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.
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.
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.
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
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
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