Lentiviral vector in gene therapy
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Lentiviral vectors in gene therapy is a method by which genes can be inserted, modified, or deleted in organisms using lentivirus.
Lentivirus are a family of viruses that are responsible for notable diseases like HIV. The Lentivirus is unique in that it has been the basis of research using viruses in gene therapy. To be effective in gene therapy, there must be insertion, alteration and/or removal of host cell genes. To do this scientists use the Lentivirus' mechanisms of infection to achieve a desired outcome to gene therapy.
To understand the capabilities of the Lentivirus as a vector, the biology of the infectious process must be understood. The Lentivirus is a retrovirus, meaning it has a single stranded RNA genome with a reverse transcriptase enzyme. Lentiviruses also have a viral envelope with protruding glycoproteins that aid in attachment to the host cell's outer membrane. The virus contains a reverse transcriptase molecule found to perform transcription of the viral genetic material upon entering the cell. Within the viral genome are DNA sequences that code for specific enzymes effecting the incorporation of the viral DNA into the host cell genome. The "gag" domain codes for the structural components of the virus like the capsid, the matrix, nucleoproteins. The "pol" domain codes for the reverse transcriptase and integrase enzymes. Lastly, the "env" domain of the viral genome encodes for the glycoproteins and envelope on the surface of the virus.
There are multiple steps involved in the infection and replication of a Lentivirus in a host cell. In the first step the virus uses its surface glycoproteins for attachment to the outer surface of a cell. More specifically, Lentiviruses attach to the CD40 ligand glycoproteins on the surface of T-lymphocyte cells. The viral material is then injected into the host cell's cytoplasm. Within the cytoplasm the viral reverse transcriptase enzyme performs reverse transcription of the viral RNA genome to create a viral DNA genome. The viral DNA is then sent into the nucleus of the host cell where it is then incorporated into the host cell's genome with the help of the viral enzyme integrase. From there the host cell performs transcription and translation to create viral particles and assemble virions which then burst from the host cell when enough are made.
Use as a vector
Some of the experimental uses for the Lentivirus vector have been in gene therapy of diseases like Diabetes mellitus, Murine haemophilia A, prostate cancer, chronic granulomatous disease, and vascular gene therapy. Therapy requires manipulation of the Lentivirus genes and structure for delivery of specific genes to alter the course of the disease. Researchers and scientists do this through the use of transinfection, which is the action of infecting a cell with a microbe to gain different genes or desired traits. HIV-derived lentiviral vectors have been used for introducing libraries of complementary DNAs, short hairpin RNAs, and cis-regulatory elements into many targets, including embryonic stem cells.
In a study designed to enhance the outcomes of vascular transplant through vascular endothelial cell gene therapy, the third generation of Lentivirus showed to be effective in the delivery of genes to moderate venous grafts and transplants in procedures like coronary artery bypass. Because the virus has been adapted to lose most of its genome, the virus becomes safer and more effective in transplanting the required genes into the host cell. A draw back to this therapy is explained in the study that long-term gene expression may require the use of promoters and can aid in a greater trans-gene expression. The researchers accomplished this by the addition of self-inactivating plasmids and creating a more universal tropism by pseudotyping a vesicular stomatis virus glycoprotein.
In chronic granulomatous disease immune functioning is deficient as a result of the loss of nicotinomide adenine dinucleotide phosphate oxidase (NADP) in phagocyte cells, which aids in lipid and nucleic acid synthesis. If this becomes deficient, the bodies immune responses become weakened. Study performed in mice emphasizes the use of lineage-specific lentiviral vectors for the production of NADP. Scientists developed this strain of lentivirus by transinfecting 293T cells with pseudotyped virus with the vesicular stomatitis G protein. The viral vector's responsibility was to increase the gene synthesis and production of NADP in these phagocytic cells. They did this to create an affinity for myeloid cells.
With prostate cancer, the Lentivirus is transformed by being bound to trastuzumab to attach to androgen-sensitive LNCaP and castration-resistant C4-2 human prostate cancer cell lines. These two cells are primarily responsible for secretion of excess human epidermal growth factor receptor 2 (HER-2), which is a hormone linked to prostate cancer. By attaching to these cells and changing their genomes, the Lentivirus can slow down, and even kill, the cancer causing cells. Researchers caused the specificity of the vector by manipulating the Fab region of the viral genome and pseudotyped it with the Sindbis virus.
Haemophilia A has also been studied in gene therapy with a lentiviral vector in mice. The vector targets the haematopoietic cells in order to increase the amount of factor VIII, which is affected in haemophilia A. But this continues to be a subject of study as the lentivirus vector was not completely successful in achieving this goal. They did this by trans-infecting the virus in a 293T cell, creating a virus known as 2bF8 expressing generation of viral vectors.
Studies have also found that injection of a lentiviral vector with IL-10 expressing genes in utero in mice can suppress, and prevent, rheumatoid arthritis and create new cells with constant gene expression. This contributes to the data on stem cells and in utero inoculation of viral vectors for gene therapy. The target for the viral vector in this study, were the synovial cells. Normally functioning synovial cells produce TNFα and IL-1.
Like many of the in utero studies, the lentiviral vector gene therapy for diabetes mellitus is more effective in utero as the stem cells that become affected by the gene therapy create new cells with the new gene created by the actual viral intervention. The vector targets the cells within the pancreas to add insulin secreting genes to help control diabetes mellitus. Vectors were cloned using a cytomegalovirus promoter and then cotransinfected in the 293T cell.
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