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Recently a novel recombinant sindbis virus displaying altered host cell specificity has been generated. Scientists inserted a nucleotide sequence coding for the IgG-binding domain of Staphyloccus aureus into the E2 viral gene. Disruption of the E2 gene renders its protein product incapable of binding laminin (hence destroying the natural viral tropism). However, the protein A domain allows the chimeric E2 product to bind monoclonal antibodies. This altered virus may prove to be a useful generic or ‘null’ vector, potentially capable of being specifically targeted to any desired cell type. This would simply necessitate pre-incubation of the virus with monoclonal antibodies raised against a surface antigen unique to the proposed target cell population (Figure 11.5). Binding of the monoclonal antibody to the protein A domain would ensue and the immobilized monoclonal antibody would dictate the cell type targeted.
Initial studies using this system have proved encouraging. The altered virus (without associated monoclonal antibody) failed to infect a wide variety of human cell lines. By initially incubating with monoclonal antibody of the appropriate specificity, however, the viral particles were capable of efficiently transducing cells expressing surface receptors such as CD4, CD33 and human leukocyte antigen (HLA).
A number of other issues must now be addressed, including determining whether the IgG-protein A affinity is sufficiently high to keep the antibody associated with the virus in vivo. The
Modified E2 protein, now containing the IgG binding domain of protein A
Figure 11.5. Generation of engineered sindbis virus capable of being targeted to bind specific cell types. (a) Simplified depiction of the virus, displaying the surface E2 protein. Genetic engineering facilitates disruption of the E2 gene by incorporation of the IgG-binding domain of protein A (b). Incubation of such engineered viral particles with most monoclonal antibody types results in effective immobilization of the antibody on the viral surface (c). Thus, the engineered viral vector should be targetable to any specific cell type, simply by its pre-incubation with monoclonal antibodies, which selectively bind a surface antigen uniquely associated with the target cell
full potential of this approach will also require more detailed characterization of surface markers uniquely associated with different cell types. However, the approach exemplifies the types of technical innovations now being introduced, which will make second-generation vectors more suited to their role in gene therapy.
Manufacture of viral vectors
Literature reports describing the large-scale manufacture of viral vectors for gene therapy application are few and far between. Development of manufacturing protocols has largely been undertaken by companies engaged in gene therapy product development and, consequently, protocol details remain confidential. The overall approach likely taken is not too dissimilar to that of therapeutic protein manufacture (Chapter 3). It involves synthesis in cells, recovery, concentration, purification and formulation steps. A likely generalized manufacturing scenario for retroviral-based vectors is outlined in Figure 11.6. The manufacture of alternative viral
NUCLEIC ACID THERAPEUTICS 475
Figure 11.6. Large-scale manufacture of retroviral vectors for use for gene therapy-based clinical protocols. Refer to text for details
vectors likely follow a similar approach. The process is initiated by the culture of packing cells in suitable animal cell bioreactors. The principles and practice of animal cell culture have been overviewed in Chapter 3. To date, bioreactor size of 1001 or less have been used, which are sufficient to satisfy clinical trial demand. The packing cells are then seeded with the replication-deficient virus, allowing vector propagation (see also Figure 11.4). Viral harvest may then be
undertaken by methods of microfiltration, which separates intact packing cells/cell debris from the vector-containing product stream. Viral vector concentration can then be undertaken by ultrafiltration (Chapter 3). Subsequent vector purification strategies employed include chromatographic approaches similar to those used to purify proteins. Ion exchange and various forms of affinity chromatography have received most attention. A detailed understanding of the interactions between chromatographic media and viral surface molecules is currently lacking and hence is the subject of ongoing research. A comprehensive understanding of such interactions would be required to optimize chromatographic purification protocols. After purification, final product analysis and formulation is undertaken. Again, few details of these steps have been openly published.
Although viral-mediated gene delivery systems currently predominate, some 20-25% of current clinical trials use non-viral-based methods of gene delivery. General advantages quoted with respect to non-viral delivery systems include: