GENE THERAPY
SOUMEN CHOWDHURY
B.PHARM, 4th YEAR
INTRODUCTION
Gene therapy typically involves the insertion of a functioning gene into cells to correcta cellular dysfunction or to provide a new cellular function.
For example, diseas such as cystic fibrosis, combined immunodeficiency syndromes,muscular ,dystrophy hemophilia, and many cancers result from the presence of defective genes.
Gene therapy can be used to correct or replace the defective genes responsible. Gene therapy has been especilly successfu in the treatment of combined
immunodeficiency syndromes, showing lasting and remarkable therapeutic benefit.
Gene therapy is a technique for correcting defective genes responsible for diseasedevelopment. Researchers may use one of several approaches for correcting faulty gene:
•A normal gene may be inserted into a nonspecific location within the genome to replace a non-functional gene. This approachs most common.
•An abnormal gene could be swapped for a normal gene through homologous recombination.
•The abnormal gene could be repaired through selective re-verse mutation, which returns the gene to its normal function.
•Gene therapy is the insertion,alteration, or removal of genes within an individual's cells and biological tissues to treat dis-eases. It is a technique for correcting defective genes that are responsible for disease development.
Gene therapy states and remains an experimental discipline and many researchers remain to be performed before the treatment will realize its potential. Majority of the gene therapy trials are being conducted in United States and Europe, with only a modest number in other countries including Australia. Scope of this approach is broad with potential in treatment of diseases caused by single gene recessive disorders (like cystic fibrosis, hemophilia muscular dystrophy, sickle cell,anae uimia etc), acquired genetic diseases such as cancer and certain viral infections
like AIDS.
GENE THERAPY HISTORY
The first clinical study using gene transfer was reported Rosenberg and his colleagues used a retroviral vector to transfer the neomycin resistance marker gene into tumor infiltrating lymphocytes obtained from five patients with metastatic melanoma.These lymphocytes then were expanded in vitro and later re-infused into the respective patient.Since this first study showed that retroviral gene transfer was safe and ,practical it led to many other studies. Indeed, since 1989, more than 900 clinical trials have been approved worldwide. What made gene therapy possible between 1963 and 1990 was the development of recombinant DNA technology. In 2003 as well as in November 2005 China approved the first gene therapy drugs for the treatment of certain malignant tumors. A first European application for the approval of a gene therapy drug for the treatment of an aggressive brain tumor was submitted to the European Agency for the Evaluation of Medicinal Products
(EMEA) in 2005. Despite continued great difficulties in the technical implementations, the successes of gene therapy can doubtlessly be confirmed today. For example, successful therapies have been developed during the past five years for patients with several hereditarily immunodeficiency diseases. These treatments are visibly beneficial to these patients with life-threatening conditions. The death of a patient in the USA in 1999as a result of a very high systemically administered dose of adenoviral vectors were tragic events that were viewed by the public as a setback for gene therapy.Nevertheless, the same principles apply to gene therapy as to other medical interventions: Effective procedures are associated with potential side effects which can be reduced by improving the procedures when the underlying mechanisms are understood. German scientists have made important contributions in this field, from basic research of the vector-host interaction to clinical studies. Among other things, in 2006 they reported on the correction of a severe immunodeficiency in adult patients through gene therapy.
ADVANTAGES OF GENE THERAPY
1. Gene therapy has the potential to eliminate and prevent hereditary disease such as citric fibrosis, ADA-SCDI and is a possible cure to heart disease,
AIDS and cancer etc.
2. In case of silence a gene. In the case of someone with HIV, had not yet developed into ADIS, scientists could have saved them the pain and suffering
of the disease by using gene therapy to silence the disease before onset.
3. These sceptics would almost certainty choose gene therapy, especially if it was
the last hope for them or one of their loved ones as it the case for many gene
therapy patients.
4. It offers the possibility of a positive medical outcome. About 3% of the births in the United States involve a condition which is potentially treatable by using gene therapy techniques. Many of the children born in this demographic die
soon after birth because of the devastating effects of their condition. Birth defects are also potentially preventable with this option, which impacts about 20% of families each year. Instead of paying for hospice care or being forced to say goodbye immediately, doctors and scientists are giving parents more hope for a brighter future because of the availability of this technology.
5. There are out-of-body treatment options available. If your doctor recommends out-of-bodygene therapy, then researchers or technicians will take bone marrow or blood to begin separating the immature cells away from the sample. Then they’ll add the necessary gene to them before injecting the cells back intothe bloodstream. Once returned to the body, the immature cells go to the bonemarrow, start maturing, and eventually multiplying to replace all of the defective cells. This technique looks to be especially helpful for people who suffer from sickle-cell disease or something similar.
6. The medical impact of gene therapy can create permanent results. Once the faulty genetics are replaced by the correct genes, this therapeutic approach earn the advantage of being a long-. lasting, sometimes permanent result.
Although this advantage doesn’t apply in every situation, many patients can reverse their bothersome symptoms in a short amount of time in either the in-
the-body or out-of-the-body approach. There is even the possibility that some of the changes could be heritable to the next generation, which would reduce their risk of suffering from a similar future.
7. It can work in combination with cell therapy techniques. Gene therapy involves the transfer of genetic materials. Cell therapy is the process of
transferring cells with relevant function into the patient when that genetic material is missing in the first place. Some protocols utilize both, which can
give patients a powerful way to recover from their medical condition. Stem cell can be isolated from a patient, and then genetically modified in tissue culture to start the expression of a new gene. Once the treatment expands to suitable number, then the patient receives the therapy as part of their overall care.
DISADVANTAGES OF GENE THERAPY
1. There could be unwanted immune system reactions. The body’s immune system might see the various viruses that we use to replace unwanted genes as invaders that must be extinguished before they cause harm. When the white blood cells attack the newly introduced genetic material, it is not unusual for a patient to experience healthissues,like inflammation, dizziness, and headaches. In severe reactions, it is even
possible for the immune response to target the body’s organs and cause them to fail.That’s why treatment options include an immunosuppressant, but this medication can make someone more susceptible to infections and illness.
2. Current gene therapy methods can sometimes target the wrong cells. Viruses have thecapability of affecting more than one type of cell in the human body during a gene therapy treatment. It is entirely possible that the altered viruses that deliver the information the cells could infect additional ones – not just the ones that contain the mutated
mutated or missing genes. When this disadvantage occurs, then the healthy cells could receive damage that may have unpredictable results. There is the possibility that the outcome could involve disease development, illness sensitivity, or even cancer.
Out-of-body gene therapy for severe combined immunodeficiency, which is the boy- in-bubble syndrome, have already experienced this disadvantage. Five of the 30 children who receive gene therapy for their condition went on to develop leukemia. One of them was unable to beat the resulting disease.
