Gene therapy using an
Adenovirus vector. A new gene is inserted into an adenovirus. If the treatment is successful, the new gene will make functional
protein to treat a disease.
Gene therapy is the use of DNA as a pharmaceutical agent to treat disease. It derives its name from the idea that DNA can be used to supplement or alter genes within an individual's cells as a therapy to treat disease. The most common form of gene therapy involves using DNA that encodes a functional, therapeutic gene in order to replace a mutated gene. Other forms involve directly correcting a mutation, or using DNA that encodes a therapeutic protein drug (rather than a natural human gene) to provide treatment. In gene therapy, DNA that encodes a therapeutic protein is packaged within a "vector", which is used to get the DNA inside cells within the body. Once inside, the DNA becomes expressed by the cell machinery, resulting in the production of therapeutic protein, which in turn treats the patient's disease.
Gene therapy was first conceptualized in 1972, with the authors urging caution before commencing gene therapy studies in humans.[1] The first FDA-approved gene therapy experiment in the United States occurred in 1990, when Ashanti DeSilva was treated for ADA-SCID.[2] Since then, over 1,700 clinical trials have been conducted using a number of techniques for gene therapy.[3]
Although early clinical failures led many to dismiss gene therapy as over-hyped, clinical successes in 2009-2011 have bolstered new optimism in the promise of gene therapy. These include successful treatment of patients with the retinal disease Leber's Congenital Amaurosis,[4][5][6][7] X-linked SCID,[8] ADA-SCID,[9] adrenoleukodystrophy,[10] and Parkinson's disease.[11] These recent clinical successes have led to a renewed interest in gene therapy, with several articles in scientific and popular publications calling for continued investment in the field.[12][13][14]
Scientists have taken the logical step of trying to introduce genes directly into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, haemophilia, muscular dystrophy and sickle cell anemia. However, this has proven more difficult than modifying bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering them to the correct site on the gene. Today, most gene therapy studies are aimed at cancer and hereditary diseases linked to a genetic defect. Antisense therapy is not strictly a form of gene therapy, but is a related, genetically-mediated therapy.
The most common form of genetic engineering involves the insertion of a functional gene at an unspecified location in the host genome.This is accomplished by isolating and copying the gene of interest, generating a construct containing all the genetic elements for correct expression, and then inserting this construct into a random location in the host organism. Other forms of genetic engineering include gene targeting and knocking out specific genes via engineered nucleases such as zinc finger nucleases, engineered I-CreI homing endonucleases, or nucleases generated from TAL effectors. An example of gene-knockout mediated gene therapy is the knockout of the human CCR5 gene in T-cells in order to control HIV infection.[15] This approach is currently being used in several human clinical trials.[16]
Gene therapy may be classified into the two following types:
In somatic gene therapy, the therapeutic genes are transferred into the somatic cells, or body, of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations. Somatic gene therapy represents the mainstream line of current basic and clinical research, where mRNA is used to treat a disease in an individual.
In germ line gene therapy, Germ cells, i.e., sperm or eggs, are modified by the introduction of functional genes, which are integrated into their genomes. This would allow the therapy to be heritable and passed on to later generations. Although this should, in theory, be highly effective in counteracting genetic disorders and hereditary diseases, many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.[17]
Gene therapy utilizes the delivery of DNA into cells, which can be accomplished by a number of methods. The two major classes of methods are those that use recombinant viruses (sometimes called biological nanoparticles or viral vectors) and those that use naked DNA or DNA complexes (non-viral methods).
Main article:
Viral vectorAll viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. Therefore this has been recognized as a plausible strategy for gene therapy, by removing the viral DNA and using the virus as a vehicle to deliver the therapeutic DNA.
A number of viruses have been used for human gene therapy, including retrovirus, adenovirus, lentivirus, herpes simplex virus, vaccinia, pox virus, and adeno-associated virus.[3]
Non-viral methods can present certain advantages over viral methods, such as large scale production and low host immunogenicity. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques that approach the transfection efficiencies of viruses.
There are several methods for non-viral gene therapy, including the injection of naked DNA, electroporation, the gene gun, sonoporation, magnetofection, and the use of oligonucleotides, lipoplexes, dendrimers, and inorganic nanoparticles.
