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Adenoasociados Virus (VAA) I Métodos de Problemas no virales The gene therapy y de sus Aplicaciones

adeno-associated virus (AAV)

are small viruses with a single-stranded DNA genome . They can insert genetic material at a specific site in the chromosome 19, with almost 100% certainty. However, the recombinant AAV , which contains no viral gene, only the therapeutic gene, not integrated into the genome. Instead, the recombinant viral genome merges its ends through the ITR (inverted terminal repeats), appearing recombination of the circular and episomal is predicted that may be the cause of long-term gene expression. We can find some drawbacks to the use of AAV, as the small amount of DNA that can carry (low capacity) and the difficulty in its preparation. This type of virus is being used, however, because it is a virus non-pathogenic (most people carry this harmless virus). Unlike adenovirus, in most patients treated with AAV will not be immune responses to eliminate the virus or cells with which they have been treated. Many trials with AAV are underway or in preparation, mainly treatment of muscle and eye diseases, the two tissues where the virus seems particularly useful. However, they are beginning to conduct clinical trials, where AAV vectors are used to introduce genes into the brain . This is possible because AAV can infect quiescent cells (not divided), such as neurons .

Herpes virus

are DNA viruses which target cells are neurons. Its complexity and how little we still know of this family of viruses, difficult to use. The big advantage is the large size of their DNA, allowing them to accept multiple therapeutic genes. One drawback is that it should be eliminated sequences encoding the lytic proteins of the virus that kills cells that it infects.

Protein "pseudotyping" viral vector

viral vectors described above have natural populations of host cells they infect efficiently. Retroviruses have limited natural host types, and although adenovirus and adeno-associated virus capable of infecting a wide range of cells efficiently, some cell types are refractory to infection by these viruses. The attack to enter a cell is mediated by the envelope protein on the surface of a virus. Retroviruses and adeno-associated virus have a single coat protein in the membrane, whereas the adenovirus are coated with a sheath of protein and fiber that extends outside the surface. The envelope protein of each of these viruses bind to molecules the cell surface, such as heparin, which is located on the surface of potential host cells, as well as specific protein receptor also induces structural changes in the protein virus, or locate the virus in endosomes , where acidification leads him to retract its coating. In any case, entry into host cells requires a favorable interaction between a protein on the surface of the virus, and a protein on the cell surface. For the purpose of gene therapy, it could limit or expand the range of cells susceptible to transduction by a gene therapy vector. Therefore, many vectors have been developed in which the viral envelope protein has been replaced by other protein coatings of other viruses, or chimeric proteins. This chimera consist parts of the viral protein necessary for incorporation into the virion and sequences, supposedly, to interfacing with specific receptors of cellular proteins. Viruses in which the protein coating has been replaced as described are referred to pseudotyped virus. For example, the most popular retroviral vector for use in gene therapy trials has been the lentivirus, human immunodeficiency virus Simian, covered with a covering of G proteins of vesicular stomatitis virus. This vector is known as VSV and can infect almost all cells through the G protein with which this vector is coated. There have been many attempts to limit tropism (the ability to infect many cells) of viral vectors for one or more populations of a host cell. This development could allow the routine administration of a relatively small amount of vector. Most attempts have used chimeric proteins to the envelope, which include fragments of antibodies . Thus, these vectors pseudotyped seem to be a great promise for the discovery of the "magic bullet" in gene therapy.

nonviral methods

Estos métodos presentan ventajas sobre los métodos virales, con una simple producción a gran escala y una baja inmunogenicidad. Anteriormente, los bajos niveles de transfección y expresión del gen mantenían a los métodos no virales en una situación de desventaja; sin embargo, los recientes avances en la tecnología de vectores han producido moléculas y técnicas de transfección con eficiencias similares a las de los virus.

ADN desnudo

Éste es el método más simple de la transfección no viral. Consiste en la inyección intramuscular of, for example, a plasmid with naked DNA . Several of these trials have yielded successful results. However, the expression was very low compared with other methods of transfection, has an unlimited size, does not integrate into the genome, and does not amplify DNA. In addition to the tests with plasmids, were tested with PCR products, and has successfully done similar or higher. This achievement, however, no better than other methods, which led to a more efficient method research of naked DNA, such as electroporation , sonoparción, or using a "gene gun" that shoots particles of DNA-coated gold into the cell using high pressure of gas.

Electroporation and microinjection , techniques and methods used in nonviral gene therapy

Oligonucleotides

Use synthetic oligonucleotides in gene therapy is the inactivation of genes involved in the disease process. There are several methods by which this is achieved. A strategy, using antisense oligonucleotides (called, in this case antisense therapy ) specific for the target gene and alter the transcription of the gene. Another uses small molecules of RNA called siRNA to signal the cell to adhere to specific and unique sequences in messenger RNA transcript of a faulty gene, disrupting the translation of defective mRNAs, and thus gene expression . Another strategy used oligodeoxynucleotides as a decoy for the transcription factors that are required in the activation of transcription of target genes. Transcription factors bind to the bait instead of the defective gene promoter, which reduces the transcription of target genes, and expression. In addition, oligonucleotides of single-stranded DNA, have been used to manage the change of a single base within a gene mutant.