3. The delivery viruses might recover their ability to create disease. There are
specific viruses used to create results during gene therapies. The carrier, which is referred to as a vector, receives genetic engineering so that it can perform its job.They can be given intravenously or injected into the specific body tissues that require the extra help. It is possible for the initial infection to develop once it is introduced in thebody, which could create problems for the information transfer. Although
scientists wouldn’t use life-threatening viruses for this process, there is a risk of
becoming sick from other issues other than the genetic concerns that doctors are trying to treat.
4. Gene therapies could cause a potential tumor. If the new genetic information from virus gets inserted in the wrong spot in your body, then there is a risk that the treatment process could lead to tumor formation. There is a risk of malignancy with this disadvantage as well. This disadvantage is present even when vectors aren’t being used to deliver information to the cells. Scientists can use stem cells for gene therapy, or the fatty particles called liposomes.
5. The cost of gene therapy is prohibitive to a number of families. The fees for gene therapy can easily exceed $1 million in the United States. If the price does not ,decline then it will create a new layer of economic segregation that pits the wealthy against everyone else. Patent laws in the U.S. reduce the immediate availability of genericsas well, which means it is up to each person to manage their costs if this optionis necessary.
6. Ethical concerns about gene therapy exist. Most people would say that the
introduction of gene therapy provides several benefits that are worth taking under consideration. There are also some who have ethical concerns about using this technology to fix illness or disease. By changing the nature of a person’s genetic profile even if it is for the better, then it works to eliminate the natural variations that occur within the human race.
APPROACHES FOR GENE THERAPY
There are two approaches to achieve gene therapy:
1) Somatic gene therapy- It involves the insertion of a functional and expressible
gene into a target somatic cell to correct a genetic disease. It represents the
mainstream line of current basic and clinical research where any modification and effects will not be inherited by the patient’s offspring or later generations.
Somatic gene therapy is viewed as a more conservative and safer approach
because it affects only the targeted cells in the patient and is not passed on to future ;generations however, somatic cell therapy is short lived because the cells of most tissue sultimately die and are replaced by new cells. In addition, transporting the gene to the target cells or tissue is also problematic. Regardless of these difficulties, ,however somatic cell gene therapy is appropriate and acceptable for many disorders.
2) Germline gene therapy- In this approach, functional genes are introduced into germ cells (sperm or egg). Therefore the changes due to therapy would be heritable.
Although this approach is highly effective in counteracting genetic and hereditary ,diseases but for safety, ethical and technical reasons, germline gene therapy is not being attempted at present. The genetic alterations in somatic cells are not carried to the next generations. Therefore, somatic gene therapy is preferred and extensively
studied with an ultimate objective of correcting human diseases.
TYPES OF GENE THERAPY
There are two types of gene therapy:
1. Ex- vivo gene therapy: This technique involves the following steps:
Isolate cells with genetic defect from a patient-
b) Grow the cells in culture
c) Introduce the therapeutic gene to correct gene defect
d) Select the genetically corrected cells and grow
e) Transplant the modified cells to the patient
2. In vivo gene therapy: The direct delivery of the therapeutic gene into the target cells of a particular tissue constitutes in vivo gene therapy. Many tissues are the potential candidates for this approach. For example liver, muscle, skin, spleen, lung, brain and blood cells etc.
The success of in vivo gene therapy mostly depends on the following parameters:
• Th efficiency of the uptake of the therapeutic gene by the target cells.
• Intracellular degradation of the gene and its uptake by nucleus.
•The expression capability of the gene.
TECHNIQUES OF GENE TRANSFER (VECTORS IN GENE
THERAPY)
The most fundamental requirement for gene therapy to be successful is to effectively delivery the therapeutic gene to the target cell. The carrier particles or molecules used to deliver genes are referred to as vectors. There are different viral and non-viral vectors for gene delivery. The ideal gene delivery vector should be very specific, capable of efficiently delivering one or more genes of the size needed for clinical application and unrecognized by the immune system. Finally, a vector should be able
to express the gene for as long as is required.
VIRAL VECTORS
RETROVIRUSES:
A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.
eg:- One of the problems of gene therapy using retroviruses is that the integrase
enzyme can insert the genetic material of the virus into any arbitrary position in the genome of the host; it randomly inserts the genetic material into a chromosome. If geneticmaterial happens to be inserted in the middle of one of the original genes of thehost cell, this gene will be disrupted (insertional mutagenesis). If the gene happens
to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This problem has recently begun to be addressed by utilizing zinc finger nucleases or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.
ADENOVIRUS
To avoid problem of inserting genes at wrong sites, some researchers have turned to other types of viruses. A class of virus with double stranded DNA genome that can cause respiratory, intestinal and eye infection (especially the common cold). When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenovirus is not incorporate into the host cell’s genetic
material. The DNA molecule is left free in the nucleus of the host cell, and the
instructions in this extra DNA molecule are transcribed just like any other gene.
Adenovirus also can infect a broader variety of cells than retrovirus, including cells that divid more slowly, such as lungs cells. However, adenovirus 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, this vector system has been promoted for treating cancer of liver and
ovaries and indeed the first gene therapy product to be licensed to treat head and neck cancer is Gendicine, adenoviral product.
ADENO-ASSOCIATE VIRUSES [AAVS]
One of the most promising potential vectors is a recently discovered virus called the, AAV which infects a broad range of cells including both dividing and non dividing cells. AAVs are small viruses from the Parvovirus family with a genome of single strandesDNA. It can insert genetic material at a specific site on chromosome 19 with near100% certainty. Researchers believe that most people carry AAV which do not cause disease and do not provoke an immune response. Scientists have demonstrated
the animal experiments using AAV to correct genetic defects.18 It is now being used in preliminary studies to treat hereditary blood disease hemophilia, muscle and eye disease. The chief drawback of AAV 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 virus inserts its genes directly into host cell’s DNA.
Researchers have also had difficulties in manufacturing large quantities of the altered virus. The production problem has recently being solved by Amsterdam Molecular Therapeutics.
NON-VIRAL METHODS
In comparison with virus-derived vectors, non-viral vectors have several advantages, such as the safety of administration without immunogenicity, almost unlimited transgene size and the possibility of repeated administration. Non-viral gene delivery systems generally consist of three categories: (a) naked DNA delivery, (b) lipid-based
and (c) polymer-based delivery.
