Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, and hereditary diseases in which a defective mutant allele is replaced with a functional one. History See also History of genetics The existence of genes was first suggested by Gregor Mendel (1822-1884 who in the 1860s studied inheritance The cell is the structural and functional unit of all known living Organisms It is the smallest unit of an organism that is classified as living and is often called Tissue is a cellular organizational level intermediate between cells and a complete organism A disease is an abnormal condition of an organism that impairs bodily functions and can be deadly A genetic disorder is a condition caused by abnormalities in Genes or Chromosomes While some diseases such as Cancer, are due to genetic abnormalities acquired A mutant is an individual organism or new genetic character arising or resulting from an instance of Mutation, which is a base-pair sequence change within the DNA An allele (ˈæliːl (UK /əˈliːl/ (US (from the Greek αλληλος allelos, meaning each other) is one member of a pair or series of different forms Although the technology is still in its infancy, it has been used with some success. Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods. Antisense therapy is a form of treatment for Genetic disorders or infections

Gene therapy using an Adenovirus vector. Adenoviruses are medium-sized (90–100 nm) Nonenveloped (naked Icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known If the treatment is successful, the new gene will make a functional protein. Proteins are large Organic compounds made of Amino acids arranged in a linear chain and joined together by Peptide bonds between the Carboxyl

Background

On September 14, 1990 at the U.S. National Institutes of Health W. French Anderson, M. Events 81 - Domitian becomes Emperor of the Roman Empire upon the death of his brother Titus. Year 1990 ( MCMXC) was a Common year starting on Monday (link displays the 1990 Gregorian calendar) The United States of America —commonly referred to as the "NIH" redirects here For other meanings of NIH see NIH (disambiguation. William French Anderson, MD (born December 31, 1936) is a US Physician, Geneticist and molecular biologist D. , and his colleagues R. Michael Blaese, M. D. , C. Bouzaid, M. D. , and Kenneth Culver, M. D. , performed the first approved gene therapy procedure on four-year old Ashanthi DeSilva. Born with a rare genetic disease called severe combined immunodeficiency (SCID), she lacked a healthy immune system, and was vulnerable to every passing germ or infection. Severe combined immunodeficiency ( SCID) or Boy in the Bubble Syndrome is a genetic disorder in which both "arms" ( B cells and T cells Children with this illness usually develop overwhelming infections and rarely survive to adulthood; a common childhood illness like chickenpox is life-threatening. Ashanthi led a cloistered existence -- avoiding contact with people outside her family, remaining in the sterile environment of her home, and battling frequent illnesses with massive amounts of antibiotics.

In Ashanthi's gene therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the lab, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream. Laboratory tests have shown that the therapy strengthened Ashanthi's immune system by 40%; she no longer has recurrent colds, she has been allowed to attend school, and she was immunized against whooping cough. This procedure was not a cure; the white blood cells treated genetically only work for a few months, after which the process must be repeated (VII, Thompson [First] 1993). As of early 2007, she was still in good health, and she was attending college. However, there is no consensus on what portion of her improvement should be attributed to gene therapy versus other treatments. Some would state that the case is of great importance despite its indefinite results, if only because it demonstrated that gene therapy could be practically attempted without adverse consequences. [1]

Although this simplified explanation of a gene therapy procedure sounds like a happy ending, it is little more than an optimistic first chapter in a long story; the road to the first approved gene therapy procedure was rocky and fraught with controversy. The biology of human gene therapy is very complex, and there are many techniques that still need to be developed and diseases that need to be understood more fully before gene therapy can be used appropriately. The public policy debate surrounding the possible use of genetically engineered material in human subjects has been equally complex. Major participants in the debate have come from the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different views to the discussion.

Scientists took the logical step of trying to introduce genes straight into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia. Cystic fibrosis (also known as CF, mucoviscoidosis, or mucoviscidosis) is a hereditary disease affecting the exocrine (mucus glands of the lungs Haemophilia (also spelled as hemophilia Muscular dystrophy (MD refers to a group of genetic, Hereditary Muscle diseases that cause progressive muscle weakness Sickle-cell disease or sickle-cell anaemia (or anemia) is a Blood disorder characterized by Red blood cells that assume an abnormal rigid However, this has been much harder than modifying simple bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering them to the correct site on the comparatively large human genome. The human genome is the Genome of Homo sapiens, which is stored on 23 chromosome pairs

Basic process

In most gene therapy studies, a "correct copy" or "wild type" gene is provided or inserted into the genome. Generally, it is not an exact replacement of the "abnormal," disease-causing gene, but rather extra, correct copies of genes are provided to complement the loss of function. A carrier called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common type of vectors are viruses that have been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. A pathogen (from Greek πάθος pathos "suffering passion" and γἰγνομαι (γεν- gignomai (gen- "I give birth to" infectious Scientists have tried to harness this ability by manipulating the viral genome to remove disease-causing genes and insert therapeutic ones.