lipoplex and poliplexes

To enhance the introduction of a new DNA in cell, it must be protected from harm and its entry into the cell must be provided. To this end new molecules, such as liposomes and polysomes were created, and have the ability to protect DNA from degradation during the transfection process . The plasmid DNA may be covered by lipids forming an organized structure like a micelle or a liposome. When the organized structure is complexed with DNA is then called lipoplex. There are three types of lipids : anionic (negatively charged), neutral, or cationic (positively charged). Initially, anionic lipids were used in the construction of lipoplex for synthetic vectors. However, these are relatively toxic incompatible with bodily fluids and have the ability to adjust to being in a specific tissue. They are complicated and time consuming to produce, so attention turned to the cationic versions. Cationic lipids, due to its positive charge, were first used to condense negatively charged DNA molecules, so as to facilitate the DNA encapsulated in liposomes. Later, it was found that the use of cationic lipids improved lipoplex stability. In addition, as a result of load , cationic liposomes interact with the cell membrane, and endocytosis is believed to be the main route by which cells absorb the lipoplex. The endosomes formed as a result of endocytosis . However, if genes can not be released into the cytoplasm by rupture of the membrane of the endosome, the liposomes and they will send all the DNA will be destroyed before the genes can achieve their tasks. Also found that although cationic lipids themselves could condense and DNA encapsulated in liposomes, the efficient transfection is low due to lack of skill in the "endosomal escape." However, when "aid lipids (fats normally electroneutral, such as DOPE) were added to form lipoplex, transfection efficiency was observed. Subsequently, it was discovered that certain lipids have the capacity to destabilize the endosome membrane to facilitate the escape of DNA , and these lipids were called fusogenic lipids. Although cationic liposomes have been used as an alternative for gene delivery vectors, the dose depends on the toxicity of cationic lipid, and it was observed that could limit their therapeutic functions. The most common use of the lipoplex has been in the transfer of genes in cancer cells, where the supplied genes have been activated suppressor genes cell tumor and decreased the activity of oncogenes. Recent studies have shown that lipoplex are useful in the epithelial cells of the respiratory system, so it can be used for the genetic treatment of respiratory diseases such as cystic fibrosis . The polymer-DNA complexes called poliplexes and most consist cationic polymers, which are regulated by ionic interactions. A major difference between the methods of action and lipoplex poliplexes poliplexes is unable to release their DNA loaded the cytoplasm .

Hybrid Methods

Because many gene transfer methods that are deficient, they have developed some hybrid methods that combine two or more techniques. The virosomes are an example, combining liposomes with inactivated HIV or influenza virus. This has proved more efficient transfer of genes in epithelial cells of the respiratory system than any other viral or liposomal methods. Other methods involved mixed with viral vectors or cationic lipids hybridized virus.

Dendrimers

A dendrimer is a macro highly branched molecule with spherical shape. The surface of the particle can be functional in many ways and some of its properties derived from its construction are determined by its surface. In particular, it is possible to construct a cationic dendrimer, ie, positive surface charge. With the presence of genetic material like DNA or RNA, direct their additional burden for the association of nucleic acid with cationic dendrimer. In reaching its destination, the dendrimer-nucleic acid complex is then taken by the cell through endocytosis. Dendrimers offer covalent structures , robust and extreme control over the structure of the molecule. Together, they have advantages over cationic lipid. Production of dendrimers has historically been expensive and slower processing, consisting of numerous slow reactions, a barrier that increases its commercial development. The company Dendritic Nanotechnologies, based in Michigan, discovered a method for producing dendrimers using chemical kinetics, a process that not only reduces costs by a magnitude of three, but also shortens the reaction of one month to several days. These new dendrimers "Prioste" can be built specifically to carry a payload of DNA or RNA transferring to cells in high efficiency and little or no toxicity .

DNA vaccines

Like all vaccines are seeking the expression of a specific viral proteins to which they intend to elicit an immune response.

In principle consisted in attenuated virus vaccines, which presented a biohazard risk. A second generation of vaccines was the introduction of proteins that produce a high immune response.

are currently developing vaccines DNA, which are safer and more effective than attenuated virus. They are also easier to transport. The only risk involved is that it can integrate into the genome. It is experiencing some diseases such as HIV, malaria and cancer, administered as liposomes, biolistic injection,.

Percentages of trials in gene therapy

Key events in the Gene therapy

2002 and earlier

The new gene therapy focuses on repair of errors mRNA derived from defective genes. This technique has the potential to treat diseases affecting the blood like thalassemia, cystic fibrosis and some cancers . (11 October 2002 )

Researchers at Case Western Reverse University and Copernicus Therapeutics are able to create tiny liposomes of 25 nanometers that can carry therapeutic DNA through pores in the nuclear membrane . (May 12, 2002)

disease sickle cell is successfully treated in mice. (March 18, 2002)

Gobea In 1993 Andrew was born with a rare and deadly genetic disease (SCID). Blood was withdrawn from the placenta of Andrew and also cord immediately after BIRTH, which contain stem cells . The allele gene encoding the adenosine deaminase, ADA, was obtained and inserted into a retrovirus. Retroviruses and stem cells were mixed. Then inserted the gene into the chromosome stem cells, and these in the blood of Andrew. For four years, T cells (white blood ), produced by stem cells were manufactured using the ADA enzyme ADA gene. After four years the child treatment requisition again.

The success of a trial for the treatment of children with SCID (Immune Deficiency Syndrome Severe Combined disease or "bubble boy") that took place between 2000 and 2002, was questioned when two of the ten children treated at Paris developed leukemia response. Clinical trials were halted temporarily in 2002, but resumed after regulatory review of protocol in the United States, the United Kingdom, France, Italy and Germany. (October 3, 2002)

2003

A team of researchers from the University of California , Los Angeles, inserted genes in brain using liposomes coated a polymer called polyethylene glycol (PEG). The transfer of genes in the brain is a significant achievement because viral vectors are too large to cross the "barrier blood-brain. " This method has the potential to treat Parkinson disease . (20 March 2003 )

by RNA interference or gene silencing is becoming a new way to treat Huntington disease . Short fragments of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If an siRNA is designed to bind to an RNA copy of a defective gene, then the abnormal protein product of this gene, will not occur. (March 13, 2003)

2006

Scientists from the National Institute of Health (Bethesda, Maryland) successfully treated a metastatic melanoma in two patients using T cells to kill and attack cancer cells . This study is the first demonstration that gene therapy can indeed be a cancer treatment.

In March 2006 , an international group of scientists announced the successful use of gene therapy to treat two adult patients infected by a disease that affects myeloid cells . The study, published in Nature Medicine, is believed to be the first to demonstrate that gene therapy can cure diseases of the myeloid system.

In May 2006, a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown of the San Raffaele Telethon Institute Against Gene Therapy (HSR-TIGET) in Milan, reported a breakthrough for gene therapy which developed a way to prevent the immune system can reject input genes. HSR-TIGET group used some genes regulated by molecules known as microRNAs. Dr. Naldini researchers observed that one could use this natural function of microRNA to selectively turn off identity of therapeutic genes into cells immune system and prevent the gene was found and destroyed. Was injected gene containing an immune cell with microRNA in mice and they did not reject the gene . Imortant This work has implications for the treatment of hemophilia and other genetic diseases for gene therapy.