NAKE PLASMID DNA
The simplest technique of non-viral gene transfer is the use of so called naked DNA. A series of approaches for naked plasmid DNA based gene delivery strategies have been reported in recent years like, naked plasmid DNA transfer method wherein a cytotoxic T-lymphocyte antigen 4- immunoglobulin (CTLA4-Ig) gene was delivered using
a naked plasmid DNA. Naked DNA was used for antiangiogenic therapy where
the fetal liver kinase-1 gene was delivered. One more interesting area that is the use of naked plasmid DNA gene delivery as electro gene therapy which is done after injection of naked plasmid DNA and delivery of electric pulses directly to the tissue the expression of gene of interest can be obtained, because of its inherent simplicity naked DNA is an attractive non-viral vector and moreover its ease of production in bacteria and manipulation using standard recombinant DNA techniques substantiates its use as non-viral gene delivery system. The other important advantage in using naked DNA gene delivery system is its ability to show very little dissemination and
transfection at distant sites following delivery and also can be administered several times as it does not show any antibody response against itself.
CATIONIC LIPIDS
Cationic liposomes are an important class of compounds suitable for carrying
negatively charged DNA. There are at present several commercial transfection
reagents that are based on cationic lipids like DOTMA(Lipofectin), DOTAP, DOSPA,
DOSPER, DDAB, DODAC, Neophectin (PCL-2), DMRIE, DC-Chol, DOGS
(Transfectam). However use of these reagents in vivo is plagued by their inherent toxicities. Cationic lipids consist of a positively charged head group, a hydrophobic tail and a linker connecting the head to the tail group. The charged head groups are usually quaternary amines, tails are saturated or unsaturated alkyl chains or cholesteryl groups. Cationic liposomes in contrast to neutral and anionic liposomes, which need DNA implementation into the vehicle, cationic liposomes naturally, create complexes with negatively charged DNA. Their positive charge, moreover, allows interactions with the negatively charged cell membrane and thus penetration into the cell is permitted. There are numerous reports on the use of various cationic lipids as non-viral gene delivery vectors. The use of cationic liposomes has made great strides between the initial report by Felgner et al. in 1987 and their use in the world’s first human gene therapy clinical trial by Nabeleta. cationic liposomes are used in recent times is
siRNA delivery. In a recent study by sato et al. siRNA complexed with galactosylated cationicliposomes for liver parenchymal cell selective delivery of siRNA has shown that siRNA did not undergo nuclease digestion and urinary excretion and moreover was delivered efficiently to the liver and was detected in parenchymal cells rather than liver non parenchymal cells. The endogenous gene (ubc13 gene) expression in
the liver was inhibited up to 80% when complexes of ubc13-siRNA and galactosylated liposomes were administered to mice. Though cationic liposomes have been extensively used as transfection agents in vitro. There, in vivo success is plagued by toxicity. In a recent study it was found that the mechanism behind the toxicity of
cationic liposomes is largely induction of apoptosis. A cDNA micro array study
showed that up regulation of 45 genes related to apoptosis, transcription regulation and immune response was due to lipofection.
PHYSICAL METHODS TO ENHANCE DELIVERY
1. 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.
2. 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.eg:- If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic cells were transduced with a corrective transgene using a retrovirus and this led to the development of T cell leukemia in 3 of 20 patients.
3. SONOPORATION
Sonoporation uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic avitation is thought to disrupt the cell membrane and allow DNA to move into cells.
4. MAGNETOFECTION
In a method termed magnetofection, DNA is complexed to a magnetic particles and a magnetis placed underneath the tissue culture dish to bring DNA complexes into contac with a cell monolayer.
CHEMICAL METHODS TO ENHANCE DELIVERY
1. 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 transcrip of the faulty gene, disrupting translation of the faulty mRNA and therefore expression of the gene.
2. LIPOPLEXES AND POLYPLEXES
To improve the delivery of the new DNA into the cell, the DNA must be protected
from damage and (positively charged). Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors.
3. DENDRIMERS
A dendrimer is a highly branched macromolecule with a spherical shape. The surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface. In particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. When in the presence of genetic material such as DNA or RNA, charge complimentarily leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex is then taken into the cell via endocytosis.
4. HYBRID METHODS
Due to every method of gene transfer having shortcomings, there have been some hybrid methods developed that combine two or more techniques. Virosomes are one example they combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other methods involve mixing other viral vector with cationic lipids or hybridising viruses.
5. ELECTRICAL METHODS
Electrotransfer are more well-established. Applying an electrical field to cells alters the resting transmembrane potential, which can induce permeability though the formation of reversible structural membrane changes (electropores). A large number of animal studies have been performed across on a range of tissues, with the main application being immunotherapy. Therapeutic levels of gene expression have been achieved, as well the cotransfer of multiple plasmids. The choice between transfection strategies compared to transduction with a virus will largely depend on the therapeutic goal. For transient gene expression or
repeat dosing scenarios, synthetic delivery system herald obvious advantages. Conversely correction of missing protein disorders which require long-term, stable gene expression may be better served by viral vectors which can lead to integration of the transgene with host DNA and more stable constitutive protein expression.
Synthetic delivery holds potential benefits in term terms of safety, low frequency of gene integration, ability to introduce larger portion of genes and ease of production.
Another consideration is the efficacy of expression: in general, viral vectors achieve higher efficiency of expression than synthetic systems. The development of artificial viral systems (synthetic viruses) remains a future strategy to harness the advantages of viral and synthetic systems.
GENE THERAPY IN DISEASES
Gene Therapy for Oral Squamous Cell Carcinoma
The current treatment strategies for oral squamous cell carcinoma (OSCC) include a combination of surgery, radiation therapy and chemotherapy. However, surgical resection of tumors frequently causes profound defects in oral functions such as speech and swallowing as well as in cosmetic aspects. Chemotherapy is associated with well-known toxicity and has demonstrated no clear impact on the survival of patients with recurrent oral cancer. Recurrence develops in approximately one third of the patients despite definitive treatment. Two thirds of the patients dying of this disease have no evidence of symptomatic distant metastasis. Therefore, local and regional disease control is paramount, underscoring an urgent need for more effective therapy. Several reports have indicated that the combination of radiation and gene therapies has synergistic suppressive effects on various cancer cells, including colorectal, ovarian, nasopharyngeal and head / neck cancer cells. Gene therapy can also be used as an adjuvant to surgery (at the resected tumor margins). This review highlights various gene therapy methods that are available for combating OSCC.
