Till now gene therapy is a new concept with lot of experimental procedure for the treatment of disease. Using stem cells in gene therapy is a relatively new idea which gives confusion for public to understand it. Stem cell in gene therapy is one of the major developing technologies in present day research. The potential goal of using stem cells in gene therapy is to treating different range of diseases, which currently have no cure1. Stem cell-based gene therapy have focused to treat different types of diseases like one third focused on cancers (e.g., ovarian, brain, breast, myeloma, leukemia, and lymphoma), one-third on human immunodeficiency virus disease (HIV-1), and one-third on so-called single-gene diseases (e.g., Gaucher's disease, severe combined immune deficiency (SCID), Fanconi anemia, Fabry disease, and leukocyte adherence deficiency)2.
Stem cells are characterized by the ability to renew themselves through mitotic cell division and differentiating into a diverse range of specialized cell types. Stem cells serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. Each new cell formed by stem cell division has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell. Recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic "somatic" or "adult" stem cells. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be "reprogrammed" genetically to assume a stem cell-like state. This new type of stem cell, called induced pluripotent stem cells.
Embryonic stem cells
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The fertilized egg (Zygote) forms a compact ball of 12 cells, the morula, in 3-5 days after rapid cell division. After 5-7 days, an embryo arises in the form of a 100 cell blastocyst5. Its outer layer is called trophoblast. Inner cell mass of trophoblast is the source of embryonic stem cells. Embryonic stem cell lines are created by separating embryo into individual cells. A single cell from the embryo is placed in a dish and provided with nutrients and growth factors that stimulate it to divide. Cell line continues to divide until it is kept in a controlled environment and provided with appropriate growth factors to prevent differentiation.
Adult stem cells
Adult stem cells have less potential of development when compared with embryonic stem cells. Life span and multiplication of adult stem cells are limited. Adult stem cells have been successfully isolated from the brain and neurons and allowed to grown in cell culture. Adult stem cell lines are majorly used for clinical research studies. Model organisms like mice and rat are used to perform adult stem cell research because humans involve invasive surgical procedures.
Gene therapy is defined as the introduction or elimination of specific genes by using molecular biology techniques to physically manipulate genetic material to alter or supplement the function of an abnormal gene by providing a copy of a normal gene, to directly repair such a gene, or to provide a gene that adds new functions or regulates the activity of other genes2. There are two types of gene therapy -Somatic and Germline.
Somatic Gene Therapy
Somatic gene therapy can be done by inserting a vector into a person's somatic cells. These vectors carry a modified gene into person's body. Somatic cells don't produce offspring's, they build up the body. There are two types of somatic gene therapy, ex vivo and in vivo. Ex vivo modifies cells outsides the body and then transplants them back into the body. In vivo is the changing the cells while they are still in the body, Somatic gene therapy do not affect any offspring of the person being treated.
Germline Gene Therapy
Germline gene therapy can be done by two stages. The first is the released egg, which can be altered before or after it is fertilized with sperm. The egg is fairly easy to manipulate and be injected with DNA. If DNA is injected into an egg, it will usually integrate into one of the chromosomes. The second stage germline gene therapy is performed at developmental stage, called blastomeres. When germline gene therapy takes places with blastomeres, the cells can be grown and manipulated in a test tube. The changes cells can then be implanted in a surrogate mother.
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Genetically engineered viruses are used to deliver the gene into cells. 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. By intravenous injection the vector can be injected into specific tissues of 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. The transplanted gene is 'switched on', when it finds right location within the cell of an affected person. The transplanted gene can then issue the instructions necessary for the cell to make the protein that was previously missing or altered. The potential success of gene therapy technology depends not only on the delivery of the therapeutic transgene into the appropriate human target cells, but also on the ability of the gene to function properly in the cell. Both requirements pose considerable technical challenges.