2007

On 1 May 2007 , Moorfields Eye Hospital and University College London's Institute of Ophthalmology announced the first trial of gene therapy for inherited retinal disease. The first operation was carried out in a British man 23 years old, Robert Johnson, in early 2007. Leber Congenital Amaurosis is an inherited disease that causes blindness by mutations in the RPE65 gene. The results of the Moorfields / UCL were published in New England Journal of Medicine. We investigated the transfection of subretinal by recombinant adeno-associated virus carrying the RPE65 gene, and found positive results. The patients showed an increase of vision, and without apparent side effects.

2008

Researchers at the University of Michigan in Ann Arbor (USA) developed a gene therapy slows down and recover before the advance of gum disease periodontal , the leading cause of tooth loss in adults. The researchers found a way to help certain cells using an inactivated virus to produce more of a natural molecule called TNF receptor . This factor is found in low amounts in patients with periodontitis . The molecule administered by gene therapy works like a sponge absorbing excessive levels of TNF, which worsens the inflammatory bone destruction in patients suffering from artritis , deterioro articular y periodontitis. Los resultados del trabajo mostraron que entre el 60 y el 80 por ciento de los tejidos periodontales se libraban de la destrucción al utilizar la terapia génica. Ésta requiere una sola administración, pero podría tener efectos durante toda la vida en los pacientes, bajo riesgo de enfermedad grave.

Problemas de la terapia génica y de sus aplicaciones

En los últimos años, se ha puesto en duda la seguridad de los ensayos realizados con la terapia génica, a raíz de que en 1999 se hiciese pública la noticia de la muerte de un paciente (Jesse Gelsinger, de 18 años), as a result of gene therapy treatment that was submitted to try to cure the deficiency of ornithine transcarbamylase suffering. The modification of the genetic material of a cell affects the cell and its descendants. The main fears are focused on genetic alterations germ line .

Some of the problems of gene therapy are:

The nature of the gene therapy itself makes patients have to undergo multiple rounds of gene therapy.

The immune response. Whenever an object stranger is introduced into human tissues, the immune system has evolved to attack the invader. The possibility that the immune system reduces the effectiveness of gene therapy exists. In addition, the immune system improved response the second time that the invader enters the body, so it is unlikely that this gene therapy can be repeated in patients.

Problems with viral vectors . Therefore may become contaminated by chemicals or by the virulent virus. Unwanted recombinations in these vectors could lead to diseases with unpredictable virulence. Also present other problems such as toxicity, immune and inflammatory responses, etc.

multigenic disorders. Disorders that arise from mutations in a single gene. Unfortunately, some of the most common disorders occurring heart disease, high blood pressure, Alzheimer disease , arthritis, diabetes ... and are caused by the combined effects of variations in many genes. These disorders may be particularly difficult to treat effectively using gene therapy.

There may be changes in the germ cells . Unintentional introduction gene in these cells expose the seed to a very high risk.

Although testing has not been transfer (spread) to other persons in contact, it may not yet be ruled out. Treated patients may have vectors in blood, feces, urine, semen ...

possibility of inducing a tumor ( mutagenesis ) . If DNA integrates into the wrong place at the genome, for example in a tumor suppressor gene, could induce a tumor. This has occurred in clinical trials for SCID (Severe Combined Immunodeficiency) X chromosome-linked , where cells hematopoietic stem of the patients are translated using a retrovirus. This led to the development of leukemia in 3 of 20 patients. However, given the risk of a malignant tumor, there are strategies, which are reflected in the table:

Gene therapy in other animals

Gene therapy is used in animals such as Pacific salmon, seeking their increased size and the consequent economic growth

The first example of gene therapy in mammals was the correction of the deficiency in the production of growth hormone in mice. The recessive mutation little (lit) produce dwarf mice. Although they present a gene apparently normal growth hormone do not produce mRNA from this gene.

The first step in correcting the defect injection consisted of five thousand copies of a DNA fragment carrier linear structural region of the gene for growth hormone in rats promoter fused to the gene of mouse metallothionein in eggs lit. The normal function of metallothionein is detoxification heavy metals, so that the regulatory region responds to the presence of heavy metals in the animal. The injected eggs were implanted in females. 1% of the mice transgenic offspring proved , and reached the greatest size.

has created a similar technology to generate transgenic varieties of Pacific salmon with a rapid rate of growth, the results have been spectacular. Was microinjected into eggs of salmon a plasmid carrying the gene for growth hormone regulated by the metallothionein promoter and a small portion of resulting fish were transgenic, weighing eleven times than non-transgenics.

Gene Therapy in popular culture

In the television series Dark Angel , the issue of gene therapy is mentioned as one of the practices in children and GM their mothers. Also in the series Alias \u200b\u200b , molecular gene therapy appears as an explanation of two identical individuals. It is a fundamental element in the plot of games like Metal Gear Solid , and plays an important role in the plot of movies like Die Another Day , James Bond in I Am Legend, Will Smith's , among many others.

source: wikipedia

Teaddy Bear Masterbate

The types of gene therapy I The first stage of Type I transfer

Gene Therapy

Per gene therapy (a cui ci if riferisce commonly done anche con il inglese di Gene Therapy ) if inserzione l'intende di materiale genetic (DNA ) within cells in order to cure diseases. This connection procedure is known as transfection .

His concept was developed as a result of the great progress of molecular biology methods developed the 80s. These techniques allowed the cloning and sequencing of several genes . This led to the precise identification of many genetic alterations in different diseases and the ability, thanks to the techniques of recombinant DNA to modify organisms (like bacteria or mushrooms) to make them express molecules of interest.

The next step consisted in the evaluation of the possibility of transfect somatic cells of an individual with a genetic disorder with a segment of DNA containing the 'allele healthy. This approach was subsequently extended to non-Mendelian diseases such as cancer , HIV infection and other diseases in which not going to replace a defective gene, but there was also one that can set in motion a phenomenon therapeutically useful.

Types of gene therapy

There are two types Gene therapy: that of the germ cells and somatic cells.

The first aims to transfect cells of the germ line as sperm and egg cells or totipotent stem cells of the earliest stages of embryo development (stage of 4-8 cells), but at present it is not put into practice both for technical reasons but mostly for the great ethical dilemmas which they may suffer.