In 1972 Friedmann and Roblin authored a paper in Science titled "Gene therapy for human genetic disease?"[1] They cite Rogers S for proposing "that exogenous 'good'" DNA be used to replace the defective DNA in those who suffer from genetic defects.[18] They also cite the first attempt to perform gene therapy as [New York Times, 20 September 1970].
The first approved gene therapy case in the United States took place on September 14, 1990, at the National Institute of Health. It was performed on a four year old girl named Ashanti DeSilva. It was a treatment for a genetic defect that left her with an immune system deficiency. The effects were only temporary, but successful.[19]
New gene therapy approach repairs errors in messenger RNA derived from defective genes. This technique has the potential to treat the blood disorder thalassaemia, cystic fibrosis, and some cancers.[20] Researchers at Case Western Reserve University and Copernicus Therapeutics are able to create tiny liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane.[21]
Sickle cell disease is successfully treated in mice.[22]
in 1992 Doctor Claudio Bordignon working at the Vita-Salute San Raffaele University, Milan, Italy performed the first procedure of gene therapy using hematopoietic stem cells as vectors to deliver genes intended to correct hereditary diseases.[23] In 2002 this work led to the publication of the first successful gene therapy treatment for adenosine deaminase-deficiency (SCID). The success of a multi-center trial for treating children with SCID (severe combined immune deficiency or "bubble boy" disease) held from 2000 and 2002 was questioned when two of the ten children treated at the trial's Paris center developed a leukemia-like condition. Clinical trials were halted temporarily in 2002, but resumed after regulatory review of the protocol in the United States, the United Kingdom, France, Italy, and Germany.[24]
In 1993 Andrew Gobea was born with severe combined immunodeficiency (SCID). Genetic screening before birth showed that he had SCID. Blood was removed from Andrew's placenta and umbilical cord immediately after birth, containing stem cells. The allele that codes for ADA was obtained and was inserted into a retrovirus. Retroviruses and stem cells were mixed, after which they entered and inserted the gene into the stem cells' chromosomes. Stem cells containing the working ADA gene were injected into Andrew's blood system via a vein. Injections of the ADA enzyme were also given weekly. For four years T-cells (white blood cells), produced by stem cells, made ADA enzymes using the ADA gene. After four years more treatment was needed.
The 1999 death of Jesse Gelsinger in a gene-therapy experiment resulted in a significant setback to gene therapy research in the United States.[25][26] As a result, the U.S. FDA suspended several clinical trials pending the re-evaluation of ethical and procedural practices in the field.[27]
In 2003 a University of California, Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol. 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.[28]
RNA interference or gene silencing may be a new way to treat Huntington's disease. Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced.[29]
Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells. This study constitutes one of the first demonstrations that gene therapy can be effective in treating cancer.[30]
In March 2006 an international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells. The study, published in Nature Medicine, is believed to be the first to show that gene therapy can cure diseases of the myeloid system.[31]
In May 2006 a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown from the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in Milan, Italy reported a breakthrough for gene therapy in which they developed a way to prevent the immune system from rejecting a newly delivered gene.[32] Similar to organ transplantation, gene therapy has been plagued by the problem of immune rejection. So far, delivery of the 'normal' gene has been difficult because the immune system recognizes the new gene as foreign and rejects the cells carrying it. To overcome this problem, the HSR-TIGET group utilized a newly uncovered network of genes regulated by molecules known as microRNAs. Dr. Naldini's group reasoned that they could use this natural function of microRNA to selectively turn off the identity of their therapeutic gene in cells of the immune system and prevent the gene from being found and destroyed. The researchers injected mice with the gene containing an immune-cell microRNA target sequence, and the mice did not reject the gene, as previously occurred when vectors without the microRNA target sequence were used. This work will have important implications for the treatment of hemophilia and other genetic diseases by gene therapy.
In November 2006 Preston Nix from the University of Pennsylvania School of Medicine reported on VRX496, a gene-based immunotherapy for the treatment of human immunodeficiency virus (HIV) that uses a lentiviral vector for delivery of an antisense gene against the HIV envelope. In the Phase I trial enrolling five subjects with chronic HIV infection who had failed to respond to at least two antiretroviral regimens, a single intravenous infusion of autologous CD4 T cells genetically modified with VRX496 was safe and well tolerated. All patients had stable or decreased viral load; four of the five patients had stable or increased CD4 T cell counts. In addition, all five patients had stable or increased immune response to HIV antigens and other pathogens. This was the first evaluation of a lentiviral vector administered in U.S. Food and Drug Administration-approved human clinical trials for any disease.[33] Data from an ongoing Phase I/II clinical trial were presented at CROI 2009.[34]
On May 1, 2007 Moorfields Eye Hospital and University College London's Institute of Ophthalmology announced the world's first gene therapy trial for inherited retinal disease. The first operation was carried out on a 23 year-old British male, Robert Johnson, in early 2007. Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. The results of the Moorfields/UCL trial were published in New England Journal of Medicine in April 2008. They researched the safety of the subretinal delivery of recombinant adeno associated virus (AAV) carrying RPE65 gene, and found it yielded positive results, with patients having modest increase in vision, and, perhaps more importantly, no apparent side-effects.
