By K. I. Berns, C. Giraud (auth.), Professor Kenneth I. Berns M.D., Ph.D., Catherine Giraud Ph.D. (eds.)
Human gene remedy holds nice promise for the healing of many genetic ailments. in an effort to in attaining one of these medication there are specifications. First, the affected gene needs to be cloned, its se quence decided and its legislation thoroughly characterised. moment, an appropriate vector for the supply of a very good replica of the affected gene needs to be on hand. 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 solid shape, skill to introduce the gene into the specified phone kind, skill to specific the brought gene in an safely regulated demeanour for a longer time period, and a scarcity of toxicity for the recipient. additionally of shock is the frequency of mobilephone transformation and, on occasion, the facility to introduce the gene into nondividing stem cells. Sev eral animal viruses were validated as power vectors, yet none has confirmed to have the entire wanted homes defined above. for instance, retroviruses are tricky to propagate in adequate titers, don't combine into nondividing cells, and are of outrage as a result of their oncogenic houses in a few hosts and since they combine at many websites within the genome and, hence, are in all probability insertional mutagens. also, genes brought by means of retroviral vectors are often expressed for really brief classes of time. A moment virus used as a vector in version structures has been adenovirus (Ad).
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Another limitation of the current technology is the need for cumbersome, labor-intensive purification of rAAV over multiple CsCI 2 gradients. Clearly, there is a great need for an efficient and easy method of packaging AAV vectors. Although no such system has been developed, the field is advancing at a rapid rate and such systems may soon be available to the gene therapy community. Several of the approaches that are being pursued are described in the remainder of this chapter. 3 Adeno-associated Virus Protein-Expressing Cell Lines For both retrovirus and Ad expression vectors, the most efficient means of producing recombinant virions is through the use of packaging cell lines (reviewed in BERKNER 1992; MORGAN and ANDERSON 1993).
J Virol 63: 4450-4454 Berns KI, Kotin RM, Labow MA (1988) Regulation of adeno-associated virus DNA replication. Biochim Biophys Acta 951: 425-429 Cheung AK, Hoggan MD, Hauswirth WW, Berns KI (1980) Integration of the adena-associated virus genome into cellular DNA in latently infected hurnan Detroit 6 cells. J Virol 33: 739-748 Chiorini JA, Weitzman MD, Owens RA, Urcelay E, Safer B, Kotin RM (1994a) Biologically active Rep proteins of adena-associated virus type 2 produced as fusion proteins in Escherichia coli.
J Virol 66: 1119-1128 Katze MG, DeCorato D, Krug RM (1986) Cellular mRNA translation is blocked at both initiation and elongation after infection by influenza virus or adenovirus. J Virol 60: 1027-1039 Khleif SN, Myers T, Carter BJ, Trempe JP (1991) Inhibition of cellular transformation by the adenoassociated virus rep gene. Virology 181: 738-741 Kotin RM (1994) Prospects for the use of adeno-associated virus as a vector for human gene therapy. Hum Gene Ther 5: 793-801 Kotin RM, Siniscalco M, Samulski RJ, Zhu X, Hunter L, Laughlin CA, McLaughlin S, Muzyczka N, Rocchi M, Berns KI (1990) Site-specific integration by adeno-associated virus.