Gen Therapy in Periodontics
Periodontal diseases have a broad spectrum of inflammatory and destructive responses, and are thought to be multifactorial in origin. Genetic variance has been considered as a major risk factor for periodontitis. With the advent of gene therapy in dentistry, significant progress has been made to control periodontal disease and reconstruct the dentoalveolar apparatus.Gene therapy is a field of Biomedicine. A broad definition of gene therapy is the genetic modification of cells for therapeutic purposes. Genes are specific sequences of bases present in the chromosome that form the basic unit of heredity. Each person’s genetic constitution is different and the changes in the genes determine the differences between individuals. Some changes
usually in a single gene, may cause serious diseases. More often, gene variants interact with the environment to predispose some individuals to various ailments. The goal of gene therapy is to transfer the DNA of interest, for example, growth factor and thrombolytic genes into cells, thereby allowing the DNA to be synthesized in these cells and its proteins (termed recombinant protein) expressed. Gene therapy may involve (1) supplying or increasing the expression of a mutant gene that is insufficiently expressed (e.g., to treat enzymatic deficiencies); (2) blocking a gene that is detrimental (e.g., using antisense constructs to inhibit tumor proliferation); or (3) adding a foreign gene to treat a situation beyond the capability of the normal genome
(e.g., introduce an enzyme into a cell or tissue that allows the tissue to become more sensitive to the effects of a pharmacologic agent).
Gene Therapy for Cystic Fibrosis Lung Disease
Gene therapy for the treatment of cystic fibrosis should be a ―natural‖: Cystic
fibrosis (CF) is a recessive disease associated with loss of function mutations in the CF transmembrane conductance regulator (CFTR) gene, which has a well-characterized gene product heterozygotes, as predicted, appear to be phenotypically perfectly normal; the level of expression of CFTR in affected cells generally appears
to be low; and the dysfunctional epithelial lining cells in the organ most affected by CF (the lung) are available for direct vector delivery via topical
administration.However, despite an impressive amount of research in this area, there is little evidence to suggest that an effective gene-transfer approach for the treatment of CF lung disease is imminent. The inability to produce such a therapy reflects in part the learning curve with respect to vector technology and the failure to appreciate the capacity of the airway epithelial cells to defend themselves against the penetration
by moieties, including gene-therapy vectors, from the outside world. This Perspective will focus on the issues that impact on moving this field forward.
Gene Therapy for Parkinson's Disease
Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease most widely recognized for the profound degeneration of mid-brain dopamine nigrostriatal neurons linked to serious motor symptoms. However, PD is far more complex than commonly appreciated, with multiple etiologic variables and pathogenic pathways,
complex pathologies and a wide range of central nervous system (CNS) and non-CNS symptoms. The drugs’ effectiveness decline with progressive pathology, leading to gradual incapacitation of patients by increased ―off‖ time (i.e., periods of no symptomatic relief) and increasing side effects such as peak-dose dyskinesias. Thus, adequate treatment of the nigrostriatal-mediated motor impairments continues to represent a significant unmet medical need, affecting over 4 million people worldwide. Though a number of solutions have been conceived to improve the function of the degenerating dopaminergic system, translating these
biopharmaceutical concepts to the clinic has been challenging due to obstacles
associated with delivering macromolecules to the central nervous system in a persistent and targeted fashion.
Gene Therapy for Infectious Diseases
Gene therapy is being investigated as an alternative treatment for a wide range of infectious diseases that are not amenable to standard clinical management. Gene therapy for infectious diseases requires the introduction of genes designed to
specifically block or inhibit the gene expression or function of gene products, such that the replication of the infectious agent is blocked or limited. In addition to this intracellular intervention, gene therapy may be used to intervene in the spread of the infectious agent at the extracellular level. This could be achieved by sustained expression in vivo of a secreted inhibitory protein or by stimulation of a specific immune response. Approaches to gene therapy for infectious diseases can be divided into three broad categories: (i) gene therapies based on nucleic acid moieties, including antisense DNA and RNA, RNA decoys and catalytic RNA moieties (ribozymes); (ii) protein approaches such as transdominant negative proteins (TNPs) and single-chain antibodies; and (iii) immunotherapeutic approaches involving genetic vaccines or pathogen-specific lymphocytes. It is further possible that combinations of the
aforementioned approaches will be used simultaneously to inhibit multiple stages of the viral life cycle. The extent to which gene therapy will be effective against
infectious agents is the direct result of several key factors: (i) selection of the
appropriate target cell or tissue for gene therapy; (ii) the efficiency of the gene
delivery system; (iii) appropriate expression, regulation and stability of the gene therapy product(s); and (iv) the efficiency of the inhibition of replication by the gene inhibition product.
Gene Therapy for Arthritis
Rheumatoid arthritis is an autoimmune disease with intra-articular inflammation and synovial hyperplasia that results in progressive degradation of cartilage and bone, in severe cases it causes systemic complications. Recently, biological agents that suppress the activities of proinflammatory cytokines have shown efficacy as antiarthritic drugs, but require frequent administration. Thus, gene transfer approaches are being developed as an alternative approach for targeted, more efficient and sustained delivery of inhibitors of inflammatory cytokines as well as other therapeutic agents. Recently, biological agents that modulate the proinflammatory activities of TNF- and IL-1 have shown efficacy as novel antiarthritic drugs. However, arthritis therapies that employ biological agents are currently limited by possible systemic side effects such as the occurrence and re-emergence of viral and bacterial infections as well as their exorbitant expense. There are several different approaches that can be utilized for the treatment of arthritis. Genes can be delivered locally at the site of disease pathology such as the joint by intra-articular injection. Alternatively,
therapeutic genes can be delivered using specific circulating cell types such as T cells or antigen-presenting cells (APCs) such as dendritic cells (DC). Although these types of cells result in more systemic delivery of therapeutically the ability of certain immune regulatory cells to home sites of inflammation can also allow for local treatments following systemic injection. It is also possible to increase the levels of circulating therapeutic proteins by delivery of the gene to tissues such as muscle or liver.
Gene Therapy in Diabetic Neuropathy
Gene therapy shows promise in treating diabetic polyneuropathy, a disorder that
commonly affects diabetics who've had the disease for many years, a new study finds. Researchers in Boston found that intramuscular injections of vascular endothelial growth factor (VEGF) gene may help patients with diabetic polyneuropathy. The study included 39 patients who received three sets of injections of VEGF gene in one leg and 11 patients who received a placebo. Loss of sensation and pain in the legs and feet, weakness, and balance problems are among the symptoms associated with
diabetic neuropathy. The loss of sensation means that ulcerations on the feet may go undetected, which can lead to amputation.
CONCLUSION
Most scientists believe the potential for gene therapy is the most exciting application of DNA science, yet undertaken. How widely this therapy will be applied, depends on the simplification of procedure. As gene therapy is uprising in the field of medicine, scientists believe that after 20 years, this will be the last cure of every genetic disease. Genes may ultimately be used as medicine and given as simple intravenous injection of gene transfer vehicle that will seek our target cells for stable, site-specific chromosomal integration and subsequent gene expression. And now that a draft of the
human genome map is complete, research is focusing on the function of each gene and the role of the faulty gene play in disease. Gene therapy will ultimately change our lives forever.
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B.PHARM, 4th YEAR
INTRODUCTION
Gene therapy typically involves the insertion of a functioning gene into cells to correcta cellular dysfunction or to provide a new cellular function.