Delivering Therapeutic Transgenes into Human Recipient
Harmless viruses (viruses that have been altered) are used as the vehicle for delivering the gene into certain human cell types to prevent from infection, in much the same way as ordinary viruses infect cells. This delivery method is fairly imprecise and limited to the specific types of human cells that the viral vehicle can infect. Some viruses commonly used as gene-delivery vehicles can only infect cells that are actively dividing. This limits their usefulness in treating diseases of the heart or brain, because these organs are largely composed of nondividing cells. Nonviral methods like direct delivering of plain DNA and DNA wrapped in liposomes are also used to directly delivery the genes into cells.
Cell Based Delivery
Cell based delivery involves the use of living cells to deliver therapeutic transgenes into the body. Delivering cells like stem cell, a lymphocyte, or a fibroblast are removed from the body, and the therapeutic transgene is introduced into them by vectors. The genetically modified cells are tested and then allowed to grow and multiply and tested in the laboratory and then they are finally infused back into the patient.
Advantages of Cell Based Therapy over Direct Therapy
Some of the advantages of cell based gene therapy are: First-The addition of the therapeutic transgene to the delivery cells takes place outside the patient; by this it's easy to select and work with cells which contain the transgene and to produce the therapeutic agent in sufficient quantity. Second-Genetically engineering or "program," the cells' level and rate of production of the therapeutic agent is carried outside of the patient. Programming the cells may vary depending on the patient, in some cases; it is desirable to program the cells to make large amounts of the therapeutic agent so that the chances that sufficient quantities are secreted and reach the diseased tissue in the patient are high. In other cases, it may be desirable to program the cells to produce the therapeutic agent in a regulated fashion. In this case, the therapeutic transgene would be active only in response to certain signals, such as drugs administered to the patient to turn the therapeutic transgene on and off.
Why Stem Cells Are Used in Some Cell-Based Gene Therapies?
One of the main reasons for using stem cells in cell-based gene therapies is that they have self-renewing property that is they can develop into any type of cell population; this may reduce or eliminate the need for repeated administrations of the gene therapy. Different types of stem cells used in gene therapy some of them are: Hematopoietic stem cells, Myoblasts (Muscle- forming stem cells), neural stem cells, Osteoblasts (Bone-forming stem cells).
Hematopoietic stem cells
In gene therapy research hematopoietic stem cells have been using as a major delivery cell of choice for many reasons. First- although they are small in number, they are readily removed from the body via the circulating blood or bone marrow of adults or the umbilical cord blood of newborn infants. Identified and manipulated of hematopoietic stem cells in the laboratory is easy and can be returned to patients relatively easily by injection. Hematopoietic stem cells give rise to many different types of blood cells such as T and B lymphocytes, natural killer cells, monocytes, macrophages, granulocytes, eosinophils, basophils, and megakaryocytes, so the therapeutic transgene present in all differentiated cells.. The clinical applications of hematopoietic stem cell-based gene therapies are organ transplantation, blood and bone marrow disorders, and immune system disorders. In addition, hematopoietic stem cells "home," or migrate, to a number of different spots in the body primarily the bone marrow, but also the liver, spleen, and lymph nodes. By this therapeutic agent for treating disorders unrelated to the blood system, such as liver diseases and metabolic disorders Gaucher's disease are possible.
Myoblasts (Muscle forming Stem cells)
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Myoblasts are potent tools for stable delivery of a gene of interest into the body, as they become an integral part of the muscle into which they are injected, in close proximity to the circulation. Some of the advantageous biological properties of myoblast are: when Myoblasts are injected into muscle, they fuse with nearby muscle fibers and become an integral part of the muscle tissue. Moreover, since muscle tissue is generally well supplied with nerves and blood, the therapeutic agents produced by the transgene are also accessible to nerves and the circulatory system. So, myoblasts may not only be useful for treating muscle disorders such as muscular dystrophy, but also possibly nonmuscle disorders such as neurodegenerative diseases, inherited hormone deficiencies, hemophilia, and cancers.