Target cells such as the patient's liver or lung cells are infected with the vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal state.

Types of gene therapy

Gene therapy may be classified into the following types:

Germ line gene therapy

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

Somatic cell gene therapy

In somatic cell gene therapy, the gene is introduced only in somatic cells, especially of those tissues in which expression of the concerned gene is critical for health. Expression of the introduced gene relieves/ eliminates symptoms of the disorder, but this effect is not heritable as it does not involve the germ line. At present, somatic cell therapy is the only feasible option, and clinical trials addressing a variety of conditions have already begun.

Broad methods

There are a variety of different methods to replace or repair the genes targeted in gene therapy. [2]

• A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
• An abnormal gene could be swapped for a normal gene through homologous recombination.
• The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
• The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

Vectors in gene therapy

Viruses

Main article: Viral vector

All viruses attack their hosts and introduce their genetic material into the host cell as part of their replication cycle. Viral vectors are a tool commonly used by molecular Biologists to deliver Genetic material into cells This process can be performed inside a living organism A virus (from the Latin virus meaning Toxin or Poison) is a sub-microscopic infectious agent that is unable This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hijacking the body's normal production machinery to serve the needs of the virus. The host cell will carry out these instructions and produce additional copies of the virus, leading to more and more cells becoming infected. Some types of viruses actually physically insert their genes into the host's genome (a defining feature of retroviruses, the family of viruses that includes HIV, is that the virus will introduce the enzyme reverse transcriptase into the host and thus use its RNA as the "instructions"). A retrovirus is any Virus belonging to the viral family Retroviridae. In Biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase Enzyme that transcribes This incorporates the genes of that virus among the genes of the host cell for the life span of that cell.

Doctors and molecular biologists realized that viruses like this could be used as vehicles to carry 'good' genes into a human cell. First, a scientist would remove the genes in the virus that cause disease. Then they would replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics). This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host's genome are left intact. This can be confusing, and requires significant research and understanding of the virus' genes in order to know the function of each. An example: A virus is found which replicates by inserting its genes into the host cell's genome. This virus has two genes- A and B. Gene A encodes a protein which allows this virus to insert itself into the host's genome. Gene B causes the disease this virus is associated with. Gene C is the "normal" or "desirable" gene we want in the place of gene B. Thus, by re-engineering the virus so that gene B is replaced by gene C, while allowing gene A to properly function, this virus could introduce the required gene - gene C into the host cell's genome without causing any disease.

All this is clearly an oversimplification, and numerous problems exist that prevent gene therapy using viral vectors, such as: trouble preventing undesired effects, ensuring the virus will infect the correct target cell in the body, and ensuring that the inserted gene doesn't disrupt any vital genes already in the genome. However, this basic mode of gene introduction currently shows much promise and doctors and scientists are working hard to fix any potential problems that could exist.

Retroviruses

The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. Ribonucleic acid ( RNA) is a Nucleic acid that consists of a long chain of Nucleotide units When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is termed reverse transcription. Reverse transcription is the process of making a double stranded DNA (deoxyribonucleic acid molecule from a single stranded RNA (ribonucleic acid template It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. In Biochemistry, a reverse transcriptase, also known as RNA-dependent DNA polymerase, is a DNA polymerase Enzyme that transcribes After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. In Cell biology, the nucleus (pl nuclei; from Latin la ''nucleus'' or la ''nuculeus'' "little nut" or kernel is a membrane-enclosed That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase. Integrase is an enzyme produced by a Retrovirus (including HIV) that enables its genetic material to be integrated into the DNA of the infected cell

Now that the genetic material of the virus is incorporated and has become part of the genetic material of the host cell, it can be said that the host cell is now modified to contain a new gene. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately.