For example, diseas such as cystic fibrosis, combined immunodeficiency syndromes,muscular ,dystrophy hemophilia, and many cancers result from the presence of defective genes.
Gene therapy can be used to correct or replace the defective genes responsible. Gene therapy has been especilly successfu in the treatment of combined
immunodeficiency syndromes, showing lasting and remarkable therapeutic benefit.
Gene therapy is a technique for correcting defective genes responsible for diseasedevelopment. Researchers may use one of several approaches for correcting faulty gene:
•A normal gene may be inserted into a nonspecific location within the genome to replace a non-functional gene. This approachs most common.
•An abnormal gene could be swapped for a normal gene through homologous recombination.
•The abnormal gene could be repaired through selective re-verse mutation, which returns the gene to its normal function.
•Gene therapy is the insertion,alteration, or removal of genes within an individual's cells and biological tissues to treat dis-eases. It is a technique for correcting defective genes that are responsible for disease development.
Gene therapy states and remains an experimental discipline and many researchers remain to be performed before the treatment will realize its potential. Majority of the gene therapy trials are being conducted in United States and Europe, with only a modest number in other countries including Australia. Scope of this approach is broad with potential in treatment of diseases caused by single gene recessive disorders (like cystic fibrosis, hemophilia muscular dystrophy, sickle cell,anae uimia etc), acquired genetic diseases such as cancer and certain viral infections
like AIDS.
GENE THERAPY HISTORY
The first clinical study using gene transfer was reported Rosenberg and his colleagues used a retroviral vector to transfer the neomycin resistance marker gene into tumor infiltrating lymphocytes obtained from five patients with metastatic melanoma.These lymphocytes then were expanded in vitro and later re-infused into the respective patient.Since this first study showed that retroviral gene transfer was safe and ,practical it led to many other studies. Indeed, since 1989, more than 900 clinical trials have been approved worldwide. What made gene therapy possible between 1963 and 1990 was the development of recombinant DNA technology. In 2003 as well as in November 2005 China approved the first gene therapy drugs for the treatment of certain malignant tumors. A first European application for the approval of a gene therapy drug for the treatment of an aggressive brain tumor was submitted to the European Agency for the Evaluation of Medicinal Products
(EMEA) in 2005. Despite continued great difficulties in the technical implementations, the successes of gene therapy can doubtlessly be confirmed today. For example, successful therapies have been developed during the past five years for patients with several hereditarily immunodeficiency diseases. These treatments are visibly beneficial to these patients with life-threatening conditions. The death of a patient in the USA in 1999as a result of a very high systemically administered dose of adenoviral vectors were tragic events that were viewed by the public as a setback for gene therapy.Nevertheless, the same principles apply to gene therapy as to other medical interventions: Effective procedures are associated with potential side effects which can be reduced by improving the procedures when the underlying mechanisms are understood. German scientists have made important contributions in this field, from basic research of the vector-host interaction to clinical studies. Among other things, in 2006 they reported on the correction of a severe immunodeficiency in adult patients through gene therapy.
ADVANTAGES OF GENE THERAPY
1. Gene therapy has the potential to eliminate and prevent hereditary disease such as citric fibrosis, ADA-SCDI and is a possible cure to heart disease,
AIDS and cancer etc.
2. In case of silence a gene. In the case of someone with HIV, had not yet developed into ADIS, scientists could have saved them the pain and suffering
of the disease by using gene therapy to silence the disease before onset.
3. These sceptics would almost certainty choose gene therapy, especially if it was
the last hope for them or one of their loved ones as it the case for many gene
therapy patients.
4. It offers the possibility of a positive medical outcome. About 3% of the births in the United States involve a condition which is potentially treatable by using gene therapy techniques. Many of the children born in this demographic die
soon after birth because of the devastating effects of their condition. Birth defects are also potentially preventable with this option, which impacts about 20% of families each year. Instead of paying for hospice care or being forced to say goodbye immediately, doctors and scientists are giving parents more hope for a brighter future because of the availability of this technology.
5. There are out-of-body treatment options available. If your doctor recommends out-of-bodygene therapy, then researchers or technicians will take bone marrow or blood to begin separating the immature cells away from the sample. Then they’ll add the necessary gene to them before injecting the cells back intothe bloodstream. Once returned to the body, the immature cells go to the bonemarrow, start maturing, and eventually multiplying to replace all of the defective cells. This technique looks to be especially helpful for people who suffer from sickle-cell disease or something similar.
6. The medical impact of gene therapy can create permanent results. Once the faulty genetics are replaced by the correct genes, this therapeutic approach earn the advantage of being a long-. lasting, sometimes permanent result.
Although this advantage doesn’t apply in every situation, many patients can reverse their bothersome symptoms in a short amount of time in either the in-
the-body or out-of-the-body approach. There is even the possibility that some of the changes could be heritable to the next generation, which would reduce their risk of suffering from a similar future.
7. It can work in combination with cell therapy techniques. Gene therapy involves the transfer of genetic materials. Cell therapy is the process of
transferring cells with relevant function into the patient when that genetic material is missing in the first place. Some protocols utilize both, which can
give patients a powerful way to recover from their medical condition. Stem cell can be isolated from a patient, and then genetically modified in tissue culture to start the expression of a new gene. Once the treatment expands to suitable number, then the patient receives the therapy as part of their overall care.
DISADVANTAGES OF GENE THERAPY
1. There could be unwanted immune system reactions. The body’s immune system might see the various viruses that we use to replace unwanted genes as invaders that must be extinguished before they cause harm. When the white blood cells attack the newly introduced genetic material, it is not unusual for a patient to experience healthissues,like inflammation, dizziness, and headaches. In severe reactions, it is even
possible for the immune response to target the body’s organs and cause them to fail.That’s why treatment options include an immunosuppressant, but this medication can make someone more susceptible to infections and illness.
2. Current gene therapy methods can sometimes target the wrong cells. Viruses have thecapability of affecting more than one type of cell in the human body during a gene therapy treatment. It is entirely possible that the altered viruses that deliver the information the cells could infect additional ones – not just the ones that contain the mutated
mutated or missing genes. When this disadvantage occurs, then the healthy cells could receive damage that may have unpredictable results. There is the possibility that the outcome could involve disease development, illness sensitivity, or even cancer.
Out-of-body gene therapy for severe combined immunodeficiency, which is the boy- in-bubble syndrome, have already experienced this disadvantage. Five of the 30 children who receive gene therapy for their condition went on to develop leukemia. One of them was unable to beat the resulting disease.
