Gene Therapy


medicine and health sciences have experienced dramatic advances in recent decades among these advances the use of genes for therapeutic purposes known as gene therapy represents a promising tool to cure some of those diseases the conventional drug therapies cannot thus gene therapy consists in the transfer of genetic material into cells or tissues to prevent or cure disease initially gene therapy was established to treat patients with hereditary diseases caused by single gene defects such as muscular dystrophy or hemophilia however the present many gene therapy efforts are also focused on curing polygenic or non inherited diseases with high prevalence such as cancer cardiovascular diseases and hepatitis C we can distinguish two types of approaches in gene therapy in vivo gene therapy is based on the introduction of a therapeutic gene into a vector which is then administered directly to the patient this factor will transfer the gene of interest in a target tissue to produce the therapeutic protein another approach ex vivo gene therapy is based on the transfer of the vector carrying the therapeutic gene into cultured cells from the patient subsequently these genetically engineered cells are reintroduced to the patient where they now Express the therapeutic protein several key aspects must be taken into account when designing a gene therapy approach first one must choose the therapeutic gene to treat the disease this gene must be transported by a vehicle or vector to the target cell that must Express the therapeutic protein however to reach the target cell one was considered the route of administration by which the vector will be introduced to the patient finally it is important to have animal models of human diseases which to test the gene therapy approach the first key element when developing a gene therapy protocol is to choose the therapeutic gene to be introduced into the body to counteract the disease there are illnesses caused by the lack or dysfunction of a single protein as in haemophilia or cystic fibrosis for these diseases the choice of the gene to transfer is easily identifiable a correct copy of the gene whose dysfunction causes the disease will be introduced however there are diseases such as diabetes cancer or AIDS whose origin is more complex either because they result from the interactions of more than one gene or because they are associated with environmental causes in these cases the truth of the therapeutic gene may be more challenging and scientists will depend on previous studies and knowledge of the disease to choose the appropriate gene or genes to be transferred in addition to the gene of interest attention must be paid at choosing the right regulatory sequences that determine where and when the production of the therapeutic protein that the gene encodes will take place these regulatory sequences are called promoters and opposition in front of the gene to be expressed some promoters direct gene expression to specific cell types such as hepatocytes in the liver cardio myocyte in the heart or muscle fibers in skeletal muscle other promoters allow gene expression simultaneously to multiple tissues the therapeutic gene is transported to the target cells within a vector an ideal vector should be able to efficiently transduce target cells without activating an immune response either against itself or the therapeutic gene over recent years a large number of vectors have been developed each one with its own characteristics however a universal vector to treat any disease does not exist the choice of one or another depends on factors such as the target tissue to manipulate whether the disease may require short-term or chronic treatment we can distinguish two large groups of vectors according to their origin the viral vectors and the non viral vectors viral vectors derive from viruses viruses or infectious agents that evolved to be highly efficient at transferring their genetic material into host cells gene therapy takes advantage of this feature of the virus to introduce therapeutic genes to target cells to this end module all of the viruses genes are replaced by the therapeutic gene turning the virus into a viral vector these file vectors are incapable of causing disease as the pathogenic genes have been eliminated the basic structure of a viral vector is formed by capsid made of structural proteins within which the nucleic acid is located some vectors also have lipid envelope the four types of viral vectors most commonly used in gene therapy include the retroviral vectors the lentiviral vectors the adenoviral vectors and the Adhan associated factors retroviral vectors and lentil viral vectors share common features as they both derived from retroviruses the two have a similar structure based on an RNA genome when a retroviral or lentil bar vector infect a target cell the RNA genome containing the therapeutic gene is retro transcribed to a double-stranded DNA in the cytoplasm through the action of reverse transcriptase enzyme carried by the vector once retro transcribed the DNA with the therapeutic gene enters the nucleus where it integrates into the host cell genome when integrated into the genome cells arising from the infected cell will also contain the therapeutic gene allowing for stable a long-term expression the main difference between retro file and length of our effectors is that in the first case the vector requires the host cell to be in the division process to be able to infect it however the length of our effectors can affect both defining cells and those that do not divide consequently the use of retroviral vectors is limited to ex vivo gene therapy approaches whereas lentiviral vectors can be used both in ex vivo and in vivo gene therapy other no viral vectors drive in turn from adenoviruses other no viruses or viruses with double-stranded