One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus in any arbitrary position in the genome of the host- it randomly shoves the genetic material into a chromosome. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). Insertional mutagenesis is Mutagenesis of dna by the insertion of one or more bases If the gene happens to be one regulating cell division, uncontrolled cell division (i. e. , cancer) can occur. Cancer (medical term Malignant Neoplasm) is a class of Diseases in which a group of cells display uncontrolled This problem has recently begun to be addressed by utilizing zinc finger nucleases^[1] or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites. Zinc finger nucleases ( ZFN s are protein chimera comprised of a Zinc finger -based DNA-binding domain and a DNA-cleavage domain

Gene therapy trials to treat severe combined immunodeficiency (SCID) were halted or restricted in the USA when leukemia was reported in three of eleven patients treated in the French Therapy X-linked SCID (XSCID) gene therapy trial. Severe combined immunodeficiency ( SCID) or Boy in the Bubble Syndrome is a genetic disorder in which both "arms" ( B cells and T cells Leukemia or leukaemia (Greek leukos λευκός, "white" aima αίμα, "blood" is a Cancer of the Blood Ten XSCID patients treated in England have not presented leukemia to date and have had similar success in immune reconstitution. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue with relative success in the USA, Italy and Japan. Adenosine deaminase (also known as ADA) is an Enzyme ( involved in purine metabolism.

Adenoviruses

Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. Adenoviruses are medium-sized (90–100 nm) Nonenveloped (naked Icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA They cause respiratory (especially the common cold), intestinal, and eye infections in humans. When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated (transient) 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. Transcription is the synthesis of RNA under the direction of DNA The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. As a result, treatment with the adenovirus will require readministration in a growing cell population although the absence of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has shown real promise in treating cancer and indeed the first gene therapy product to be licensed to treat cancer is an adenovirus.

Adeno-associated viruses

Adeno-associated viruses, from the parvovirus family, are small viruses with a genome of single stranded DNA. Adeno-associated virus (AAV is a small Virus which infects humans and some other primate species Parvovirus, commonly abbreviated to parvo, is a Genus of the Parvoviridae family linear non-segmented single stranded DNA viruses The wild type AAV can insert genetic material at a specific site on chromosome 19 with near 100% certainty. But the recombinant AAV, that does not contain any viral genes and only the therapeutic gene does not integrate into the genome. Instead the recombinant viral genome fuses at its ends via the ITR (inverted terminal repeats) recombination to form circular, episomal forms which are predicted to be the primary cause of the long term gene expression. There are a few disadvantages to using AAV, including the small amount of DNA it can carry (low capacity) and the difficulty in producing it. This type of virus is being used, however, because it is non-pathogenic (most people carry this harmless virus). A pathogen (from Greek πάθος pathos "suffering passion" and γἰγνομαι (γεν- gignomai (gen- "I give birth to" infectious In contrast to adenoviruses, most people treated with AAV will not build an immune response to remove the virus and the cells that have been successfully treated with it. Several trials with AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases; the two tissues where the virus seems particularly useful. However, clinical trials have also been initiated where AAV vectors are used to deliver genes to the brain. This is possible because AAV viruses can infect non-dividing (quiescent) cells, such as neurons in which their genomes are expressed for a long time.

