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发表于 2015-5-6 12:02:02
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How are HIV lentiviral vectors used?
* Delivery Into Patients' Target Cells
The HIV-based vector can be delivered directly into the body without in vitro manipulations of the patient’s cells (Adler, Gifford, and Sumner). Additionally, lentiviral vectors have been shown to be superior to murine retroviral vectors. Ex vivo manipulations that activate stem cells with growth factors to induce cell division must be carried for the retrovirus to be able to enter the stem cells. However, it has been shown that ex vivo stem cell stimulation is not necessary with lentiviral vectors, so the vectors can be inserted directly into the patient and will find their way to the target cell (Amado and Chen, 1999).
Previous gene therapy using retroviral vectors required that cells be dividing, limiting therapy to proliferating cells in vivo or ex vivo. In the ex vivo method, the target cells are removed from the patient's body, treated to stimulate replication and then transduced with the vector before being returned to the patient. However, with lentiviral vectors there is no need for ex vivo treatment, and the target cells need not be dividing. The HIV-based vector is simply injected into a patient, upon which it seeks out its target cells based on cell membrane receptor proteins. Immune responses to the lentiviruses have not been found (Peel, 1998).
* Uses for HIV Lentiviral Vectors
Scientists have recently been using the HIV lentiviral vector to repair neurons. HIV is also being developed as a delivery system to provide successful gene therapy in many diseases such as metabolic diseases, cancer, viral infection, cystic fibrosis, muscular dystrophy, hemophilia, retinitis pigmentosa, and maybe even Alzheimer’s disease (Adler, Gifford, and Sumner; Naldini et al.; Amado and Chen, 1999; Planelles).
* Concerns With Using HIV Lentiviral Vectors
There is still concern with using lentiviral vectors for safety reasons. One concern involves the possibility that the HIV could self-replicate and could be produced during manufacture of the vector in the packaging cell line or in the target cells by a process of recombination. Thus, the person undergoing gene therapy would also be infected with HIV in addition to the new therapeutic gene. A self-replicating infectious vector could cause cancer by inserting itself into the host genome and activate a neighboring proto-oncogene, thus causing mutagenesis (Amado and Chen, 1999).
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Current research involving lentiviral vectors
Because scientists have shown that lentiviruses, such as HIV, are successful and efficient gene delivery vehicles, the field has now turned its attention to producing vectors with built-in safety features to prevent the development of replication competent lentiviruses (RCL). However, even the earliest studies with HIV lentiviral vectors did not generate RCL in vitro or in vivo (Amado and Chen, 1999), but precautions are still very important.
* Safety Modifications
HIV lentiviral vectors are being produced whose packaging plasmid does not contain the necessary HIV genes. This does not interfere with efficient vector production and is a great increase in safety because potential RCL’s cannot use the HIV genes necessary for replication of HIV in humans. The drawback to these vectors is that they cannot transduce macrophages because the accessory gene vpr is needed for HIV infection of this type of cells. Thus, scientists are showing that the type of lentiviral vector necessary is dependent on the type of cell chosen as target, so the HIV vectors will be made with different accessory genes (Amado and Chen, 1999).
Researchers at the Salk Institute are creating HIV lentiviral vectors that are self-inactivating. The scientists are working on packaging a defective HIV genome that contains only the necessary elements for gene transduction into a virion that has a broad host range. HIV normally targets human CD4 (helper T cells) through interactions with membrane-bound target proteins, but to broaden the host cell targets a surrogate targeting molecule (VSV-G) was inserted into the viral membrane. The HIV genome was modified to produce a minimal construct and the cytomegalovirus promoter and green fluorescence protein as a marker were added (Sikorski and Peters, 1998). A deletion in the LTR region at the end of the virus genome is also created. These are unique cis-acting sequences that are essential to the virus life cycle. The deletion inactivates the LTR promoter and eliminates the production of vector RNA. The gene to be transferred by the vector is expressed from an exogenous viral or cellular promoter that is inserted into the lentiviral vector. Inactivation of the promoter activity of the LTR reduces the possibility of insertional mutagenesis as the lentiviral products are integrated into the host genome. Also, as expression of the vector RNA is eliminated, the potential for RCL production in the target cell is further minimized (Amado and Chen, 1999).
Other safety methods include using specific internal promoters that regulate gene expression either temporally or with tissue or cell specificity so as to prohibit gene expression that would cause replication of HIV in the gene therapy target cell (Amado and Chen, 1999).
