Vectors lentiviruses

There are severable methodologies for expressing siRNAs. One method for stable and controlled expression of siRNAs from the context of the cell is via the use of len-tiviral vectors. Lentiviruses, unlike retroviruses such as Moloney murine leukemia virus (MoMLV), tend to preferentially integrate downstream of active promoters within the active transcriptional unit, potentially limiting their overall oncogenicity... 

To circumvent this problem, vectors that are based on lentiviruses have been developed. In contrast to prototypic retroviruses, lentiviruses do not require cell division for integration. Gene-therapy vectors have been developed from a broad spectrum of lentiviruses including human immunodeficiency vims (HIV), simian and feline immunodeficiency vims as well as visna/maedi vims. The most widely used lentiviral vector system is based on HIV-1. These vectors can efficiently transduce a broad spectrum of dividing and nondividing cells including neurons, hepatocytes, muscle cells, and hematopoietic stem cells [1,2]. 

One of the most commonly used vectors, these viruses naturally insert their RNA, in the form of reverse-transcribed DNA, into the chromosomes of dividing cells. Most retroviruses can traverse the nuclear membranes of dividing cells only an exception is the lentivirus (of which HIV is an example). 

There is a wide variety of vectors used to deliver DNA or oligonucleotides into mammalian cells, either in vitro or in vivo. The most common vector systems are based on viral [retroviruses (9, 10), adeno-associated virus (AAV) (11), adenovirus (12, 13), herpes simplex virus (HSV) (14)] andnonviral [cationic liposomes (15,16), polymers and receptor-mediated polylysine-DNA] complexes (17). Other viral vectors that are currently under development are based on lentiviruses (18), human cytomegalovirus (CMV) (19), Epstein-Barr virus (EBV) (20), poxviruses (21), negative-strand RNA viruses (influenza virus), alphaviruses and herpesvirus saimiri (22). Also a hybrid adenoviral/retroviral vector has successfully been used for in vivo gene transduction (23). A simplified schematic representation of basic human gene therapy methods is described in Figure 13.1. 

GFP gene under control of CMV to retinal cells by injection into the subretinal space of eyes in rats the GFP gene was efficiently expressed in both photoreceptor cells and retinal pigment epithelium predominant expression in photoreceptor cells was achieved using the rhodopsin promoter. The transduction efficiency was high and photoreceptor cells in >80% of the area of whole retina were expressing GFP (76). Intron-containing constructs have been successfully introduced into recent versions of lentivirus vectors (77). 

Recently, a series of lentivirus vectors were developed for transduction of hepa-tocytes in vivo (78). Various promoters, such as the human CMV, the human phos-phoglycerate kinase (PGK) and the mouse albumin promoter, were introduced into the FlIV-1-based vector. These vectors showed enhanced nuclear translocation in hepatocytes and improved transgene expression. Interestingly, targeted expression to the liver could be accomplished by the use of the albumin promoter. Therapeutic levels of human factor IX were achieved after a single injection. 

Kafri, T. (2001). Lentivirus vectors difficulties and hopes before clinical trials. Cum Opin. Mol. Then, 3, 316-326. 

Lentiviruses can be very effective vectors for gene therapy since they can change the expression of genes in target cells for up to 6 months. They are useful for nondividing and terminally differentiated cells including muscle cells, hepato-cytes, neurons, macrophages, retinal photoreceptors and hematopoietic stem cells. However, lentiviruses cannot enter quiescent cells in which reverse transcription is blocked. 

A newer generation of retroviral vectors, based on the lentivirus, hold the same advantages of retroviral vectors with the added ability to transduce non-dividing cells efficiently. Pre-clinical studies with lentiviral vectors for the treatment of hemophilia have shown great promise. Using a feline immunodeficiency virus (FlV)-based vector, Stein et al. showed that 9 of 12 treated hemophilia A mice could express greater than 5 ng/ml factor VIII, six of which... 

Sinn, P. L., Hickey, M. A., Staber, P. D., Dylla, D. E., Jeffers, S. A., Davidson, B. L., Sanders, D. A. and McCray, P. B., Jr. (2003). Lentivirus vectors pseudotyped with filoviral envelope glycoproteins transduce airway epithe-lia from the apical surface independently of folate receptor alpha. J. Virol. 77, 5902-5910. 

Bjorklund, A. et al. (2000). Towards a neuroprotective gene therapy for Parkinson s disease Use of adenovirus, AAV and lentivirus vectors for gene transfer of GDNF to the nigrostriatal system in the rat Parkinson model. Brain Res. 886(1-2), 82-98. 

Waddington, S. N., Mitrophanous, K. A., Ellard, F. M. et al. (2003). Longterm transgene expression by administration of a lentivirus-based vector to the fetal circulation of immuno-competent mice. Gene Ther. 10(15), 1234—1240. 

The lentiviral vectors have safety concerns similar to the retroviral vector-based gene therapy products (1) the production of a replication competent lentivirus (RCL) during manufacturing and (2) the potential for insertional mutagenesis, resulting in oncogene activation. One concern unique to the lentiviral vectors is the possibility that the vector can be mobilized in vivo,... 

The nature of the vector under investigation should be considered in the design of preclinical studies. For example, for preclinical evaluations of vectors that have a documented potential to integrate, such as gammaretrovirus and lentivirus, or possess the potential for latency and reactivation, such as the... 

Evans JT, Gai cia JV (2000) Lentivirus vector mobilization and spread by human immunodeficiency virus. Hum Gene Ther 11 2331-2339. 

Keywords Active immunization Adenovirus Adeno-associated virus Alzheimer s disease Amyloid-beta Chemokine Cytokine Lentivirus Herpes simplex vims Isotypes Parkinson s disease Passive immunization Single-chain antibodies Thl cells Th2 cells Transgene Vector... 

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