Envelope protein pseudotyping of viral vectors

The viral vectors described above have natural host cell populations that they infect most efficiently. Retroviruses have limited natural host cell ranges, and although adenovirus and adeno-associated virus are able to infect a relatively broader range of cells efficiently, some cell types are refractory to infection by these viruses as well. A retrovirus is any Virus belonging to the viral family Retroviridae. Adenoviruses are medium-sized (90–100 nm) Nonenveloped (naked Icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA Adeno-associated virus (AAV is a small Virus which infects humans and some other primate species Attachment to and entry into a susceptible cell is mediated by the protein envelope on the surface of a virus. Retroviruses and adeno-associated viruses have a single protein coating their membrane, while adenoviruses are coated with both an envelope protein and fibers that extend away from the surface of the virus. The envelope proteins on each of these viruses bind to cell-surface molecules such as heparin sulfate, which localizes them upon the surface of the potential host, as well as with the specific protein receptor that either induces entry-promoting structural changes in the viral protein, or localizes the virus in endosomes wherein acidification of the lumen (anatomy) induces this refolding of the viral coat. Heparan sulfate (HS is a linear Polysaccharide found in all animal tissues In Biochemistry, a receptor is a Protein molecule embedded in either the Plasma membrane or Cytoplasm of a cell to which a mobile signaling In Biology, an endosome is a membrane-bound compartment inside cells roughly 300-400 nm in diameter when fully mature A lumen (Lat lūmen, an opening or light (pl lumina is the inside space or lining of a tubular structure such as an artery or intestine In either case, entry into potential host cells requires a favorable interaction between a protein on the surface of the virus and a protein on the surface of the cell. For the purposes of gene therapy, one might either want to limit or expand the range of cells susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses, or by chimeric proteins. Such chimera would consist of those parts of the viral protein necessary for incorporation into the virion as well as sequences meant to interact with specific host cell proteins. Viruses in which the envelope proteins have been replaced as described are referred to as pseudotyped viruses. For example, the most popular retroviral vector for use in gene therapy trials has been the lentivirus Simian immunodeficiency virus coated with the envelope proteins, G-protein, from Vesicular stomatitis virus. Lentivirus ( lenti-, Latin for " slow " is a Genus of slow Viruses of the Retroviridae family Simian immunodeficiency virus ( SIV) is a Retrovirus that is found in numerous strains in Primates; the specific strains infecting Humans GTP chemical structurepng|thumb|180px| Guanosine triphosphate]] G proteins short for guanine nucleotide-binding proteins, are a family of Proteins involved Vesicular stomatitis virus (VSV is a Virus in the family Rhabdoviridae; the well-known Rabies virus belongs to the same family This vector is referred to as VSV G-pseudotyped lentivirus, and infects an almost universal set of cells. This tropism is characteristic of the VSV G-protein with which this vector is coated. Many attempts have been made to limit the tropism of viral vectors to one or a few host cell populations. This advance would allow for the systemic administration of a relatively small amount of vector. The potential for off-target cell modification would be limited, as well as many concerns from the medical community. Most attempts to limit tropism have used chimeric envelope proteins bearing antibody fragments. Antibodies (also known as immunoglobulins, abbreviated Ig) are Gamma globulin Proteins that are found in Blood or other Bodily These vectors show great promise for the development of "magic bullet" gene therapies.

Non-viral methods

Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses. Transfection is the process of introducing nucleic acids into cells by non-viral methods. Gene expression is the process by which inheritable information from a Gene, such as the DNA sequence, is made into a functional Gene product, such

Naked DNA

This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection. Naked DNA is Histone -free DNA that is passed from cell to cell during a gene transfer process called transformation or Transfection. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success. This success, however, does not compare to that of the other methods, leading to research into more efficient methods for delivery of the naked DNA such as electroporation and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas. Electroporation, or electropermeabilization, is a significant increase in the Electrical conductivity and permeability of the cell plasma membrane caused The gene gun or the Biolistic Particle Delivery System originally designed for Plant transformation, is a device for injecting cells with genetic information

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. Sense, when applied in a Molecular biology context is a general concept used to compare the polarity of Nucleic acid molecules such as DNA or RNA Another uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. Small interfering RNA ( siRNA) sometimes known as short interfering RNA or silencing RNA, is a class of 20-25 Nucleotide -long double-stranded Messenger ribonucleic acid ( mRNA) is a molecule of RNA encoding a chemical "blueprint" for a Protein product A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression.

Lipoplexes and polyplexes

To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and its entry into the cell must be facilitated. To this end new molecules, lipoplexes and polyplexes, have been created that have the ability to protect the DNA from undesirable degradation during the transfection process.

Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome. A micelle (rarely micella, plural micelles) is an aggregate of Surfactant molecules dispersed in a liquid Colloid. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids, anionic (negatively charged), neutral, or cationic (positively charged). Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors. However, in spite of the facts that there is little toxicity associated with them, that they are compatible with body fluids and that there was a possibility of adapting them to be tissue specific; they are complicated and time consuming to produce so attention was turned to the cationic versions.