3. The delivery viruses might recover their ability to create disease. There are
specific viruses used to create results during gene therapies. The carrier, which is referred to as a vector, receives genetic engineering so that it can perform its job.They can be given intravenously or injected into the specific body tissues that require the extra help. It is possible for the initial infection to develop once it is introduced in thebody, which could create problems for the information transfer. Although
scientists wouldn’t use life-threatening viruses for this process, there is a risk of
becoming sick from other issues other than the genetic concerns that doctors are trying to treat.
4. Gene therapies could cause a potential tumor. If the new genetic information from virus gets inserted in the wrong spot in your body, then there is a risk that the treatment process could lead to tumor formation. There is a risk of malignancy with this disadvantage as well. This disadvantage is present even when vectors aren’t being used to deliver information to the cells. Scientists can use stem cells for gene therapy, or the fatty particles called liposomes.
5. The cost of gene therapy is prohibitive to a number of families. The fees for gene therapy can easily exceed $1 million in the United States. If the price does not ,decline then it will create a new layer of economic segregation that pits the wealthy against everyone else. Patent laws in the U.S. reduce the immediate availability of genericsas well, which means it is up to each person to manage their costs if this optionis necessary.
6. Ethical concerns about gene therapy exist. Most people would say that the
introduction of gene therapy provides several benefits that are worth taking under consideration. There are also some who have ethical concerns about using this technology to fix illness or disease. By changing the nature of a person’s genetic profile even if it is for the better, then it works to eliminate the natural variations that occur within the human race.
APPROACHES FOR GENE THERAPY
There are two approaches to achieve gene therapy:
1) Somatic gene therapy- It involves the insertion of a functional and expressible
gene into a target somatic cell to correct a genetic disease. It represents the
mainstream line of current basic and clinical research where any modification and effects will not be inherited by the patient’s offspring or later generations.
Somatic gene therapy is viewed as a more conservative and safer approach
because it affects only the targeted cells in the patient and is not passed on to future ;generations however, somatic cell therapy is short lived because the cells of most tissue sultimately die and are replaced by new cells. In addition, transporting the gene to the target cells or tissue is also problematic. Regardless of these difficulties, ,however somatic cell gene therapy is appropriate and acceptable for many disorders.
2) Germline gene therapy- In this approach, functional genes are introduced into germ cells (sperm or egg). Therefore the changes due to therapy would be heritable.
Although this approach is highly effective in counteracting genetic and hereditary ,diseases but for safety, ethical and technical reasons, germline gene therapy is not being attempted at present. The genetic alterations in somatic cells are not carried to the next generations. Therefore, somatic gene therapy is preferred and extensively
studied with an ultimate objective of correcting human diseases.
TYPES OF GENE THERAPY
There are two types of gene therapy:
1. Ex- vivo gene therapy: This technique involves the following steps:
Isolate cells with genetic defect from a patient-
b) Grow the cells in culture
c) Introduce the therapeutic gene to correct gene defect
d) Select the genetically corrected cells and grow
e) Transplant the modified cells to the patient
2. In vivo gene therapy: The direct delivery of the therapeutic gene into the target cells of a particular tissue constitutes in vivo gene therapy. Many tissues are the potential candidates for this approach. For example liver, muscle, skin, spleen, lung, brain and blood cells etc.
The success of in vivo gene therapy mostly depends on the following parameters:
• Th efficiency of the uptake of the therapeutic gene by the target cells.
• Intracellular degradation of the gene and its uptake by nucleus.
•The expression capability of the gene.
TECHNIQUES OF GENE TRANSFER (VECTORS IN GENE
THERAPY)
The most fundamental requirement for gene therapy to be successful is to effectively delivery the therapeutic gene to the target cell. The carrier particles or molecules used to deliver genes are referred to as vectors. There are different viral and non-viral vectors for gene delivery. The ideal gene delivery vector should be very specific, capable of efficiently delivering one or more genes of the size needed for clinical application and unrecognized by the immune system. Finally, a vector should be able
to express the gene for as long as is required.
VIRAL VECTORS
RETROVIRUSES:
A class of viruses that can create double-stranded DNA copies of their RNA genomes. These copies of its genome can be integrated into the chromosomes of host cells. Human immunodeficiency virus (HIV) is a retrovirus.
eg:- One of the problems of gene therapy using retroviruses is that the integrase
enzyme can insert the genetic material of the virus into any arbitrary position in the genome of the host; it randomly inserts the genetic material into a chromosome. If geneticmaterial happens to be inserted in the middle of one of the original genes of thehost cell, this gene will be disrupted (insertional mutagenesis). If the gene happens
to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This problem has recently begun to be addressed by utilizing zinc finger nucleases or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.
ADENOVIRUS
To avoid problem of inserting genes at wrong sites, some researchers have turned to other types of viruses. A class of virus with double stranded DNA genome that can cause respiratory, intestinal and eye infection (especially the common cold). When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenovirus is not incorporate into the host cell’s genetic
material. The DNA molecule is left free in the nucleus of the host cell, and the
instructions in this extra DNA molecule are transcribed just like any other gene.
Adenovirus also can infect a broader variety of cells than retrovirus, including cells that divid more slowly, such as lungs cells. However, adenovirus 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, this vector system has been promoted for treating cancer of liver and
ovaries and indeed the first gene therapy product to be licensed to treat head and neck cancer is Gendicine, adenoviral product.
ADENO-ASSOCIATE VIRUSES [AAVS]
One of the most promising potential vectors is a recently discovered virus called the, AAV which infects a broad range of cells including both dividing and non dividing cells. AAVs are small viruses from the Parvovirus family with a genome of single strandesDNA. It can insert genetic material at a specific site on chromosome 19 with near100% certainty. Researchers believe that most people carry AAV which do not cause disease and do not provoke an immune response. Scientists have demonstrated
the animal experiments using AAV to correct genetic defects.18 It is now being used in preliminary studies to treat hereditary blood disease hemophilia, muscle and eye disease. The chief drawback of AAV 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 virus inserts its genes directly into host cell’s DNA.
Researchers have also had difficulties in manufacturing large quantities of the altered virus. The production problem has recently being solved by Amsterdam Molecular Therapeutics.
NON-VIRAL METHODS
In comparison with virus-derived vectors, non-viral vectors have several advantages, such as the safety of administration without immunogenicity, almost unlimited transgene size and the possibility of repeated administration. Non-viral gene delivery systems generally consist of three categories: (a) naked DNA delivery, (b) lipid-based
and (c) polymer-based delivery.
