1887

Abstract

Efforts to achieve cell type-specific transduction of retroviral vectors for gene therapy have centred on modification of the envelope protein (Env). Typically, addition of a ligand to Env gives binding to the new or target receptor, but little or no infection, and affects the subunit association of the modified Env. We previously discovered two point mutations that increase targeted infection by over 1000-fold when added to an Env modified by N-terminal insertion of the receptor-binding domain from amphotropic murine leukemia virus Env. Here, we asked whether these mutations would similarly increase transduction by Env modified with a clinically relevant ligand, human interleukin-13 (IL-13L). Addition of the point mutations stabilized the weak subunit association observed in some IL-13L-modified Env proteins, but infection via the target IL-13 receptor still did not occur. Fluorescence-based cell–cell fusion assays and studies with a membrane-curving agent revealed that defects in membrane fusion differed with the site of ligand insertion. When IL-13 was inserted into the N terminus of Env, membrane fusion was blocked prior to membrane-lipid mixing, regardless of whether flanking flexible linkers were added. Unexpectedly, insertion of IL-13 in the proline-rich region showed evidence of initiation of fusion and fusion-peptide exposure, but fusion was blocked at a subsequent step prior to fusion-pore formation. Thus, the site of ligand insertion influenced initiation of membrane fusion and its progression. These observations suggest that a novel site for ligand insertion must be identified before clinically useful targeted transduction will be achieved.

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2008-04-01
2019-11-18
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vol. , part 4, pp. 1049 – 1058

Surface expression of IL-13Rα2

Addition of the two point mutations did not increase transduction of NIH 3T3 cells

Positive- and negative-control samples in the fluorescent-based cell–cell fusion assays

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