Cationic lipids, due to their positive charge, were first used to condense negatively charged DNA molecules so as to facilitate the encapsulation of DNA into liposomes. Cationic liposomes are structures that are made of positively charged Lipids and are increasingly being researched for use in Gene therapy due to their favourable interactions Later it was found that the use of cationic lipids significantly enhanced the stability of lipoplexes. Also as a result of their charge, cationic liposomes interact with the cell membrane, endocytosis was widely believed as the major route by which cells uptake lipoplexes. Endocytosis is a process where cells absorb material ( Molecules such as proteins from the outside by engulfing it with their Cell membrane. Endosomes are formed as the results of endocytosis, however, if genes can not be released into cytoplasm by breaking the membrane of endosome, they will be sent to lysosomes where all DNA will be destroyed before they could achieve their functions. It was also found that although cationic lipids themselves could condense and encapsulate DNA into liposomes, the transfection efficiency is very low due to the lack of ability in terms of “endosomal escaping”. However, when helper lipids (usually electroneutral lipids, such as DOPE) were added to form lipoplexes, much higher transfection efficiency was observed. Later on, it was figured out that certain lipids have the ability to destabilize endosomal membranes so as to facilitate the escape of DNA from endosome, therefore those lipids are called fusogenic lipids. Although cationic liposomes have been widely used as an alternative for gene delivery vectors, a dose dependent toxicity of cationic lipids were also observed which could limit their therapeutic usages.

The most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. Recent studies have shown lipoplexes to be useful in transfecting respiratory epithelial cells, so they may be used for treatment of genetic respiratory diseases such as cystic fibrosis. In biology and medicine epithelium is a tissue composed of cells that line the cavities and surfaces of structures throughout the body

Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis, the process by which the polyplex enters the cell) such as inactivated adenovirus must occur. However, this isn't always the case, polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan. Chitosan is a linear Polysaccharide composed of randomly distributed β-(1-4-linked D-glucosamine (deacetylated unit and N-acetyl-D-glucosamine (acetylated

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. A virosome is a unilamellar phospholipid bilayer vesicle with a mean diameter of 150 nm A liposome is a tiny bubble ( vesicle) made out of the same material as a cell membrane. Human immunodeficiency virus ( HIV) is a Lentivirus (a member of the Retrovirus family that can lead to acquired immunodeficiency syndrome The Orthomyxoviridae (Derivation of name orthos is Greek for straight myxa is Greek for Mucus) are a family of RNA viruses that This has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. In biology and medicine epithelium is a tissue composed of cells that line the cavities and surfaces of structures throughout the body Other methods involve mixing other viral vectors with cationic lipids or hybridising viruses.

Dendrimers

A dendrimer is a highly branched macromolecule with a spherical shape. Dendrimers are repeatedly branched Molecules The huge number of papers on dendritic architectures such as dendrimers dendronized hyperbranched and brush-polymers has generated The term macromolecule by definition implies "large Molecule " 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 complimentarity 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.

In recent years the benchmark for transfection agents has been cationic lipids. Limitations of these competing reagents have been reported to include: the lack of ability to transfect a number of cell types, the lack of robust active targeting capabilities, incompatibility with animal models, and toxicity. Dendrimers offer robust covalent construction and extreme control over molecule structure, and therefore size. Together these give compelling advantages compared to existing approaches.

Producing dendrimers has historically been a slow and expensive process consisting of numerous slow reactions, an obstacle that severely curtailed their commercial development. The Michigan based company Dendritic Nanotechnologies discovered a method to produce dendrimers using kinetically driven chemistry, a process that not only reduced cost by a magnitude of three, but also cut reaction time from over a month to several days. These new "Priostar" dendrimers can be specifically constructed to carry a DNA or RNA payload that transfects cells at a high efficiency with little or no toxicity.

Major developments in gene therapy

2002 and earlier

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. Thalassemia (from Greek θαλασσα thalassa sea + αίμα haima blood British spelling "thalassaemia" is an inherited Autosomal recessive Cystic fibrosis (also known as CF, mucoviscoidosis, or mucoviscidosis) is a hereditary disease affecting the exocrine (mucus glands of the lungs See Subtle gene therapy tackles blood disorder at NewScientist. com (October 11, 2002). Events 1138 - A massive earthquake struck Aleppo, Syria. 1531 - Huldrych Zwingli is killed See also 2002 (disambiguation Year 2002 ( MMII) was a Common year starting on Tuesday of the Gregorian calendar.

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. Case Western Reserve University is a private research university located in Cleveland Ohio, United States, with some residence halls on the south end of campus See DNA nanoballs boost gene therapy at NewScientist. com (May 12, 2002). Events 1191 - Richard I of England marries Berengaria of Navarre. See also 2002 (disambiguation Year 2002 ( MMII) was a Common year starting on Tuesday of the Gregorian calendar.

