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 AIDS, which infect by inserting DNA into their host cells' genome. Many such viruses have been the basis of research using viruses in gene therapy, but the lentivirus is unique in its ability to infect non-dividing cells, and therefore has a wider range of potential applications. Lentiviruses can become endogenous, integrating their genome into the host germline genome, so that the virus is henceforth inherited by the host's descendants. 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.
Lentivirus
To understand the capabilities of a lentiviral vector, one has to consider the biology of the infection process. The lentivirus is a retrovirus, meaning it has a single strandedRNA 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 RNA sequences that code for specific proteins that facilitate the incorporation of the viral sequences into the host cell genome. The "gag" gene codes for the structural components of the viral nucleocapsid proteins: the matrix, the capsid and the nucleocapsid proteins. 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 CD4 glycoproteins on the surface of a host's target cell. 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 incorporated into the host cell's genome with the help of the viral enzyme integrase. From now on, the host cell starts to transcribe the entire viral RNA and express the structural viral proteins, in particular those that form the viral capsid and the envelope. The lentiviral RNA and the viral proteins then assemble and the newly formed virionsleave the host cell when enough are made.
One method of gene therapy involves modifying a virus to act as a vector to insert beneficial genes into cells. Unlike other retroviruses, which cannot penetrate the nuclear envelope and can therefore only act on cells while they are undergoing mitosis, lentiviruses can infect cells whether or not they are dividing. Many cell types, like neurons, do not divide in adult organisms, so lentiviral gene therapy is a good candidate for treating conditions that affect those cell types. Some experimental applications of lentiviral vectors have been done in gene therapy in order to cure diseases like Diabetes mellitus, Murine haemophilia A, prostate cancer, chronic granulomatous disease, and vascular diseases. Therapy requires manipulation of the lentivirus genes and structure for delivery of specific genes to alter the course of the disease. Parts of the viral genome must be removed so that the virus can't replicate itself. It is replaced with a gene to permanently incorporate into the host cell's genome using genetically modified virus. 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.
Applications
Vascular transplants
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 drawback 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.
Chronic Granulomatous Disease
In chronic granulomatous disease immune functioning is deficient as a result of the loss of nicotinomide adenine dinucleotide phosphate oxidase in phagocyte cells, which catalyzes the production of superoxide free radicals. If this becomes deficient, the phagocytes can't kill effectively the engulfed bacrteria, so granulomas can be formed. 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.
Prostate cancer
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, 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.
Rheumatoid arthritis
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.
Diabetes mellitus
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.