The second type, however, it is proposed that only the somatic cells, without affecting, therefore, the germ line and today is the most studied and tried. The Gene therapy of somatic cells, in turn, is divided into two groups: ex vivo gene therapy and the in vivo .

ex vivo gene therapy

is the type that was first put into practice and involves the collection of somatic cells of the person concerned. They then are placed in laboratory culture. During this time are also transfected with the gene of interest, added to an appropriate carrier (often used viral vectors), and are then infused into the body of the subject or reinmpiantate. This procedure is certainly the most longest and most expensive of the two, but allows you to select and amplify the cells of interest and also has a better efficiency.

mode is currently the most widely used but is reserved only to those cases where it is possible to withdraw, put the cells in culture and put them back in the body.

in vivo gene therapy

is implemented in all those cases in which the cells can not be cultured, or removed and replanted, such as the brain or heart and most of the internal organs, also represents a model treatment with high compliance and inexpensive but, at present, more difficult to implement. In this case, the gene or oligonucleotide of interest is inserted into the body through a suitable carrier, directly by local or systemic. The systems being studied are of three types: lipoplessi , poliplessi , lipopoliplessi . These are formed through the electrostatic interaction existing between the DNA (negatively charged) and nanoparticles (positively charged). Nanoparticles can be kind of lipid, respectively ( cationic liposomes), or polymer ( polycations) or a supramolecular system format from liposomes and polycations. Potentially all three types of non-viral vectors could replace the existing viral vectors and physical.

The first stage

order for gene therapy can be performed is necessary to know the pathophysiology of the disease in question and identify any altered genes or those involved in the process or treatment. The methods of molecular biology and genetics ensures these results certainly times faster than before. Once the gene of interest it is amplified, cloned and sequenced.

This makes it possible collect all the information necessary to understand its role and its possibilities.

Types of transfer

Once the gene of interest is inserted into the cell may happen that it will face with integration into the genome phone or who stays outside, forming a particle episomale .

integration into the genome allows replication of the gene and its transfer to daughter cells resulting from duplication of the parent cell.

episomiale The particle, however, is not affected duplication, so it is not passed on to daughter cells. You can remedy this situation, however, gene therapy involving the origin of replication, a DNA sequence that allows the attachment of polymerase phones, which means that the episome is transmitted to daughter cells.

These three types of transfer can be useful in the treatment of different diseases. Having to do with genetic diseases, in fact, necessary to use a gene that is replicated in a stable manner for which it has to integrate into the host genome (for example, by using a retrovirus), or it can be accepted as a particle containing episomiale source replication.

In other cases, however, the therapeutic gene is required only for a certain period of time for which you can add it as a free particle episomiale origin of replication.

Methodology of gene transfer

The decisive part of the gene therapy method is to be taken to effect the transfer of therapeutic gene (transfection ). The different systems used to carry out this process are currently divided into viral and nonviral.

transfer non-viral

The methods used to transfer the DNA without the use of viruses include the injection of naked DNA, liposomes insertion through the insertion through the use of cationic polymers or via particle bombardment (gene gun).

injection of naked DNA is the process smoother and easier and also allows transfer of large gene constructs. Consists of injecting a therapeutic gene, linked to a plasmid directly into the cell through the use of a micropipette . The disadvantage of this method is that we must inject the DNA in each cell, one by one. The yield Furthermore, it is decidedly low.

Liposomes are spherical vesicles whose wall is composed of a phospholipid bilayer. Using cationic liposomes they can be complex to the DNA, which neutral pH introduces a negative charge. The complex DNA-liposome can fuse with the cell membrane but in most cases is internalized by endocytosis . Subsequently, the DNA is released into the cytoplasm , enters the nucleus and is expressed . Unfortunately, this process is inefficient because it is seen that only 0.1% of the introduced DNA is expressed. To avoid this in the liposomes were also included proteins and antibodies that may increase the effectiveness of the procedure to minimize the degradation of DNA and facilitating the proper guidance of the vesicle.

Very similar is the procedure that applies to transfection using cationic polymers, because polymers with many positive charges interact with DNA, which, as mentioned, is a polyanion at physiological pH, causing the condensation and protecting it from aggressive chemical and enzymatic, as well as ionizing radiation. Although the DNA-polycation complexes are internalized by endocytosis from the cell, and may be actively targeted to specific cell lines or tissues using antibodies or other molecules direzionanti.

The fourth method is to use power tools or special high-pressure, known as gene gun (gene gun), which allow you to send microscopic particles in the cell 's gold or tungsten coated with DNA. At present there are no human studies of this method but only on animals.

transfer viral

It is based on the use of appropriate recombinant virus.

viruses have a high tendency to infect cells and to incorporate the its DNA is in the form of supplementing episome. Compared to non-viral transfer systems, therefore, have much higher efficiency. The viruses to be used, however, should enjoy some of the features:

• recombinant virus particles, compared to wild-type ( wild-type virus that is not recombined) should be compared to the replication defective
• the virus must have some undesirable quality (such as production of toxic compounds or activation of the immune system)
• there must be enough room for the therapeutic gene (size constraints).

The viruses currently studied as vectors for gene therapy are:

• the retrovirus, lentivirus
• the ,
• the adenovirus ,
• the adenoassociati virus, herpesvirus
• the .

Retroviruses

They were the first virus to be studied in gene therapy, whose parent is the murine leukemia virus in humans is not associated with any disease. A retrovirus has two strands of RNA complexed with various proteins, a capsid and a lipid envelope, derived from the infected host cell. It binds to specific receptors located on cell membrane, which triggers a mechanism that leads to fusion with the viral lipid envelope of the cell. In this way the virus is released into the cytoplasm and then the RNA is released dall'involucro capsid and can thus serve as a template for a RNA dependent DNA polymerase (the reverse transcriptase) that synthesizes it, a strand of DNA that, with the work of a viral integrase, is integrated into the host genome.

General layout of the genome of retroviruses

The genome of a retrovirus is composed of three genes: gag , pol and env .

Gag encodes the viral capsid proteins that are responsible for the assembler and the virion dell'incapsidazione of genetic material. Pol encodes the reverse transcriptase and env is responsible for the synthesis of proteins located on the wrapping lipid necessary for the interaction with specific receptors.

At both ends of the material viral genetic sequences are those non-coding Long Terminal Repeat (LTR sequences, long terminal repeat) containing the information needed to package the RNA to form the virion (packaging signal , ψ) and to regulate the transcription and 'Integration of the DNA.