In May 2008, three groups reported positive results using gene therapy to treat Leber's Congenital Amaurosis (LCA), a rare inherited retinal degenerative disorder that causes blindness in children. The patients had a defect in the RPE65 gene, which was replaced with a functional copy using adeno-associated virus.
The LCA trials were conducted independently by groups in the United Kingdom, Florida, and Pennsylvania. The first study to be announced was on 1 May 2007 Moorfields Eye Hospital and University College London's Institute of Ophthalmology. The first operation was carried out on a 23 year-old British male, Robert Johnson, in early 2007.[35] Two other groups, one at the University of Florida and another at the University of Pennsylvania, conducted independent clinical trials with similar results.
In all three clinical trials, patients recovered functional vision without apparent side-effects.[4][5][6][7] These studies, which used adeno-associated virus, have spawned a number of new studies investigating gene therapy for human retinal disease.
In September 2009, the journal Nature reported that researchers at the University of Washington and University of Florida were able to give trichromatic vision to squirrel monkeys using gene therapy, a hopeful precursor to a treatment for color blindness in humans.[36] In November 2009, the journal Science reported that researchers succeeded at halting a fatal brain disease, adrenoleukodystrophy, using a vector derived from HIV to deliver the gene for the missing enzyme.[37]
A paper by Komáromy et al. published in April 2010, deals with gene therapy for a form of achromatopsia in dogs. Achromatopsia, or complete color blindness, is presented as an ideal model to develop gene therapy directed to cone photoreceptors. Cone function and day vision have been restored for at least 33 months in two young dogs with achromatopsia. However, the therapy was less efficient for older dogs.[38]
In 2007 and 2008, a man being treated by Gero Hütter was cured of HIV by repeated Hematopoietic stem cell transplantation (see also Allogeneic stem cell transplantation, Allogeneic bone marrow transplantation, Allotransplantation) with double-delta-32 mutation which disables the CCR5 receptor; this cure was not completely accepted by the medical community until 2011.[39] This cure required complete ablation of existing bone marrow which is very debilitating.
In August 2011, two of three subjects of a pilot study were confirmed to have been cured from chronic lymphocytic leukemia (CLL). The study carried out by the researchers at the University of Pennsylvania used genetically modified T cells to fight the disease. [40]
Human HGF plasmid DNA therapy of cardiomyocytes is being examined as a potential treatment for coronary artery disease as well as treatment for the damage that occurs to the heart after myocardial infarction.[41][42]
For the safety of gene therapy, the Weismann barrier is fundamental in the current thinking. Soma-to-germline feedback should therefore be impossible. However, there are indications[43] that the Weismann barrier can be breached. One way it might possibly be breached is if the treatment were somehow misapplied and spread to the testes and therefore would infect the germline against the intentions of the therapy.
Some of the problems of gene therapy include:
- Short-lived nature of gene therapy – Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. 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 – Anytime a foreign object is introduced into human tissues, the immune system has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced response to invaders that it has seen before makes it difficult for gene therapy to be repeated in patients.
- Problems with viral vectors – Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient: 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 of 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.
- Chance of inducing a tumor (insertional mutagenesis) - 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 stem 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.[44] One possible solution for this is to add a functional tumor suppressor gene onto the DNA to be integrated; however, this poses its own problems, since the longer the DNA is, the harder it is to integrate it efficiently into cell genomes.
