Strang, Blair Lewis; (2003) Development of pseudotyped HIV vectors. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Vectors based on human immunodeficiency virus type 1 (HIV-1) offer a means for the delivery of therapeutic transgenes into a wide variety of cell types, both dividing and non-dividing. Virus entry is mediated through interactions between the viral envelope and its cellular receptor(s). For the benefit of gene transfer efficiency and safety it is practical and necessary to complement the vector particle with an envelope protein from another virus (vector pseudotyping) rather than using an HIV-1 envelope that has a limited host-range and cell tropism. The ability of HIV-1 based vectors to be pseudotyped with gammaretroviral envelope glycoproteins was examined. Modifications to the cytoplasmic tails of the envelope glycoproteins from gibbon ape leukaemia virus (GALV) and the feline endogenous retrovirus RD114 resulted in an increase in HIV-1 vector titre compared to pseudotyping with the wild-type envelope glycoproteins. Attempts were made to understand how these modifications affect vector titre. Pseudotyping of HIV-1 based vectors with alphaviral envelopes glycoproteins was also investigated, and it was found that the envelope glycoproteins of Ross River virus (RRV) and Semliki Forest virus (SFV) could pseudotype HIV-1 vectors. Stable HIV-1 vector producer cells were established by introducing envelopes suitable for HIV-1 pseudotyping into a novel HIV-1 stable pre-packaging cell line, STAR, which continuously produces HIV-1 Gag-Pol proteins. Pseudotyped vectors harvested from these producer cells were characterised with regard to future ex vivo and in vivo use as the envelope borne by the vector can dictate certain properties of the virion. These data include the stability of each pseudotype in human sera and at 31°C. Finally, attempts were made to transduce primary human cell cultures, particularly dendritic cells, with different HIV-1 pseudotypes. Although pseudotyped HIV-1 vectors could transduce human dendritic cells to some degree, transduction of dendritic cells with virus from STAR cells appeared to be limited by some facet of the codon-optimised packaging construct used in this cell line.

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