Sickle cell disease is successfully treated in mice. See Murine Gene Therapy Corrects Symptoms of Sickle Cell Disease from March 18, 2002, issue of The Scientist. Sickle-cell disease or sickle-cell anaemia (or anemia) is a Blood disorder characterized by Red blood cells that assume an abnormal rigid Events 37 - The Roman Senate annuls Tiberius ' will and proclaims Caligula emperor See also 2002 (disambiguation Year 2002 ( MMII) was a Common year starting on Tuesday of the Gregorian calendar.

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. Severe combined immunodeficiency ( SCID) or Boy in the Bubble Syndrome is a genetic disorder in which both "arms" ( B cells and T cells 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. (V. Cavazzana-Calvo, Thrasher and Mavilio 2004; see also 'Miracle' gene therapy trial halted at NewScientist. com, October 3, 2002). Events 42 BC - First Battle of Philippi: Triumvirs Mark Antony and Octavian fight an indecisive battle with Caesar's See also 2002 (disambiguation Year 2002 ( MMII) was a Common year starting on Tuesday of the Gregorian calendar.

In 1993 Andrew Gobea was born with a rare, normally fatal genetic disease - severe combined immunodeficiency (SCID). Severe combined immunodeficiency ( SCID) or Boy in the Bubble Syndrome is a genetic disorder in which both "arms" ( B cells and T cells 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. Adenosine deaminase (also known as ADA) is an Enzyme ( involved in purine metabolism. 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. 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.

2003

In 2003 a University of California, Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol (PEG). The University of California Los Angeles (generally known as UCLA) is a public research university located in Westwood Los Angeles, California, United A liposome is a tiny bubble ( vesicle) made out of the same material as a cell membrane. A polymer is a large Molecule ( Macromolecule) composed of repeating Structural units typically connected by Covalent Chemical bonds Poly( Ethylene glycol) (PEG also known as poly( Ethylene oxide) (PEO or polyoxyethylene (POE is the most commercially important type of polyether The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the "blood-brain barrier. The blood-brain barrier (BBB is a metabolic or cellular structure in the Central nervous system (CNS that restricts the passage of various chemical substances and microscopic " This method has potential for treating Parkinson's disease. Parkinson's disease (also known as Parkinson disease or PD) is a degenerative disorder of the Central nervous system that often impairs the sufferer's See Undercover genes slip into the brain at NewScientist. com (March 20, 2003). Events 1600 - The Linköping Bloodbath takes place on Maundy Thursday in Linköping, Sweden. Year 2003 ( MMIII) was a Common year starting on Wednesday of the Gregorian calendar.

RNA interference or gene silencing may be a new way to treat Huntington's. RNA interference ( RNAi) is a mechanism that inhibits Gene expression at the stage of translation or by hindering the transcription of specific Gene silencing is a general term describing Epigenetic processes of Gene regulation. 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. Small interfering RNA ( siRNA) sometimes known as short interfering RNA or silencing RNA, is a class of 20-25 Nucleotide -long double-stranded See Gene therapy may switch off Huntington's at NewScientist. com (March 13, 2003). Events 1138 - Cardinal Gregorio Conti is elected Antipope as Victor IV, succeeding Anacletus II. Year 2003 ( MMIII) was a Common year starting on Wednesday of the Gregorian calendar.

2006

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. "NIH" redirects here For other meanings of NIH see NIH (disambiguation. Bethesda is an Unincorporated area in southern Montgomery County Maryland, just Northwest of Washington D This study constitutes the first demonstration that gene therapy can be effective in treating cancer. The study results have been published in Science (October 2006).

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. Milan (Milano Milan (listen) is one of the largest cities in Italy, located in the plains of Lombardy. Italy (Italia officially the Italian Republic, (Repubblica Italiana is located on the Italian Peninsula in Southern Europe, and on the two largest ^[1] 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. An immune system is a collection of mechanisms within an Organism that protects against Disease by identifying and killing Pathogens and Tumor To overcome this problem, the HSR-TIGET group utilized a newly uncovered network of genes regulated by molecules known as microRNAs. In Genetics, microRNAs ( miRNA) are single-stranded RNA molecules of about 21–23 Nucleotides in length which regulate Gene expression 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 spectacularly, 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 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 term myeloid suggests an origin in the Bone marrow or Spinal cord, or a resemblance to the marrow or spinal cord The study, published in Nature Medicine, is believed to be the first to show that gene therapy can cure diseases of the myeloid system.