Three patients' deaths have been reported in gene therapy trials, putting the field under close scrutiny. The first was that of Jesse Gelsinger in 1999,[45] which represented a major setback in the field. One X-SCID patient died of leukemia following gene therapy treatment in 2003.[2] In 2007, a rheumatoid arthritis patient died from an infection in a gene therapy trial; a subsequent investigation concluded that the death was not related to her gene therapy treatment.[46]
Preventive gene therapy is the repair of a gene with a mutation associated with a progressive disease, prior to the expression of a medical condition, in order to prevent that expression. There are a num/ref> Similar to organ transplantation, gene therapy has been plagued by the problem of immune rejection. So far, delivery of the 'normal' gene has been difficult because the immune system recognizes the new gene as foreign and rejects the cells carrying it. To overcome this problem, the HSR-TIGET group utilized a newly uncovered network of genes regulated by molecules known as microRNAs. Dr. Naldini's group reasoned that they could use this natural function of microRNA to selectively turn off the identity of their therapeutic gene in cells of the immune system and prevent the gene from being found and destroyed. The researchers injected mice with the gene containing an immune-cell microRNA target sequence, and the mice did not reject the gene, as previously occurred when vectors without the microRNA target sequence were used. This work will have important implications for the treatment of hemophilia and other genetic diseases by gene therapy.
In November 2006 Preston Nix from the University of Pennsylvania School of Medicine reported on VRX496, a gene-based immunotherapy for the treatment of human immunodeficiency virus (HIV) that uses a lentiviral vector for delivery of an antisense gene against the HIV envelope. In the Phase I trial enrolling five subjects with chronic HIV infection who had failed to respond to at least two antiretroviral regimens, a single intravenous infusion of autologous CD4 T cells genetically modified with VRX496 was safe and well tolerated. All patients had stable or decreased viral load; four of the five patients had stable or increased CD4 T cell counts. In addition, all five patients had stable or increased immune response to HIV antigens and other pathogens. This was the first evaluation of a lentiviral vector administered in U.S. Food and Drug Administration-approved human clinical trials for any disease.ber of considerations:[47]
- It is hard to get U.S. FDA approval to treat a pre-symptomatic condition because it is hard to predict the complications that may arise, so it is hard to give a risk/benefit analysis. This is an obstacle to long-term therapies.
- It is easier to gain approval for short-term therapies to treat expressed conditions rather than prevent them.
- It is not known whether the repair of a mutation will help to treat a condition which has already progressed beyond the initial consequences of that mutation.
- In the TV series Dark Angel gene therapy is mentioned as one of the practices performed on transgenics and their surrogate mothers at Manticore, and in the episode Prodigy, Dr. Tanaka uses a groundbreaking new form of gene therapy to turn Jude, a premature, vegetative baby of a crack/cocaine addict, into a boy genius.
- Gene therapy is a crucial plot element in the video game Metal Gear Solid, where it has been used to illegally enhance the battle capabilities of soldiers within the US military, and their Next Generation Special Forces units.
- Gene therapy plays a major role in the sci-fi series Stargate Atlantis, as a certain type of alien technology can only be used if one has a certain gene which can be given to the members of the team through gene therapy involving a mouse retrovirus.
- Gene therapy also plays a major role in the plot of the James Bond movie Die Another Day, where a scientist has developed a means of altering peoples' entire appearances through the use of DNA samples acquired from others- generally homeless people that would not be missed- that are subsequently injected into the bone marrow, the resulting transformation apparently depriving the subjects of the ability to sleep.
- Gene therapy plays a recurring role in the present-time sci-fi television program ReGenesis, where it is used to cure various diseases, enhance athletic performance and produce vast profits for bio-tech corporations. (e.g. an undetectable performance-enhancing gene therapy was used by one of the characters on himself, but to avoid copyright infringement, this gene therapy was modified from the tested-to-be-harmless original, which produced a fatal cardiovascular defect)
- Gene therapy is the basis for the plot line of the film I Am Legend.[48]
- Gene therapy is an important plot key in the game Bioshock where the game contents refer to plasmids and [gene] splicers.
- The book Next by Michael Crichton unravels a story in which fictitious biotechnology companies experiment with gene therapy.
- In the television show Alias, a breakthrough in molecular gene therapy is discovered, whereby a patient's body is reshaped to identically resemble someone else. Protagonist Sydney Bristow's best friend was secretly killed and her "double" resumed her place.
- In the 2011 film Rise of the Planet of the Apes, a fictional gene therapy called ALZ-112 was a drug that was a possible cure for Alzheimer's disease, the therapy increased the host's intelligence and made their irises green, along with the revised therapy called 113 which increased intelligence in apes yet was a deadly, interinal virus.
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