Viral vectors fusion proteins

This is in contrast to viruses, where the virus particles also show active transport when present in the cytosol after fusion with the plasma membrane or endosomal membrane [60-62], This is due to the ability of specific proteins of the virus particle to bind motor proteins. Single-particle tracking reveals that the quantitative intracellular transport properties of internalized non-viral gene vectors (e.g., polyplexes) are similar to that of viral vectors (e.g., adenovirus) [63]. Suk et al. showed that over 80% of polyplexes and adenoviruses in neurons are subdiffusive and 11-13% are actively transported. However, their trafficking pathways are substantially different. Polyplexes colocalized with endosomal compartments whereas adenovirus particles quickly escaped endosomes after endocytosis. Nevertheless, both exploit the intracellular transport machinery to be actively transported. 

Laboratory techniques such as biolistics (gene gun) and DNA delivery to protoplasts are not discussed in this section, as they are inadequate for producing quantitative amounts of purified protein. The speed of the viral vectors does provide material in the time-frame of several weeks therefore, this production system can provide both rapid and scaleable production. This system is described in the previous sections it is still not clear how well the viral expression system can tolerate large proteins. Most of the reports concern epitopes or small proteins. In addition, the potential need for fusions with coat protein for efficient expression might also limit this system. The lack of reports to refute these concerns after many years suggests that there are serious limitations to using viral vectors for expressing some proteins. 

Figure 7.1-3. The ideal synthetic (nonviral) gene delivery vector. After dense DNA packaging is accomplished (e.g., by protamine sulfate), the surface of synthetic particles (which is usually positively charged) needs to be shielded (e.g., by poly (ethylene-glycol) [PEG]) so that they do not attach to blood elements or to each other and, therefore, have an extended circulating plasma half-life (1) (passive targeting to leaky vessels ). The surface of the particles will contain specific ligands for active targeting to selected cells/ tissues (2). By engineering viral fusion proteins to the particle coat, cell entry is facilitated...

Inside the endosome the pH (5.0-6.2) is more acidic and poses the problem of nucleic acid degradation. In the case of viral vectors, their inherent property to undergo conformational changes in the coat proteins promotes endosomal membrane fusion, which helps in protecting them from the endosomal environment, but in the case of nonviral vectors, lysosomotropic agents like chloroquine, membrane-destabilizing peptides such as synthetic N-terminal peptides of rhinovirus VP-1 or influenza virus HA-2 are attached to the cationic complex to mediate endosomal release. 

Fig. 2. Schematic of the baculoviral recombination system used in this work, (a) Disruption of the essential 1,629 gene renders the baculoviral genome replication-deficient, (b) Linearised baculoviral genomic DNA and transfer vector are co-transfected into insect cells, (c) Flomologous recombination regenerates an intact 1,629 gene, enabling viral replication. Expression of the BCCP fusion protein is driven from the polh promoter.

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