2007

On 1 May 2007 Moorfields Eye Hospital and University College London's Institute of Ophthalmology announced the world's first gene therapy trial for inherited retinal disease. Events 305 - Diocletian and Maximian retire from the office of Roman Emperor. Year 2007 ( MMVII) was a Common year starting on Monday of the Gregorian calendar in the 21st century. University College London ( UCL) is a multi-faculty university institution based in the United Kingdom and a constituent college of the University of London The first operation was carried out on a 23 year-old British male, Robert Johnson, in early 2007. The United Kingdom of Great Britain and Northern Ireland, commonly known as the United Kingdom, the UK or Britain,is a Sovereign state located Robert Johnson In early 2007 Robert Johnson became the first person in the world to undergo Gene therapy treatment on the Eye. ^[2] Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. Leber's congenital amaurosis (LCA is a rare inherited eye disease that appears at birth or in the first few months of life and affects around 1 in 80000 The results of the Moorfields/UCL trial were published in New England Journal of Medicine in April 2008. The New England Journal of Medicine ( N Engl J Med or NEJM) is an English-language Peer-reviewed Medical journal published 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. ^[3]

Problems and ethics

For the safety of gene therapy, the Weismann barrier is fundamental in the current thinking. The Weismann barrier is the principle that hereditary information moves only from genes to body cells and never in reverse Soma-to-germline feedback should therefore be impossible. The Weismann barrier is the principle that hereditary information moves only from genes to body cells and never in reverse However, there are indications [3] that the Weissman 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 it has seen before makes it difficult for gene therapy to be repeated in patients. An immune system is a collection of mechanisms within an Organism that protects against Disease by identifying and killing Pathogens and Tumor
• Problems with viral vectors - Viruses, while 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. In biology mutations are changes to the Nucleotide sequence of the Genetic material of an organism 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. Heart disease is an Umbrella term for a variety for different diseases affecting the Heart. Hypertension, also referred to as high blood pressure, HTN or HPN, is a medical condition in which the Blood pressure is chronically elevated Alzheimer's disease ( AD) also called Alzheimer disease or simply Alzheimer's, is the most common form of Dementia. Arthritis (from Greek arthro-, joint + -itis, inflammation plural arthritides is a group of conditions involving damage to the Joints of the body Diabetes mellitus (ˌdaɪəˈbiːtiːz or /ˌdaɪəˈbiːtəs/ /məˈlaɪtəs/ or /ˈmɛlətəs/ often referred to simply as diabetes ( Ancient Greek: grc 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. See also Cancer A tumor or tumour is the name for a swelling or lesion formed by an abnormal growth of cells (termed neoplastic Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known In classical genetics the genome of a Diploid Organism including Eukarya refers to a full set of chromosomes or genes in a Gamete, thereby A tumor suppressor gene, or antioncogene is a Gene that protects a cell from one step on the path to cancer 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. ^[4]
• Religious concerns - among people who believe that humans were created in God's image, some may consider the alteration of an individual's genes as tampering or corrupting God's work. "Creationism" can also refer to Creation myths in general or to a concept about the origin of the soul.

Deaths have occurred due to gene therapy, including that of Jesse Gelsinger. Jesse Gelsinger ( June 18 1981 - September 17 1999) was the first person publicly identified as having died in a clinical trial for Gene

from:http://www.ornl.gov/sci/techresources/Human_Genome/medicine/genetherapy.shtml

In popular culture

• Gene therapy is a crucial plot element in the video game Metal Gear Solid, where it has been used to enhance the battle capabilities of enemy soldiers. is a stealth-action Video game directed by Hideo Kojima. The game was developed by Konami Computer Entertainment Japan and first published

• 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 is given to the members of the team through gene therapy. Stargate Atlantis (often abbreviated as SGA) is an American - Canadian Science fiction Television program, part of the

• Gene therapy also plays a major role in the plot of the James Bond movie Die Another Day. James Bond 007 is a Fictional character created in 1953 by writer Ian Fleming, who featured him in twelve Novels and two Short story Die Another Day is a 2002 film the 20th in the James Bond series, and the fourth and final film to star Pierce Brosnan as fictional MI6

• The Yellow Bastard from Frank Miller's Sin City was also apparently the recipient of gene therapy. Sin City is the title for a series of comics by Frank Miller, told in a Film noir -like style (now known as Neo noir)