Myoblasts mediated gene therapy was successful in correcting liver and spleen abnormalities associated with a lysosomal storage disease in mice. Stable production of the human clotting factor IX deficient in hemophilia at therapeutic concentrations in mice for at least eight months is achieved. Engineered myoblasts are used to secrete erythropoietin (a hormone that stimulates red blood cell production) were successful in reversing a type of anemia associated with end-stage renal disease in a mouse model of renal failure.
Another animal study of myoblast-mediated gene transfer is carried by using a mouse model of familial amyotrophic lateral sclerosis (ALS, also known as Lou Gehrig's disease) a fatal disorder characterized by progressive degeneration of the brain and spinal cord nerves that control muscle activity. Myoblasts containing the transgene for a human nerve growth factor is injected into the muscles of the ALS mice before the onset of disease symptoms and motor neuron degeneration. The transgene remained active in the muscle for up to 12 weeks, and, most importantly, the gene therapy successfully delayed the onset of disease symptoms, slowed muscle atrophy, and delayed the deterioration of motor skills.
Neural stem cells
Many experiments are going on rodents by using neural stem cells as vehicles for cell-based gene therapy for brain tumors known as gliomas. Gliomas are virtually impossible to treat because the tumor cells readily invade the surrounding tissue and migrate extensively into the normal brain. The researchers genetically modified human neural stem cells to produce a protein cytosine deaminase that converts a nontoxic precursor drug into an active form that kills cancer cells. The engineered neural stem cells were then injected into the brains of mice with human-derived gliomas. Within two weeks of the gene therapy and systemic treatment with the precursor drug, the tumors had shrunk by 80 percent. The animal studies also revealed that neural stem cells were able to quickly and accurately "find" glioma cells, regardless of whether the stem cells were implanted directly into the tumors, implanted far from the tumors (but still within the brain), or injected into circulating blood outside the brain.
Microglia are often found near damaged tissue in Alzheimer's disease patients. A couple of studies showed that microglia not only eliminating Î²-amyloid aggregates via phagocytosis but also killing nearby neurons by causing inflammation and the release of neurotoxic proteases. These two functions of microglia are controlled by different cell-surface receptors, thus providing a way for how to clear Î²-amyloid (AÎ²) plaques without destroying healthy neurons that are in close proximity.
Osteoblasts (Bone-forming stem cells)
Recent preliminary study of examining a gene therapy approach to bone repair and regeneration, researchers genetically engineered Osteoblasts to produce a bone growth factor. The Osteoblasts were added to a biodegradable matrix that could act as a "scaffold" for new bone formation. Within a month after the cell-impregnated scaffold was implanted into mice, new bone formation was detectable. By these results, Osteoblasts offers a new hope for effective alternative to conventional bone-grafting techniques.
The Food and Drug Administration (FDA) has not yet approved any human gene therapy product for sale. Current gene therapy is experimental and has not proven very successful in clinical trials. First gene therapy clinical trial began in 1990. In 1999, 18-year-old Jesse Gelsinger participating in a gene therapy trial for ornithine transcarboxylase deficiency (OTCD). He died from multiple organ failures 4 days after starting the treatment. His death is believed to have been triggered by a severe immune response to the adenovirus carrier.
In January 2003, the FDA placed a temporary halt on all gene therapy trials using retroviral vectors in blood stem cells. A second child treated in a French gene therapy trial had developed a leukemia-like condition. Both this child and another who had developed a similar condition in August 2002 had been successfully treated by gene therapy for X-linked severe combined immunodeficiency disease (X-SCID) or "bubble baby syndrome." In April of 2003 the FDA eased the ban on gene therapy trials using retroviral vectors in blood stem cells.
Recent developments in gene therapy research
Nanotechnology and gene therapy yields treatment to torpedo cancer. March, 2009. The School of Pharmacy in London is testing a treatment in mice, which delivers genes wrapped in nanoparticles to cancer cells to target and destroy hard-to-reach cancer cells18.
