Identification of the block in targeted retroviral-mediated gene transfer

Abstract

A chimeric retroviral vector (33E67) containing a CD33-specific single-chain antibody was generated in an attempt to target cells displaying the CD33 surface antigen. The chimeric envelope protein was translated, processed, and incorporated into viral particles as efficiently as wild-type envelope protein. The viral particles carrying the 33E67 envelope protein could bind efficiently to the CD33 receptor on target cells and were internalized, but no gene transfer occurred. A unique experimental approach was used to examine the basis for this postbinding block. Our data indicate that the chimeric envelope protein itself cannot participate in the fusion process, the most reasonable explanation being that this chimeric protein cannot undergo the appropriate conformational change that is thought to be triggered by receptor binding, a suggested prerequisite to subsequent fusion and core entry. These results indicate that the block to gene transfer in this system, and probably in most of the current chimeric retroviral vectors to date, is the inability of the chimeric envelope protein to undergo this obligatory conformational change.

Latest Developments in Gene Transfer Technology: Achievements, Perspectives, and Controversies over Therapeutic Applications

“…The latter issue has never been tackled, whereas the engineering of ecotropic-based retroviral vectors with altered cell tropism has attracted much attention [5], but all the attempts had little success. The chimeric retroviral particles that have been produced have a low transduction capacity [5], or even fail the gene transfer process [192]. To date, the ex vivo retroviral-mediated gene transfer model is more realistic than the in vivo one, although it is not optimal for gene therapy applications.…”

Surface Modification of Retroviral Vectors for Gene Therapy

“…The fused parts may be complete ligands (Kasahara et al 1994), peptides (Gollan and Green 2002;Morizono et al 2009a) or single-chain antibodies (Anliker et al 2010;Somia et al 1995). However, limits have been shown to apply to this technique, as structural or functional elements are typically disturbed by their introduction, leading to loss of infectivity, since cellular uptake is inhibited at the level of envelope-cell membrane fusion (Galanis et al 2001;Ryu et al 2008;Zhao et al 1999). When a chimeric Env molecule, containing a CD33 specific single-chain antibody, was generated to target CD33 positive cells, the collected data indicated, that the chimeric protein could not initiate fusion of the virus and cell membranes during infection (Zhao et al 1999).…”

“…However, limits have been shown to apply to this technique, as structural or functional elements are typically disturbed by their introduction, leading to loss of infectivity, since cellular uptake is inhibited at the level of envelope-cell membrane fusion (Galanis et al 2001;Ryu et al 2008;Zhao et al 1999). When a chimeric Env molecule, containing a CD33 specific single-chain antibody, was generated to target CD33 positive cells, the collected data indicated, that the chimeric protein could not initiate fusion of the virus and cell membranes during infection (Zhao et al 1999). The most widely accepted explanation for this is that the chimeric proteins are unable to undergo a mandatory conformational change which activates the fusion activity (see also section 3.5.).…”

“…Upon this first contact, TM activates fusogenic properties, which allow viral and cellular membranes to fuse, resulting in viral entry. The interaction of SU and TM is highly sensitive to changes in SU and already small changes can disturb the activation of the TM activity (Zhao et al 1999). Subsequently, modifications introduced to the Env proteins are tolerated badly, quite often leading to severe reduction in infectivity.…”

Surface-engineering of lentiviral vectors

“…It seems that although the fusogenic potential of chimeric glycoproteins incorporated on the viral particles is intact, in the absence of the retroviral receptor, the interaction of the displayed ligand with its target cellsurface molecule is generally not able to activate the fusion functions of the chimeras [83]. Infectivity of the recombinant viruses is therefore inhibited at a postbinding block [75,100]. Thus, the poor fusion activity of chimeric glycoproteins is attributed to the loss of coupling between retargeted binding and fusion activation.…”