As with all respect to the replication defective recombinant virus must be taken of particular systems to allow for adequate production.

Scheme of the packaging used for the production of recombinant retroviruses

In the case of retroviruses using cell lines (in most cases are murine 3T3 fibroblasts ) transfected with a gene segment containing the genes gag, pol and env and LTR sequences except for packaging sequence. The cells so transfected (such cells packaging) are able to produce the viral proteins but are not able to assemble to form a mature virion. They form what is called VLP (Virion-Like Particle), a capsid-free viral genome, can recognize its receptor, and tie it, but not comppiere a production cycle of infection.

These cells are then infected with a retrovirus containing the LTR sequences, that of packaging and the therapeutic gene but not gag, pol and env. Such a virus would not be able to replicate themselves but they produce using cells packaged the viral proteins needed, which in turn recognize the sequence of virus RNA packaging defects will assemble resulting in mature infectious virions. Using such a system is capable of cell lines capable of producing 0.1 to 1.0 viral particles per cell for hours obtaining evidence of recombinant viruses between 10 3 - 10 ⁷ infectious particles per ml of culture.

The use of retroviruses has advantages such as their attitude to infection of several cell lines, the high efficiency in the integration of the therapeutic gene in the genome.

The disadvantages in the use of retroviruses is their fragility, which makes the complex process of purification from the culture medium. The retroviral genome also can be integrated into the cell only when the nuclear membrane is absent and therefore only replicating cells can be infected. Another problem arises from the random integration of viral DNA, which can lead to the activation or deactivation of some genes with risk of phenomena insertional mutagenesis.

It should be noted, finally, that the space the two LTR sequences allows the insertion of a gene with a maximum length of 8 kb.

lentiviruses

I belong to the lentivirus family of retroviruses which share morphology and replication cycle but unlike the previous ones, can also infect non-replicating cells, making them good candidates to change the expression of cells to differenzizione terminal, such as the heart or central nervous system, and facilitates the processes of ex vivo transfection as the cells placed in culture do not require stimuli that lead them to divide. The DNA obtained by reverse transcriptase, in fact, a complex with viral proteins, forming a complex, said preiniziazione, which allows the passage through the nuclear membrane. This mechanism, however, is not the only one i was identified as a regulatory sequence polipurinica Central (CPPT, central polypurinic tract), located in the polymerase gene, which promotes the translocation in the cell nucleus. Recently, moreover, was given a residue of valine located 165 of the integrase gene as a factor that can facilitate entry into the nucleus to a greater extent of CPPT.

Among the treated virus was also studied HIV and this has meant that there have been many studies to construct vectors and cell lines of packing that prevents recombination that restores the wild-type .

The construction of viral vectors provides a modified genome sequences that exist only on integration, reverse transcription and RNA including the sequence of all'incapsidamento packaging. The cell line, however, is transfected with two plasmids: one packaging encoding capsid protein and another which surface glycoproteins in which the sequence of the protein gp120 was replaced with that of the G glycoprotein of the virus vescicolostomatite , which increases the cell lines that can be infected and facilitates the purification of virions by centrifugation .

After the packaging plasmid many genes have been eliminated leaving only gag, pol , tat and rev . Finally, it followed the use of packaging plasmids in which the tat gene is completely eliminated.

The potential risk of lead an infectious viral particle and autonomously replicating has led scholars to give rise to a carrier autoinattivantesi (self-inactivating SIN ). This type of construct is based on the fact that reverse transcription may be lost with the other sequences essential for replication. This is achieved by eliminating part dell'LTR in 5 'and engaging with it at the dell'LTR U3 region in 3', delete the sequences are related to the TATA box and those for the binding of transcription factors phones NF-kβ and Sp1 . All this means that once took the reverse transcription, DNA has produced both inactive LTR sequences and consequently transcription becomes impossible while the inserted therapeutic gene is transcribed through the action of an internal promoter (typically derived from cytomegalovirus, CMV). This same promoter is coupled to both the 5 'region, creating a hybrid CMV-LTR sequence, both plasmids in which the cell line was transfected for packing, completely changed upon their LTR sequences. The use of the sequence of CMV promotes transcription, obviating the absence of tat, and reduces the chance yet to form a wild-type virion . The use of SIN vectors also reduces the risk of insertional mutagenesis in the absence of a functional LTR prevents any activation of protooncogenes downstream of it.

At present, however, there are no human studies have used recombinant lentivirus.

adenoviruses

Adenoviruses are double-stranded DNA virus, not enclosed by a lipid envelope in icosahedral symmetry. They are associated mainly with human respiratory infections. Adenoviruses used for gene therapy in the group C which includes serotypes 1, 2, 5 and 6.

The life cycle of an adenovirus includes a link to specific cellular receptors that allow viral entry via endocytosis. The endosome is then fused with a lysosome and the consequent change in pH probably promotes a conformational change of capsid followed by a dismantling of the vesicle and the release of viral DNA is transported into the nucleus where it remains in the form episomiale.

General layout of the genome of adenovirus.

The genome of the adenovirus is apprissimativamente of 36 kb and it can be identified in coding regions for genes expressed early (early , E) and late (late , L). Either side of the genome sequences are so-called inverted terminal (ITR, Inverted Terminal Repeat ) which are necessary for virus replication.

are expressed in the cell nucleus to the early genes E1 (called immediate early) that allow the transactivation of the E2 and E4 genes that determine the lock cellular protein synthesis and are involved in viral DNA replication. Once it is initiated replication, late genes are activated that encode structural proteins that are assembled in the cell nucleus by trapping adenovirus viral DNA. The cell then undergoes lysis.

The recombinant adenovirus vectors are used as a deletion at least the E1 region, which makes the virus defective for replication. Cells are used as packing embryonic kidney cells cells (293) that were transfected with the E1 region. Infecting these cells with the defective virus replication and it makes for the production of new recombinant virions to a very high yield of about 10 12 - 10 13 particles / ml.

Generally recombinant adenoviruses have a deletion of both the E1 region of the E3. It is seen, however, that some infected cells express the genes from this vector remaining low in sufficient quantities, however, perevocare a cytotoxic response capable of eliminating them.

Subsequent studies have made it possible to obtain recombinant vectors which also have a deletion of the E2 or E4 regions, but this resulted in a reduction of gene expression.