Results of world's first gene therapy for inherited blindness show sight improvement. 28 April 2008. UK researchers from the UCL Institute of Ophthalmology and Moorfields Eye Hospital NIHR Biomedical Research Centre have announced results from the world's first clinical trial to test a revolutionary gene therapy treatment for a type of inherited blindness. The findings are a landmark for gene therapy technology and could have a significant impact on future treatments for eye disease.
Researchers at the National Cancer Institute (NCI), part of the National Institutes of Health, successfully reengineer immune cells, called lymphocytes, to target and attack cancer cells in patients with advanced metastatic melanoma. This is the first time that gene therapy is used to successfully treat cancer in humans.
Using adult stem cell injections to reset the immune systems of patients with early-onset Type 1 diabetes was done by Northwestern University researcher, it was announced April 11. After the therapy, patients no longer needed to take insulin for up to 35 months. In the study, patients with Type 1 diabetes were treated with a high dose of immune suppression drugs followed by an intravenous injection of their own blood stem cells, which had previously been removed and treated.
University of California, Los Angeles, research team gets genes into the brain using liposome's coated in a polymer call polyethylene glycol (PEG). The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the "blood-brain barrier." This method has potential for treating Parkinson's disease.
Problems have to overcome for successful Gene Therapy
Short-lived nature of gene therapy - the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable, before gene therapy can become a permanent cure for any condition. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
Immune response - the immune system is designed in a way that it can attack the invader whenever a foreign object is introduced into human tissues. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a potential risk. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.
Problems with viral vectors - Viruses, which are used to carry therapeutic gene creates lot of potential problems to the patient like toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.
Future Goals of Stem cell based Gene Therapy
Using embryonic stem cells in gene therapy may avoid immune reactions speculated by John Gearhart of Johns Hopkins University and Peter Rathjen at the University of Adelaide.
Establish an extensive "bank" of embryonic stem cell lines, each with a different set of MHC genes. Then, an embryonic stem cell that is immunologically compatible for a patient could be selected, genetically modified, and triggered to develop into the appropriate type of adult stem cell that could be administered to the patient. Genetically modifiing MHC genes of an embryonic stem cell, it may also be possible to create a "universal" cell that would be compatible with all patients. Another approach might be to "customize" embryonic stem cells such that cells derived from them have a patient's specific MHC proteins on their surface and then to genetically modify them for use in gene therapy.
More research is needed to determine whether the differentiated stem cells retain the advantages, such as longer life span, of the embryonic stem cells from which they were derived. Because of the difficulty in isolating and purifying many of the types of adult stem cells, embryonic stem cells may still be better targets for gene transfer. The versatile embryonic stem cell could be genetically modified, and then, in theory, it could be induced to give rise to all varieties of adult stem cells. Also, since the genetically modified stem cells can be easily expanded, large, pure populations of the differentiated cells could be produced and saved. Even if the differentiated cells were not as long-lived as the embryonic stem cells, there would still be sufficient genetically modified cells to give to the patient whenever the need arises again.
Using stem cells in gene therapy is one of the major developing fields of research. Stem cells play a major role in treating various diseases because these are the cells which have the ability to develop into different types of cells in our body. Gene therapy in combination with stem cells gives lot of opportunities to cure or prevent diseases. Various types of stem cells are used in gene therapy to treat specific diseases like neural stem cells for brain diseases, blood stem cells for treating blood disorders. We can also develop organs in laboratory by using stem cells and can use for organ transplantation and therapeutic gene is transferred into stem cells which are injected into patient. Along with advantages of stem cell based gene therapy some problems are also arising if we overcome problems like immune rejection, Multigene disorders, viral vector problems and short lived nature of gene therapy we can prevent majority of diseases. If we can achieve this, we can give new life to people who don't know name of the disease and what it causes to them. So stem cells in gene therapy may bring new life to patients who are suffering from diseases which don't have permanent cure till now.