NAKE PLASMID DNA
The simplest technique of non-viral gene transfer is the use of so called naked DNA. A series of approaches for naked plasmid DNA based gene delivery strategies have been reported in recent years like, naked plasmid DNA transfer method wherein a cytotoxic T-lymphocyte antigen 4- immunoglobulin (CTLA4-Ig) gene was delivered using
a naked plasmid DNA. Naked DNA was used for antiangiogenic therapy where
the fetal liver kinase-1 gene was delivered. One more interesting area that is the use of naked plasmid DNA gene delivery as electro gene therapy which is done after injection of naked plasmid DNA and delivery of electric pulses directly to the tissue the expression of gene of interest can be obtained, because of its inherent simplicity naked DNA is an attractive non-viral vector and moreover its ease of production in bacteria and manipulation using standard recombinant DNA techniques substantiates its use as non-viral gene delivery system. The other important advantage in using naked DNA gene delivery system is its ability to show very little dissemination and
transfection at distant sites following delivery and also can be administered several times as it does not show any antibody response against itself.
CATIONIC LIPIDS
Cationic liposomes are an important class of compounds suitable for carrying
negatively charged DNA. There are at present several commercial transfection
reagents that are based on cationic lipids like DOTMA(Lipofectin), DOTAP, DOSPA,
DOSPER, DDAB, DODAC, Neophectin (PCL-2), DMRIE, DC-Chol, DOGS
(Transfectam). However use of these reagents in vivo is plagued by their inherent toxicities. Cationic lipids consist of a positively charged head group, a hydrophobic tail and a linker connecting the head to the tail group. The charged head groups are usually quaternary amines, tails are saturated or unsaturated alkyl chains or cholesteryl groups. Cationic liposomes in contrast to neutral and anionic liposomes, which need DNA implementation into the vehicle, cationic liposomes naturally, create complexes with negatively charged DNA. Their positive charge, moreover, allows interactions with the negatively charged cell membrane and thus penetration into the cell is permitted. There are numerous reports on the use of various cationic lipids as non-viral gene delivery vectors. The use of cationic liposomes has made great strides between the initial report by Felgner et al. in 1987 and their use in the world’s first human gene therapy clinical trial by Nabeleta. cationic liposomes are used in recent times is
siRNA delivery. In a recent study by sato et al. siRNA complexed with galactosylated cationicliposomes for liver parenchymal cell selective delivery of siRNA has shown that siRNA did not undergo nuclease digestion and urinary excretion and moreover was delivered efficiently to the liver and was detected in parenchymal cells rather than liver non parenchymal cells. The endogenous gene (ubc13 gene) expression in
the liver was inhibited up to 80% when complexes of ubc13-siRNA and galactosylated liposomes were administered to mice. Though cationic liposomes have been extensively used as transfection agents in vitro. There, in vivo success is plagued by toxicity. In a recent study it was found that the mechanism behind the toxicity of
cationic liposomes is largely induction of apoptosis. A cDNA micro array study
showed that up regulation of 45 genes related to apoptosis, transcription regulation and immune response was due to lipofection.
PHYSICAL METHODS TO ENHANCE DELIVERY
1. 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.
2. 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.eg:- If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic cells were transduced with a corrective transgene using a retrovirus and this led to the development of T cell leukemia in 3 of 20 patients.
3. SONOPORATION
Sonoporation uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic avitation is thought to disrupt the cell membrane and allow DNA to move into cells.
4. MAGNETOFECTION
In a method termed magnetofection, DNA is complexed to a magnetic particles and a magnetis placed underneath the tissue culture dish to bring DNA complexes into contac with a cell monolayer.
CHEMICAL METHODS TO ENHANCE DELIVERY
1. 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 transcrip of the faulty gene, disrupting translation of the faulty mRNA and therefore expression of the gene.
2. LIPOPLEXES AND POLYPLEXES
To improve the delivery of the new DNA into the cell, the DNA must be protected
from damage and (positively charged). Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors.
3. DENDRIMERS
A dendrimer is a highly branched macromolecule with a spherical shape. The surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface. In particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. When in the presence of genetic material such as DNA or RNA, charge complimentarily leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex is then taken into the cell via endocytosis.
4. HYBRID METHODS
Due to every method of gene transfer having shortcomings, there have been some hybrid methods developed that combine two or more techniques. Virosomes are one example they combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other methods involve mixing other viral vector with cationic lipids or hybridising viruses.
5. ELECTRICAL METHODS
Electrotransfer are more well-established. Applying an electrical field to cells alters the resting transmembrane potential, which can induce permeability though the formation of reversible structural membrane changes (electropores). A large number of animal studies have been performed across on a range of tissues, with the main application being immunotherapy. Therapeutic levels of gene expression have been achieved, as well the cotransfer of multiple plasmids. The choice between transfection strategies compared to transduction with a virus will largely depend on the therapeutic goal. For transient gene expression or
repeat dosing scenarios, synthetic delivery system herald obvious advantages. Conversely correction of missing protein disorders which require long-term, stable gene expression may be better served by viral vectors which can lead to integration of the transgene with host DNA and more stable constitutive protein expression.
Synthetic delivery holds potential benefits in term terms of safety, low frequency of gene integration, ability to introduce larger portion of genes and ease of production.
Another consideration is the efficacy of expression: in general, viral vectors achieve higher efficiency of expression than synthetic systems. The development of artificial viral systems (synthetic viruses) remains a future strategy to harness the advantages of viral and synthetic systems.
GENE THERAPY IN DISEASES
Gene Therapy for Oral Squamous Cell Carcinoma
The current treatment strategies for oral squamous cell carcinoma (OSCC) include a combination of surgery, radiation therapy and chemotherapy. However, surgical resection of tumors frequently causes profound defects in oral functions such as speech and swallowing as well as in cosmetic aspects. Chemotherapy is associated with well-known toxicity and has demonstrated no clear impact on the survival of patients with recurrent oral cancer. Recurrence develops in approximately one third of the patients despite definitive treatment. Two thirds of the patients dying of this disease have no evidence of symptomatic distant metastasis. Therefore, local and regional disease control is paramount, underscoring an urgent need for more effective therapy. Several reports have indicated that the combination of radiation and gene therapies has synergistic suppressive effects on various cancer cells, including colorectal, ovarian, nasopharyngeal and head / neck cancer cells. Gene therapy can also be used as an adjuvant to surgery (at the resected tumor margins). This review highlights various gene therapy methods that are available for combating OSCC.