Currently, however, produced a third generation of recombinant vectors (Helper Dependent or gutless or high Capacity) in which all the viral genes were removed and remained the only regions ITR and packaging sequence to next to the gene of interest. However, to maintain constant the size of the genome (36Kb), the deleted viral genes were replaced by DNA intron (Intron ) of different nature (Human, phage, etc...) This has decreased the problems of immunogenicity on the long term, as the CTL-mediated immune response, did not increase the security of vecttori regarding the immediate toxicity, namely that given by the violent inflammatory response that occurs in the first hours after administration of carrier and is thought to be due largely to the capsid proteins.

The production of this type of virus uses cells stably transfected with E1 and a call CRE recombinase, which is able to cut the packaging signal of the helper virus containing all viral genes except E1, which has the function to provide for the transcription of all the capsid proteins of the helper dependent vector. The helper virus can not be packaged in contrast to the missing signal from the protein folding as excise CRE. Modern systems allow the production of helper dependent vectors at very high concentrations> 10 of 13 viral particles per ml.

Viruses adenoassociati

General layout of the genome of the virus adenoassociati.

adenoassociati viruses belong to the family parvovirus, have a genome consisting of a single-stranded DNA molecule of about 5 kb, has an icosahedral capsid and a lack of housing lipidco. At the time were not associated with any disease and can infect both replicating cells that do not.

virus adenoassociati The name derives from the fact that they are unable to replicate autonomously but need of another virus which is usually an adenovirus or a herpesvirus. In the absence of helper virus adenoassociati DNA virus that integrates itself into the host cell in a precise region of chromosome 19 (19q 13.3-q ter).

adenoassociato The genome of a virus consists of two genes: rep neccessarie encoding proteins for the control of viral replication and cap that gives rise to structural proteins of the capsid. On either side of the filament are long sequences of DNA about 145 bp ITR of each required to regulate the replication and encapsidation of the virus.

The carrier-based adenoassociati recombinant virus is constructed by replacing the therapeutic gene to rep and cap as ITR sequences contain all the information necessary for the integration and packaging . The production of such a carrier, achieved a transfected cell line (the 293) with a plasmid containing genes cap and rep and subsequently infected with an adenovirus helper defective for E1. Unfortunately, the recombinant virus compared to wild-type not always integrated in the chromosome 19 and is sometimes episomiale. Viruses adenoassociati also does not elicit an immune response in them but you can not insert more segments of 4.7 kb.

The herpesvirus

of herpesviruses, double-stranded DNA virus with icosahedral capsid and the presence of a lipid envelope, the virus is used herpes simplex type 1 (HSV-1) .

is of a neurotropic virus able to establish a lytic cycle but also to persist in the form episomiale the host cell. The genome of HSV-1 consists of a double-stranded DNA of 152 kb that contains at least 80 genes.

When he starts the lytic cycle is expressed VmW65 protein that activates the immediate early genes (IP0, ICP4, ICP22, ICP27 and ICP47) that act as factors transattivanti for other early genes that encode products necessary for replication and nucleotide metabolism. They are subsequently activated late genes encoding structural proteins. The cycle ends with the lysis of the cell.

To get a HSV-1 vector were used two approaches.

The first is the use of an amplicon , a plasmid containing an origin of replication of bacteria (usually Escherichia coli ), a HSV-1 (Oris), the sequence of packaging of HSV-1 and the gene to be inserted. Everything is placed in a cell line infected with a helper virus containing structural and regulatory genes are missing.

The second approach is the use of a recombinant virus obtained by removing one or more immediate early genes and by producing particles from cells expressing the missing proteins. This approach is burdened by the fact that the carrier is produced in this way essere neurotossico.

source: wikipedia

Chemistry Of Levaquin

Fibrosis Gene Therapy The quística The Los vectores en la The gene therapy Inmunodeficiencia combinado Severa (SCID) Gene Therapy

Gene Therapy

The gene therapy involves inserting a functional copy of a normal defective or absent gene in the genome a individual in cells of the tissues of the individual with the aim of restoring normal tissue function and eliminate the symptoms of disease in general and hereditary diseases in particular, and to make a marking. Although the technique still is in development (which is why its implementation is carried out in controlled clinical trials), has been used with some success. Although at first it was a technique referred exclusively to treat genetic diseases, the fact is that currently being proposed for almost any disease, being one of the most promising mechanism.

In the 80's, advances in molecular biology had allowed human genes were located in the genome and cloned . Scientists looking for a simple method of producing proteins - such as insulin , deficit of the molecule in type 1 diabetes - introducing human genes into the bacterial DNA. The modified bacteria then produce the corresponding protein, can be cultivated and injected into patients who could not produce it naturally.

On 14 September 1990 , researchers National Institutes of Health the U.S.. UU. performed the first approved gene therapy procedure on a patient for four years, Ashanthi DeSilva, who had a rare genetic disease called severe combined immunodeficiency (SCID), characterized by the absence a competent immune system, so it was vulnerable to infection. Children with this disease usually develop severe infections and rarely reach adulthood, so that common childhood diseases like chickenpox are dangerous to your survival. Ashanthi had to be insulated, as should avoid contact with people outside their family, maintain a sterile environment in your home, and fight infections with antibiotics many .

Applications

genetic Marked

• : The marking is not intended to cure genetic the patient but is used to improve the treatment of a particular disease. An example would be the development of vectors for clinical trials.

• monogenic disease therapy: Used on metabolic diseases in which there may or may not be efficient administration of a protein deficit. It provides the gene defective or absent.

• Therapy acquired diseases, such diseases among the most prominent is the cancer. Using different strategies such as the insertion of certain genes in the cell suicide tumor or the insertion of tumor antigens to enhance immune response .

Procedure

The procedure Ashanthi gene therapy, doctors removed white blood the child's body, let the cells grow in the laboratory by inserting the missing gene in cells, and then introduced genetically modified white blood cells within the patient's bloodstream. Laboratory tests have shown that the therapy strengthened Ashanthi immune system, since not reappeared recurrent infections, can attend school, leading a normal life and even be vaccinated against whooping cough . This procedure was not curative, as the genetically treated WBCs are effective only for a few months, after which the process should be repeated (VII, Thompson [first] 1993).

Although this simplified explanation of gene therapy procedure was a success, involved no more than an optimistic first chapter in a long history, the way to the first procedure approved gene therapy was complicated and fraught with controversy.

