved. These three regions are flanked by two long terminal repeat sequences and LTRs are essential for the initiation of viral DNA synthesis, integration and regulation of its expression. To use retroviruses as viral vectors for gene therapy eliminates the genes responsible for replication and replace these regions gag, pol and env by the transgene followed by a marker gene (to detect infected cells). Viral genome ends are only LTR y la región psi, que precede a gag. Así se crean los vectores retrovirales genómicos (VRGs). ¿Cómo producirlos a gran escala ahora si carecen de los genes necesarios para ello? Introduciéndolos en líneas celulares empaquetadoras, que contienen plásmidos ayudantes con las secuencias gag, pol y env. Las secuencias gag-pol (no pueden separarse) y env se aportan en plásmidos separados para reducir los riesgos de que se produzcan virus capaces de replicarse por recombinación entre los distintos fragmentos. Al usar tres plásmidos distintos (gag-pol, env y el VRG) se necesitan tres eventos de recombinación homóloga para producir virus que sean capaces de replicarse. Es poco probable que esto ocurra. Últimamente is studying the use of self-inactivating vectors LTRs once their sequences were integrated into the genome of the target cell. This prevents the integrated sequences are packaged in the event that occurred in the organism a retrovirus infection, with the consequent spread of the virus that is integrated by the organism and its infection to other nonspecific cells.
Once the retrovirus genetic material is incorporated and has become part of the genetic material of the host cell, whether this is then divided, its progeny will contain all the new genes. Although sometimes the retrovirus genes are not expressed immediately. One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert genetic material of retroviruses is not arbitrary positions in the host genome, and if genetic material is inserted in the middle of the original gene, this gene will disturbed ( insertion mutagenesis). If, for example, the gene regulates cell division, it will be uncontrolled. Gene therapy trials using retroviral vectors to treat severe combined immunodeficiency X-linked (X-SCID) represent the most successful application of therapy to date. Thus, more than twenty patients have been treated in France and Britain, with a high rate of reconstitution of the immune system . However, similar tests were restricted in the United States when it reported the appearance of leukemia patients. To date, four cases of children know French and one British have developed leukemia as a result of insertional mutagenesis of retroviral vectors, and all but one of these children responded well to conventional treatment for leukemia. Currently, gene therapy to treat SCID continues to be successful in USA, Britain , Italy and Japan .
Adenovirus
are viruses that carry their genetic material in the form of double-stranded DNA. Cause human infections respiratory, intestinal and others that affect the eyes (especially the common cold). When these viruses infect the host cell, introducing the DNA molecule. The adenovirus genetic material is incorporated into the host's genetic material. The DNA molecule remains free host nucleus and the instructions on the extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division , so that the descendants of the cells do not have the extra gene. As a result, treatment with the adenovirus will require a growing population of cells, however, his absence in the host genome should prevent insertion mutagenesis.
source: wikipedia
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