DNA genome after contacting the cell the adenoviral vector enters in vesicles or endosomes by process called endocytosis after being released from the endosome the adenoviral vector DNA enters the cell nucleus where it remains in an extra chromosomal form adenoviral vectors can infect both dividing cells and quiescent cells these vectors are capable of producing high levels of the therapeutic protein however with the first-generation adenoviral vectors which only some of the viral genes have been removed the host immune system can recognize the remaining viral proteins and destroy transduced cells throughout Lee after the infection as a result the expression of the therapeutic gene is usually short termed however in the next generation identify factors called gutless all of our genes have been deleted from the vector so a long term expression of the gene of interest is possible finally as an Associated vectors derive from a virus that does not cause disease in humans the genome of a dissociated viruses is formed by single-stranded DNA after entering the cell by endocytosis the virus particle releases the DNA molecule into the nucleus where it becomes a molecule of double-stranded DNA which is maintained in an extra chromosomal form like adenoviral and length of our effectors as an Associated vectors can affect both dividing cells and cells that do not divide they are also capable of producing high levels of the therapeutic protein in the target cell but unlike adenoviral vectors this expression can be maintained for years in tissues with a low rate of cell division such as the liver and the skeletal muscle the major limitation of these vectors is their small size since they can only package a therapeutic gene of up to four point five kilobases while the retro and lentivirus actors can package up to eight kilobases and goodness adenoviral vectors up to 37 kilobases as its name implies non-viral gene therapy uses any kind of vector that is not derived from a virus in most applications the therapeutic gene is part of a larger structure of double-stranded DNA called the plasmid in its simplest form non-viral gene therapy consists in the transfer of plasmids directly into tissues where they will be captured by target cells although with very low efficiency for this reason different physical and chemical methods that allow an increased efficiency of the nucleic acid delivery into the cell have been developed physical methods increase the cell membrane permeability to plasmids by using electrical pulses which is known as electrode transference or by applying sound waves which is known as sone operation with regard to chemical methods plasmids can be covered with cationic lipids or polymers to form organized structures called the perplexes and polar plexus respectively in both cases these structures protect and stabilize the nucleic acid increase its uptake by the cell once in the cell nucleus the plasmids are maintained in an extra chromosomal form the use of non viral vectors has certain advantages over viral vectors for example there is no limit in the size of the therapeutic gene to transfer and also no immune responses are triggered against the vector so it can be readmitted however the invivo transfer efficiency obtained is usually lower than that of viral vectors depending on the disease to be treated the therapeutic gene must be directed to target cells in specific tissues for example in the case of cystic fibrosis the epithelial cells of the lungs are the main target of treatment as the disease primarily affects the respiratory system in the case of muscular dystrophy however the cells that must receive the treatment are the muscle fibers on the other hand there are some types of diseases such as hemophilia caused by mutations and proteins that act on the blood in these cases tissues with a high capacity for protein secretion into the bloodstream must be chosen such as the liver thus cell producing the functional protein acts as a factory to deliver the therapeutic protein into the bloodstream resulting the treatment of the disease once designed to produce the vector containing the therapeutic gene must reach the target cell to do this it must be administered by the most appropriate route once again the route of choice will depend on factors such as the vector of itself the target tissue or organ to genetically manipulate or the disease to be treated for example add a Navarro and add an Associated vector introduction into the bloodstream result in hepatic transduction for this reason if a local distribution of the vector is desired in the tissue other than the liver it must be injected directly into the target tissue such as an intramuscular injection to manipulate the skeletal muscle an intracranial administration to transfer genes to the brain an intra-articular injection for the manipulation of joints or even intramural for the treatment of solid tumors a key point in designing a gene therapy approach is to have animal models of human diseases to test new therapies once it has been demonstrated that a new gene therapy approach is effective and safe in laboratory animals such as mice or rats it must be tested in larger animal models such as dogs or non-human primates this phase prior to the application of the gene therapy protocol in patients is known as the preclinical phase which can be very lengthy and expensive finally the clinical phase is reached which takes place in an ordered series of stages during which the safety of the procedure and its therapeutic efficacy must be determined to do this as with any medicine the responsible regulatory agencies such as the Food and Drug Administration in the United States or the European Medicines Agency in Europe control both the design and implementation of the clinical phase

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