Gen Therapy in Periodontics
Periodontal diseases have a broad spectrum of inflammatory and destructive responses, and are thought to be multifactorial in origin. Genetic variance has been considered as a major risk factor for periodontitis. With the advent of gene therapy in dentistry, significant progress has been made to control periodontal disease and reconstruct the dentoalveolar apparatus.Gene therapy is a field of Biomedicine. A broad definition of gene therapy is the genetic modification of cells for therapeutic purposes. Genes are specific sequences of bases present in the chromosome that form the basic unit of heredity. Each person’s genetic constitution is different and the changes in the genes determine the differences between individuals. Some changes
usually in a single gene, may cause serious diseases. More often, gene variants interact with the environment to predispose some individuals to various ailments. The goal of gene therapy is to transfer the DNA of interest, for example, growth factor and thrombolytic genes into cells, thereby allowing the DNA to be synthesized in these cells and its proteins (termed recombinant protein) expressed. Gene therapy may involve (1) supplying or increasing the expression of a mutant gene that is insufficiently expressed (e.g., to treat enzymatic deficiencies); (2) blocking a gene that is detrimental (e.g., using antisense constructs to inhibit tumor proliferation); or (3) adding a foreign gene to treat a situation beyond the capability of the normal genome
(e.g., introduce an enzyme into a cell or tissue that allows the tissue to become more sensitive to the effects of a pharmacologic agent).
Gene Therapy for Cystic Fibrosis Lung Disease
Gene therapy for the treatment of cystic fibrosis should be a ―natural‖: Cystic
fibrosis (CF) is a recessive disease associated with loss of function mutations in the CF transmembrane conductance regulator (CFTR) gene, which has a well-characterized gene product heterozygotes, as predicted, appear to be phenotypically perfectly normal; the level of expression of CFTR in affected cells generally appears
to be low; and the dysfunctional epithelial lining cells in the organ most affected by CF (the lung) are available for direct vector delivery via topical
administration.However, despite an impressive amount of research in this area, there is little evidence to suggest that an effective gene-transfer approach for the treatment of CF lung disease is imminent. The inability to produce such a therapy reflects in part the learning curve with respect to vector technology and the failure to appreciate the capacity of the airway epithelial cells to defend themselves against the penetration
by moieties, including gene-therapy vectors, from the outside world. This Perspective will focus on the issues that impact on moving this field forward.
Gene Therapy for Parkinson's Disease
Parkinson’s disease (PD) is a chronic, progressive neurodegenerative disease most widely recognized for the profound degeneration of mid-brain dopamine nigrostriatal neurons linked to serious motor symptoms. However, PD is far more complex than commonly appreciated, with multiple etiologic variables and pathogenic pathways,
complex pathologies and a wide range of central nervous system (CNS) and non-CNS symptoms. The drugs’ effectiveness decline with progressive pathology, leading to gradual incapacitation of patients by increased ―off‖ time (i.e., periods of no symptomatic relief) and increasing side effects such as peak-dose dyskinesias. Thus, adequate treatment of the nigrostriatal-mediated motor impairments continues to represent a significant unmet medical need, affecting over 4 million people worldwide. Though a number of solutions have been conceived to improve the function of the degenerating dopaminergic system, translating these
biopharmaceutical concepts to the clinic has been challenging due to obstacles
associated with delivering macromolecules to the central nervous system in a persistent and targeted fashion.
Gene Therapy for Infectious Diseases
Gene therapy is being investigated as an alternative treatment for a wide range of infectious diseases that are not amenable to standard clinical management. Gene therapy for infectious diseases requires the introduction of genes designed to
specifically block or inhibit the gene expression or function of gene products, such that the replication of the infectious agent is blocked or limited. In addition to this intracellular intervention, gene therapy may be used to intervene in the spread of the infectious agent at the extracellular level. This could be achieved by sustained expression in vivo of a secreted inhibitory protein or by stimulation of a specific immune response. Approaches to gene therapy for infectious diseases can be divided into three broad categories: (i) gene therapies based on nucleic acid moieties, including antisense DNA and RNA, RNA decoys and catalytic RNA moieties (ribozymes); (ii) protein approaches such as transdominant negative proteins (TNPs) and single-chain antibodies; and (iii) immunotherapeutic approaches involving genetic vaccines or pathogen-specific lymphocytes. It is further possible that combinations of the
aforementioned approaches will be used simultaneously to inhibit multiple stages of the viral life cycle. The extent to which gene therapy will be effective against
infectious agents is the direct result of several key factors: (i) selection of the
appropriate target cell or tissue for gene therapy; (ii) the efficiency of the gene
delivery system; (iii) appropriate expression, regulation and stability of the gene therapy product(s); and (iv) the efficiency of the inhibition of replication by the gene inhibition product.
Gene Therapy for Arthritis
Rheumatoid arthritis is an autoimmune disease with intra-articular inflammation and synovial hyperplasia that results in progressive degradation of cartilage and bone, in severe cases it causes systemic complications. Recently, biological agents that suppress the activities of proinflammatory cytokines have shown efficacy as antiarthritic drugs, but require frequent administration. Thus, gene transfer approaches are being developed as an alternative approach for targeted, more efficient and sustained delivery of inhibitors of inflammatory cytokines as well as other therapeutic agents. Recently, biological agents that modulate the proinflammatory activities of TNF- and IL-1 have shown efficacy as novel antiarthritic drugs. However, arthritis therapies that employ biological agents are currently limited by possible systemic side effects such as the occurrence and re-emergence of viral and bacterial infections as well as their exorbitant expense. There are several different approaches that can be utilized for the treatment of arthritis. Genes can be delivered locally at the site of disease pathology such as the joint by intra-articular injection. Alternatively,
therapeutic genes can be delivered using specific circulating cell types such as T cells or antigen-presenting cells (APCs) such as dendritic cells (DC). Although these types of cells result in more systemic delivery of therapeutically the ability of certain immune regulatory cells to home sites of inflammation can also allow for local treatments following systemic injection. It is also possible to increase the levels of circulating therapeutic proteins by delivery of the gene to tissues such as muscle or liver.
Gene Therapy in Diabetic Neuropathy
Gene therapy shows promise in treating diabetic polyneuropathy, a disorder that
commonly affects diabetics who've had the disease for many years, a new study finds. Researchers in Boston found that intramuscular injections of vascular endothelial growth factor (VEGF) gene may help patients with diabetic polyneuropathy. The study included 39 patients who received three sets of injections of VEGF gene in one leg and 11 patients who received a placebo. Loss of sensation and pain in the legs and feet, weakness, and balance problems are among the symptoms associated with
diabetic neuropathy. The loss of sensation means that ulcerations on the feet may go undetected, which can lead to amputation.
CONCLUSION
Most scientists believe the potential for gene therapy is the most exciting application of DNA science, yet undertaken. How widely this therapy will be applied, depends on the simplification of procedure. As gene therapy is uprising in the field of medicine, scientists believe that after 20 years, this will be the last cure of every genetic disease. Genes may ultimately be used as medicine and given as simple intravenous injection of gene transfer vehicle that will seek our target cells for stable, site-specific chromosomal integration and subsequent gene expression. And now that a draft of the
human genome map is complete, research is focusing on the function of each gene and the role of the faulty gene play in disease. Gene therapy will ultimately change our lives forever.
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