By K. I. Berns, C. Giraud (auth.), Professor Kenneth I. Berns M.D., Ph.D., Catherine Giraud Ph.D. (eds.)
Human gene treatment holds nice promise for the therapy of many genetic ailments. with a view to in achieving this sort of healing there are requisites. First, the affected gene has to be cloned, its se quence decided and its law thoroughly characterised. moment, an appropriate vector for the supply of a superb replica of the affected gene needs to be to be had. For a vector to be of use a number of attributes are hugely fascinating: those contain skill to hold the intact gene (although this can be both the genomic or the cDNA shape) in a strong shape, skill to introduce the gene into the specified cellphone sort, skill to precise the brought gene in an correctly regulated demeanour for a longer time period, and an absence of toxicity for the recipient. additionally of outrage is the frequency of cellphone transformation and, every so often, the power to introduce the gene into nondividing stem cells. Sev eral animal viruses were confirmed as power vectors, yet none has confirmed to have the entire wanted homes defined above. for instance, retroviruses are tough to propagate in enough titers, don't combine into nondividing cells, and are of outrage due to their oncogenic houses in a few hosts and since they combine at many websites within the genome and, therefore, are probably insertional mutagens. also, genes brought through retroviral vectors are often expressed for fairly brief sessions of time. A moment virus used as a vector in version platforms has been adenovirus (Ad).
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Additional resources for Adeno-Associated Virus (AAV) Vectors in Gene Therapy
J Virol 66: 1236-1240 Owens RA, Trempe JP, Chejanovsky N, Carter BJ (1991) Adeno-associated virus rep proteins produced in insect and mammalian expression systems: wild-type and dominant-negative mutant proteins bind to the viral replication origin. Virology 184: 14-22 Owens RA, Weitzman MD, Kyostio SR, Carter BJ (1993) Identification of a DNA-binding domain in the amino terminus of adeno-associated virus Rep proteins. J Virol 67: 997-1005 The Roles of AAV Rep Proteins in Gene Expression and Targeted Integration 33 Pilder S, Moore M, Logan J, Shenk T (1986) The adenovirus E1 B-55K transforming polypeptide modulates transport or cytoplasmic stabilization of viral and host cell mRNAs.
1995). These possibilities are not mutually exclusive, and given the pleiotropic effects of the Rep proteins, it is likely that Rep proteins mediate their antiproliferative effects through several mechanisms. The chances of developing an efficient packaging cell line have been improved with the isolation of 293 cell-based lines that inducibly express the AAV Rep proteins (YANG et al. 1994). The plasmid used to isolate these cell lines contained a 719 bp DNA fragment of the mouse metallothionein I transcription promoter attached to the rep gene from nucleotide 263-2233 of the AAV genome.
A, B The DNA substrates for Rep68 and Rep78 binding. The Inverted terminal repeat (itr) of adeno-associated virus (AAV) and the integration site on chromosome 19, respectively. C, D Rep endonuclease activity on the viral and cellular substrates. Rep remains covalently attached to the 5' end at the nick site. E Association of a cellular replication complex with Rep at the nicked site. DNA synthesis initiating at the free 3'-OH at the nick site with Rep functioning as a helicase for lead strand synthesis through the itr.