• In the The Dark Knight Strikes Again, Dick Grayson, the first Robin, becomes a victim of extensive gene therapy for years by Lex Luthor to become The Joker. Batman The Dark Knight Strikes Again (also referred to as DK2) is a Batman mini-series by Frank Miller with Lynn Richard John "Dick" Grayson is a fictional Superhero that appears in Comic books published by DC Comics. Robin (also referred to as The Boy Wonder) is the name of several Fictional characters appearing in Comic books published by DC Comics, originally Lex Luthor is a fictional character that appears in comic books published by DC Comics. The Joker is a fictional character appearing in Comic books published by DC Comics.

• 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. I Am Legend is a 2007 post-apocalyptic Science fiction film directed by Francis Lawrence and starring Will Smith.

• Gene therapy is an important plot key in the game Bioshock where the game contents refer to plasmids and [gene] splicers. BioShock is a video game by 2K Boston/2K Australia —previously known as Irrational Games A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA

• The book Next by Michael Crichton unravels a story in which fictitious biotechnology companies which experiment with gene therapy are involved.

• 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. Sydney Anne Bristow (born 17 April 1975) played by Jennifer Garner, is the main character on the television series Alias

See also

• Antisense therapy
• DNA
• Genetic engineering
• Life extension
• List of life extension related topics
• Technology assessment
• Therapeutic gene modulation
• Pharmacological gene therapy

References

• Durai, Sundar; Mala Mani, Karthikeyan Kandavelou, Joy Wu, Matthew H. Porteus, and Srinivasan Chandrasegaran (October 2005). "Zinc finger nucleases: custom-designed molecular scissors for genome engineering of plant and mammalian cells". Nucleic Acids Research 33 (18): 5978–5990. Nucleic Acids Research or NAR is a Peer reviewed Scientific journal published by Oxford University Press. doi:10.1093/nar/gki912. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document. PMID 16251401.  

• Gardlik, Roman; Roland Pálffy, Július Hodosy, Ján Lukács, Ján Turňa and Peter Celec (April 2005). "Vectors and delivery systems in gene therapy". Medical Science Monitor 11 (4): 110–121. PMID 15795707.  

• Staff (November 18, 2005). Gene Therapy (FAQ). Human Genome Project Information. Oak Ridge National Laboratory. Oak Ridge National Laboratory ( ORNL) is a multiprogram science and technology National laboratory managed for the United States Department of Energy by Retrieved on 2006-05-28. Year 2006 ( MMVI) was a Common year starting on Sunday of the Gregorian calendar. Events 585 BC - A Solar eclipse occurs as predicted by Greek philosopher and scientist Thales, while Alyattes is battling

• Salmons B, Gunzburg WH (1993) "Targeting of retroviral vectors for gene therapy". Human Gene Therapy 4(2):129-141.

• Baum C, Dullmann J, Li Z, Fehse B, Meyer J, Williams DA, von Kalle C. Side effects of retroviral gene transfer into hematopoietic stem cells. Blood. 2003 Mar 15;101(6):2099-114

• Horn PA, Morris JC, Neff T, Kiem HP. Stem cell gene transfer--efficacy and safety in large animal studies. Molecular Therapy, 2004 Sep;10(3):417-31

• Wang, Hongjie; Dmitry M. Shayakhmetov, Tobias Leege, Michael Harkey, Qiliang Li, Thalia Papayannopoulou, George Stamatoyannopolous, and André Lieber (September 2005). "A capsid-modified helper-dependent adenovirus vector containing the beta-globin locus control region displays a nonrandom integration pattern and allows stable, erythroid-specific gene expression". Journal of Virology 79 (17): 10999-11013. doi:10.1128/JVI.79.17.10999-11013.2005. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document.  

External links

• Gene Therapy: Molecular Bandage? University of Utah's Genetic Science Learning Center
• Gene Therapy-Types
• The American Society of Gene Therapy
• The European Society of Gene Therapy
• 2003 news relating to gene therapy
• Research Group at Cambridge, UK working on overcoming current hurdles to successful gene therapy
• Cell Therapy News
• Council for Responsible Genetics
• Molecular Medicine and Gene Therapy at Lund University
• Dossier on gene therapy and clinical trials
• Gene Therapy Net
• The Gene Therapy Review