Scientists tried

introduce genes directly into human cells, focusing on diseases caused by single gene defects such as cystic fibrosis , hemophilia, muscular dystrophy and sickle cell anemia . However, this process has been much more complicated than the one conducted in modified bacteria, mainly due to problems arising from the passage of large sections of DNA in the human genome a comparatively larger.

In most gene therapy studies, a gene "normal" is inserted into the genome to replace an "abnormal." A carrier called a vector is used to deliver the therapeutic gene to target cells of the patient. Currently, the most common vector are virus that have been genetically altered to carry normal human DNA. Viruses have evolved ways of encapsulating and delivering their genes to human cells in a pathogen. Scientists have tried to replicate this ability by manipulating the viral genome to remove disease-causing genes and insert therapeutic ones.

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

Achievements

is theoretically possible transfected somatic cells (most cells of the body) or germ cells (such as gametes, and their precursors). All human gene therapy has so far focused on somatic cells while germ cell use in humans remains highly controversial just a perspective. So that the introduced gene is normally transmitted the offspring, must not only be inserted into the cell, but also be incorporated into the chromosomes by genetic recombination .

somatic gene therapy can be divided into two categories: ex vivo (where cells are modified outside the body and then transplanted again) and in vivo (where genes are modified in cells directly the body).

There are a variety of different methods to replace or repair the altered genes in gene therapy.

A normal gene can be inserted into a nonspecific location within genome to replace a nonfunctional gene. This approach is the most common. An abnormal gene could be exchanged for a normal gene with homologous recombination. The abnormal gene could be repaired with selective reverse mutation, which returns the gene to normal function. The regulation (the degree to which a gene is active or inactive) ...

Standards to receive gene therapy

The rules for receiving gene therapy are well established and include several requirements:

• The gene must be isolated and should be available for transfer, usually by cloning .

• must be an effective means of gene transfer . For now, many trials using retroviral vectors , but also used other methods such as adenoviral vectors and physical and chemical techniques.

• The target tissue must be accessible for gene transfer. The first generation of gene therapy processes used white blood cells or their precursors as the target tissue.

• There should be no any form of effective therapy available , and gene therapy should not harm the patient.

Diseases treated by gene therapy

Although gene therapy was originally developed to treat diseases caused by a single gene (monogenic), this technique was quickly adapted to treat acquired diseases such as cancer and infectious diseases like AIDS . Are currently also trying several hereditary diseases such as severe combined immunodeficiency (SCDI, English Severe Combined Inmunodeficiency ), the Familial hypercholesterolemia the cystic fibrosis and muscular dystrophy, among others.

Cystic Fibrosis

In cystic fibrosis (CF, cystic fibrosis English ) called Δ508 deletion (mutation) is found in 70 percent of all copies of the mutant gen. The CF is a disease autosomal recessive associated with a defect in a protein called conductance regulator Cystic fibrosis transmembrane regulator (CFTR), which regulates the transport of ions in the plasma membrane . CF affects approximately 1 in 2000 persons of northern European descent. One of the most vectors have been used in the fight against fibrosis has been the retroviral or retroviral. A cDNA for conductance regulator gene for cystic fibrosis transmembrane regulator (CFTR) was constructed by the overlap of three cDNA clones, and a complete cDNA was cloned into a retroviral vector. CF cells were infected with the vector , carrying a CFTR gene, and cell "virus", expressing the CFTR gene.

Schematic of the proposed structure of the CF .

experiments showed that the flow of chlorine (one of the ions that pass through the membrane) were detected only in cells CF CFTR. Significantly, the response of CF cells with CFTR is comparable with normal human tracheal responses and encourages the hope that gene therapy is effective in cystic fibrosis , using retroviral vectors directly on the lung epithelium through aerosols.

Hypercholesterolemia

is an inherited disease caused by mutations in receptor low density lipoprotein (LDLR), and those patients who are homozygous for mutation of LDLR gene generally die by a heart attack before age 20. In this experiment, retroviral vectors carrying the gene for human LDLR used to infect cultures of hepatocytes (liver tissue cells). Cell lines were obtained and were able to degrade low density lipoproteins at a level or less normal. So far this experiment has only been used in cell culture, although one of the safest to try individuals.

Severe Combined Immunodeficiency (SCID)

gene therapy began in 1990 with the treatment of a girl named Ashanti De Silva who had the disease.

Affected individuals do not have a functional immune system and usually die of infections. Ashanti had an autosomal SCID, and is due to a defect in the gene encoding the enzyme adenosine deaminase (ADA). Its gene therapy began with the isolation of a type of white blood cells called T. These cells, which were part of the immune system, mixed with retroviral vector carrying a normal ADA gene copy inserted. The virus infected thousands of T cells and a normal copy of the ADA gene was inserted into the genome some of them. After mixing these cells with the vector and the occurrence of the infection, T cells were cultured in the laboratory and analyzed to ensure that the transferred ADA gene was expressed. The last step of this gene therapy was the injection of billions of these cells T, GM, in your bloodstream. Some of these T cells migrated to the bone marrow Ashanti, and began to divide and produce ADA. 25% to 30% of T cells expressing the Ashanti protein ADA, which is sufficient to allow a normal life. Another example is that of David, "bubble boy" who had to live in sterile chamber to avoid any risk of infection because your immune system produces defenses having the mutation of the gene coding for the enzyme adenosine deaminase.

Duchenne Muscular Dystrophy

The dystrophin gene is responsible for Duchenne muscular dystrophy .

The Duchenne Muscular Dystrophy (DMD) is a genetic disease that begins in early childhood, causing progressive loss of muscle strength and volume. Appears when a gene on the X chromosome stops making essential muscle protein, dystrophin the . This gene is one of the largest in the human body (it takes almost a 1% of the X chromosome).

The March 28, 2006, at Children's Hospital in Columbus, Ohio, began a trial in six boys with DMD who received replacement genes for an essential muscle protein via injection into the biceps of an arm . This therapy was developed based on adeno-associated virus, specifically designed to carry the dystrophin gene into muscle cells.

