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Progresses towards safe and efficient gene therapy vectors

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  • You are covered by the eBay Money Back Guarantee if you receive an item that is not as described in the listing. Payment details. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene.

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    As a result, treatment with the adenovirus will require readministration in a growing cell population although the absence of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has been promoted for treating cancer and indeed the first gene therapy product to be licensed to treat cancer, Gendicine , is an adenovirus.

    Gendicine, an adenoviral pbased gene therapy was approved by the Chinese food and drug regulators in for treatment of head and neck cancer. Concerns about the safety of adenovirus vectors were raised after the death of Jesse Gelsinger while participating in a gene therapy trial. Since then, work using adenovirus vectors has focused on genetically crippled versions of the virus. The viral vectors described above have natural host cell populations that they infect most efficiently. Retroviruses have limited natural host cell ranges, and although adenovirus and adeno-associated virus are able to infect a relatively broader range of cells efficiently, some cell types are resistant to infection by these viruses as well.

    Attachment to and entry into a susceptible cell is mediated by the protein envelope on the surface of a virus. Retroviruses and adeno-associated viruses have a single protein coating their membrane, while adenoviruses are coated with both an envelope protein and fibers that extend away from the surface of the virus.

    The envelope proteins on each of these viruses bind to cell-surface molecules such as heparin sulfate , which localizes them upon the surface of the potential host, as well as with the specific protein receptor that either induces entry-promoting structural changes in the viral protein, or localizes the virus in endosomes wherein acidification of the lumen induces this refolding of the viral coat.

    Types of Viral Vector for Gene Therapy

    In either case, entry into potential host cells requires a favorable interaction between a protein on the surface of the virus and a protein on the surface of the cell. For the purposes of gene therapy, one might either want to limit or expand the range of cells susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses, or by chimeric proteins.

    Such chimera would consist of those parts of the viral protein necessary for incorporation into the virion as well as sequences meant to interact with specific host cell proteins. Viruses in which the envelope proteins have been replaced as described are referred to as pseudotyped viruses. For example, the most popular retroviral vector for use in gene therapy trials has been the lentivirus Simian immunodeficiency virus coated with the envelope proteins, G-protein , from Vesicular stomatitis virus.

    This vector is referred to as VSV G-pseudotyped lentivirus , and infects an almost universal set of cells. This tropism is characteristic of the VSV G-protein with which this vector is coated. Many attempts have been made to limit the tropism of viral vectors to one or a few host cell populations. This advance would allow for the systemic administration of a relatively small amount of vector.

    The potential for off-target cell modification would be limited, and many concerns from the medical community would be alleviated. Most attempts to limit tropism have used chimeric envelope proteins bearing antibody fragments. These vectors show great promise for the development of "magic bullet" gene therapies.

    Vectors in gene therapy

    A replication-competent vector called ONYX is used in replicating tumor cells. Apoptosis was mainly the result of the ability of EIA to inactivate p In p53 - cells, deletion of E1B 55kd has no consequence in terms of apoptosis, and viral replication is similar to that of wild-type virus, resulting in massive killing of cells. A replication-defective vector deletes some essential genes. These deleted genes are still necessary in the body so they are replaced with either a helper virus or a DNA molecule.

    The transfer construct also carries the sequences which are necessary for the general functioning of the viral genome: packaging sequence, repeats for replication and, when needed, priming of reverse transcription. These are denominated cis-acting elements, because they need to be on the same piece of DNA as the viral genome and the gene of interest. Trans-acting elements are viral elements, which can be encoded on a different DNA molecule.

    For example, the viral structural proteins can be expressed from a different genetic element than the viral genome. The herpes simplex virus is a human neurotropic virus. This is mostly examined for gene transfer in the nervous system. The wild type HSV-1 virus is able to infect neurons and evade the host immune response, but may still become reactivated and produce a lytic cycle of viral replication. Therefore, it is typical to use mutant strains of HSV-1 that are deficient in their ability to replicate. Though the latent virus is not transcriptionally apparent, it does possess neuron specific promoters that can continue to function normally.

    Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses. This is the simplest method of non-viral transfection.

    Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success. Cellular uptake of naked DNA is generally inefficient.

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    Research efforts focusing on improving the efficiency of naked DNA uptake have yielded several novel methods, such as electroporation , sonoporation , and the use of a " gene gun ", which shoots DNA coated gold particles into the cell using high pressure gas.

    Electroporation is a method that uses short pulses of high voltage to carry DNA across the cell membrane. This shock is thought to cause temporary formation of pores in the cell membrane, allowing DNA molecules to pass through. Electroporation is generally efficient and works across a broad range of cell types. However, a high rate of cell death following electroporation has limited its use, including clinical applications.

    More recently a newer method of electroporation, termed electron-avalanche transfection, has been used in gene therapy experiments. By using a high-voltage plasma discharge, DNA was efficiently delivered following very short microsecond pulses.

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    Compared to electroporation, the technique resulted in greatly increased efficiency and less cellular damage. The use of particle bombardment, or the gene gun , is another physical method of DNA transfection. In this technique, DNA is coated onto gold particles and loaded into a device which generates a force to achieve penetration of the DNA into the cells, leaving the gold behind on a "stopping" disk.

    Sonoporation uses ultrasonic frequencies to deliver DNA into cells. The process of acoustic cavitation is thought to disrupt the cell membrane and allow DNA to move into cells. In a method termed magnetofection , DNA is complexed to magnetic particles, and a magnet is placed underneath the tissue culture dish to bring DNA complexes into contact with a cell monolayer.

    Hydrodynamic delivery involves rapid injection of a high volume of a solution into vasculature such as into the inferior vena cava , bile duct , or tail vein. Because delivery of naked DNA or RNA to a cell is an inefficient process, most gene transfer is carried out using a vector, or gene delivery vehicle. These vehicles have generally been engineered from viruses by deleting some or all of the viral genome and replacing it with the therapeutic gene of interest under the control of a suitable promoter Table 91e Gene transfer strategies can thus be described in terms of three essential elements: 1 a vector; 2 a gene to be delivered, sometimes called the transgene; and 3 a physiologically relevant target cell to which the DNA or RNA is delivered.

    The series of steps in which the vector and donated DNA enter the target cell and express the transgene is referred to as transduction. Gene delivery can take place in vivo, in which the vector is directly injected into the patient, or, in the case of hematopoietic and some other target cells, ex vivo, with removal of the target cells from the patient, followed by return of the gene-modified autologous cells to the patient after manipulation in the laboratory.

    The latter approach effectively combines gene transfer techniques with cellular therapies Chap. Gene transfer is one of the most powerful concepts in modern molecular medicine and has the potential to address a host of diseases for which there are currently no available treatments. Clinical trials of gene therapy have been under way since ; a recent landmark in the field Forgot Password? What is MyAccess? Otherwise it is hidden from view.

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