* Use of Non-Human Lentiviral Vectors
By using non-human lentiviruses, scientists hope to bypass the issue of host infection by the gene therapy vector. Researchers are developing non-human lentiviruses such as the feline immunodeficiency virus (FIV) to be used in gene therapy (Amado and Chen, 1999). FIV infects two to twenty percent of domestic cats worldwide and causes a disease similar to human AIDS. While humans have been exposed to this virus through cat bites, humans have never been shown to be infected by the virus. It has been shown that evolutionarily FIV diverged early on from HIV and other lentiviruses. Researchers at the University of San Diego, though, have found that while nonprimate lentiviruses may provide safer alternatives to HIV they have highly restricted host range of infection. However, promoter substitution of FIV enabled an env-deleted, three plasmid, human cell-FIV lentiviral vector system to express high levels of FIV proteins and FIV vectors in human cells. The researchers replaced the U3 element within the 5’ LTR of FIV with the human cytomegalovirus early gene promoter. Pseudotyped FIV vectors were shown to be able to efficiently transduced dividing, growth-arrested, and postmitotic human targets. The researchers also showed that human cells supported mechanisms of the FIV life cycle needed for efficient lentiviral vector transduction. It is the U3 element in FIV that is the only restriction to the productive phase of FIV replication in human cells. The researchers concluded that lentivirus-specific properties of FIV vectors are retained in human cells, and they speculate that eventually FIV vector will have advantages in human clinical use. Additionally, vectors derived from FIV may represent a safer alternative to HIV vectors, even those with deleted nonstructural proteins, because they cannot induce HIV-reactive antibodies in recipients. Overall, FIV has experimental advantages over HIV (Poeschla, Wong-Staal, and Looney, 1998).
Researchers at the University of North Carolina at Chapel Hill are working with equine infectious anemia virus (EIAV) to be used as a lentiviral vector in humans. EIAV is a lentivirus that normally infects horses, donkeys, and mules. It has been shown to be able to infect mature macrophages, and thus has the potential to infect quiescent cells, and has relatively simple genome organization. The researchers constructed separate plasmids encoding EIAV proteins, a viral envelop, and an EIAV vector. They attempted to broaden the host range of the vector to human cells by using non-EIAV enhancer/promoter elements to drive expression and a non-EIAV envelop glycoprotein. They succeeded in transducing up to about 60 to 70 percent of human CFT1 cells which were placed in a culture dish. This is still quite a bit lower than the transduction level obtained using murine retroviruses, but more work with EIAV will hopefully increase the efficiency of this procedure. In addition, the fact that both EIAV-based and HIV vector can mediate gene transfer and expression to non-dividing human cells suggests that nuclear targeting mechanisms of equine and human lentiviruses are functionally conserved (Olsen, 1998).
* Lentiviral Vectors for Hematopoietic Stem Cells
Many recent studies with lentiviral vectors have focused on modifying the hematopoietic stem cell which has the capacity to self-renew and to differentiate into all of the mature cells of the blood and immune systems. Thus, by introducing therapeutic genes into stem cells many diseases that affect these systems could be treated (Amado and Chen, 1999).
* Gene Therapy for Cystic Fibrosis
Researchers at the Institute for Gene Therapy at the University of Pennsylvania evaluated a replication-deficient vector based on HIV for gene transfer directly into the lung to correct the genetic defects of cystic fibrosis (CF). They expanded the target range of the vector by adding the vesticular stomatitis virus G protein into the HIV vector envelop. LacZ was the reporter gene in the HIV-based vector, so the level of transduction was assessed based on the expression of lacZ. The researchers were successful at transducing nondividing airway epithelial cells in vitro, whereas they were unsuccessful when using murine-based retroviral vectors. Thus, the vector corrected the CF defect in proliferating airway cells. There were complications with differentiated epithelial lung cells as the vectors did not effectively transduce these cells. The blockage appeared to be at the level of entry, the researchers reported. Further experimentation is being conducted to examine the problems of cell entry into differentiated cells (Goldman, et al., 1997).
* Liver-Directed Gene Therapy
Initial research aimed at delivering genes to the liver in vivo with HIV-based lentiviral vectors showed promising results, reported Ganjam Kalpana of Albert Einstein College of Medicine this year. This scientist developed a crippled version of HIV and used it as a vehicle for in vivo gene therapy on low-density lipoprotein receptor-deficient Watanabe heritable hyperlipidemic rabbits. A eukaryotic humanized gene fluorescent protein gene was cloned into the transfer vector to act as the reporter gene for successful cell transduction. The HIV vector was highly superior to previous methods of gene therapy using retroviral vectors which were highly invasive to the patient. There was also no host mediated cellular immune response to the lentiviral vector (Kalpana, 1999). This is another application to HIV-based gene therapy vectors that has been shown to be successful.
* Therapy Against Retinitis Pigmentosa
Retinitis pigmentosa is an inherited genetic disease which causes the retina to degenerate leading to loss of visual field and night blindness. Genetic defects of photoreceptor cells of the visual system are the cause of this disease. A vector for gene therapy of retinitis pigmentosa should only target photoreceptor cells, which are located in the outer nuclear layer of the retina. Miyoshi, Takahashi, Gage, and Verma conducted an experiment using an HIV-based vector with a gfp-gene (green fluorescent protein) as a reporter. The vector was injected into rat retina. It was shown that the HIV-based vector did achieve long-term gene expression in the photoreceptor cells when a rhodopsin-promoter was used in the vector. This is only active in the photoreceptor cells, so the vector only targets these cells and not others in the retina. Thus, the researchers were successful in performing gene therapy on their rat patients (Schmidt, HIV as a Vector For Gene Therapy).
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发表于 2015-5-6 11:54:40
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