Cancer

Standard treatments for this disease have been the chemotherapy and radiotherapy . Gene therapy is presented as a possible weapon for action against this disease. Essentially, gene therapy will focus on the stimulation of natural immunity to tumor cells, remove or infer growth drugs, insert a tumor suppressor gene or slowing drug resistance. Virus used lytic cycle and is activated only in tumor cells, with the help of promoters are activated only in these cases. If infected normal cells, this promoter can not be activated and the introduced gene is not activated. But it may be another case and is to be carried out the necessary lytic cycle expression of certain tumor genes. In the second case, is placed under a promoter of the tumor cell, a gene that expresses a toxic enzyme. This enzyme will kill the cell that contains it and it will come out by removing neighboring cells, which are possibly also tumor.

vectors in gene therapy

The great diversity of situations in which gene therapy could be applied makes impossible the existence of only one type of vector . However, you can define characteristics for the "ideal vehicle" and then adapt specific situations.

• Reproducibe.
• Stable.
• for the insertion of genetic material regardless of size.
• allowing transduction in both dividing and quiescent cells.
• which enables integration of a therapeutic gene specific.
• To recognize and act on specific cells. That
• therapeutic gene expression can be regulated.
• that lack elements that induce an immune response.
• That may be fully characterized.
• Safe. To minimize potential side effects.
• Easy to produce and store.
• reasonable cost.

The system consists of "vector" itself, which is the means of transport, and the "cargo", the transported material. The latter in turn consists of two components: The "effector" which is the gene to be introduced, and the "medium", which are the elements that control the expression of this gene. The most important is the developer, but there may be others: enhancers and repressors sequences, isolation, integration, packaging , homologous recombination, immunostimulatory sequences, ...

can be separated into two types of vectors for gene therapy: viral vectors and nonviral vectors. Viral vectors have high transformation efficiency, but low specificity of tissue-infect all cells have receptors for the viruses used. By contrast, non-viral vectors are highly tissue-specific or protected inject naked DNA with various molecules directly into the tissue, but we want to infect your transformation efficiency is low.

Virus

All virus attach to their hosts and introduce their genetic material in the host cell as part of its replication cycle . This genetic material contains "instructions" core of how to produce more copies of these viruses, making the normal body becomes a machine that serves to meet the needs of the virus. The host cell will carry out these instructions and produce additional copies of the virus. This will lead to a greater number of infected cells. Some viruses insert their genes physically in the host genome (a typical feature of retrovirus , such as HIV , introducing transcriptase enzyme reverse the host and also uses its RNA to give "instructions"). This makes mixing the genes of the virus with host cell genes during the life of that cell.

has been shown that viruses of this type can be used as vectors to carry genes "benign" in a human cell. This will eliminate the virus genes that cause disease and then be replaced by the genes that encode the desired effect (eg, insulin production in the case of diabetics ). This procedure should be such that genes inserted the virus with the host genome intact.

There are many problems that prevent successful gene therapy using viral vectors, such as:

• Difficulty prevention effects.
• We must ensure that the virus infects cells of the body and the inserted gene does not disrupt any other critical gene in the genome.

Retrovirus

The genetic material of retroviruses is in the form of RNA molecules, while their host's DNA. When a retrovirus infects a cell host, introduces its RNA together with some enzymes mainly reverse transcriptase (RT) and integrase (IN). This RNA molecule should produce a DNA copy of of RNA molecule to be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is called reverse transcription. Is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is free in the host cell's nucleus, should be incorporated into the genome. This process is mediated by the integrase enzyme of the virus. The viral genome contains essentially three regions, gag, pol and env, which encode capsid proteins, viral proteins (RT, IN, and protease inhibitors) and proteins involved. These three regions are flanked by two long terminal repeat sequences and LTRs are essential for the initiation of viral DNA synthesis, integration and regulation of its expression. To use retroviruses as viral vectors for gene therapy eliminates the genes responsible for replication and replace these regions gag, pol and env by the transgene followed by a marker gene (to detect infected cells). Viral genome ends are only LTR y la región psi, que precede a gag. Así se crean los vectores retrovirales genómicos (VRGs). ¿Cómo producirlos a gran escala ahora si carecen de los genes necesarios para ello? Introduciéndolos en líneas celulares empaquetadoras, que contienen plásmidos ayudantes con las secuencias gag, pol y env. Las secuencias gag-pol (no pueden separarse) y env se aportan en plásmidos separados para reducir los riesgos de que se produzcan virus capaces de replicarse por recombinación entre los distintos fragmentos. Al usar tres plásmidos distintos (gag-pol, env y el VRG) se necesitan tres eventos de recombinación homóloga para producir virus que sean capaces de replicarse. Es poco probable que esto ocurra. Últimamente is studying the use of self-inactivating vectors LTRs once their sequences were integrated into the genome of the target cell. This prevents the integrated sequences are packaged in the event that occurred in the organism a retrovirus infection, with the consequent spread of the virus that is integrated by the organism and its infection to other nonspecific cells.

Once the retrovirus genetic material is incorporated and has become part of the genetic material of the host cell, whether this is then divided, its progeny will contain all the new genes. Although sometimes the retrovirus genes are not expressed immediately. One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert genetic material of retroviruses is not arbitrary positions in the host genome, and if genetic material is inserted in the middle of the original gene, this gene will disturbed ( insertion mutagenesis). If, for example, the gene regulates cell division, it will be uncontrolled. Gene therapy trials using retroviral vectors to treat severe combined immunodeficiency X-linked (X-SCID) represent the most successful application of therapy to date. Thus, more than twenty patients have been treated in France and Britain, with a high rate of reconstitution of the immune system . However, similar tests were restricted in the United States when it reported the appearance of leukemia patients. To date, four cases of children know French and one British have developed leukemia as a result of insertional mutagenesis of retroviral vectors, and all but one of these children responded well to conventional treatment for leukemia. Currently, gene therapy to treat SCID continues to be successful in USA, Britain , Italy and Japan .

Adenovirus

are viruses that carry their genetic material in the form of double-stranded DNA. Cause human infections respiratory, intestinal and others that affect the eyes (especially the common cold). When these viruses infect the host cell, introducing the DNA molecule. The adenovirus genetic material is incorporated into the host's genetic material. The DNA molecule remains free host nucleus and the instructions on the extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division , so that the descendants of the cells do not have the extra gene. As a result, treatment with the adenovirus will require a growing population of cells, however, his absence in the host genome should prevent insertion mutagenesis.

source: wikipedia