Additional viral-based vectors

A number of additional viral types may also prove useful as vectors in the practice of gene therapy. Chief amongst these are the adenoviruses. Adeno-associated virus, the herpes virus and a number of other viruses are also being considered (Table 11.2). 

While short-term, high-level gene expression may be appropriate for some gene therapy applications, it would be of less use for the treatment of, for example, genetic diseases, where long-term gene expression would be required. This could be achieved in theory by repeat administration of the adenoviral vector. However, adenoviruses prompt a strong immune response, which limits the efficacy of repeat administration. 

Additional viruses that may prove of some use as future viral vectors include adeno-associated virus and herpes virus. Adeno-associated virus is a very small, single-stranded DNA (ssDNA) virus—its genome consists of only two genes. It does not have the ability to replicate autonomously and can do so only in the presence of a co-infecting adenovirus (or other selected viruses). 

An additional virus, which has more recently gained some attention as a possible vector, is that of the sindbis virus. A member of the alphavirus family, this ssRNA virus can infect a broad range of both insect and vertebrate cells. The mature virion particles consist of the RNA genome complexed with a capsid protein C. This, in turn, is enveloped by a lipid bilayer in which two additional viral proteins, El and E2, are embedded. The E2 polypeptide appears to mediate viral binding to the surface receptors of susceptible cells (the major mammalian cell surface receptor it targets appears to be the highly conserved, widely distributed laminin receptor). 

The sindbis virus is simple, robust, capable of infecting non-dividing cells and generally supports high levels of gene expression. However, it does display a broad host range and, hence, lacks the inherent targeting specificity characteristic of an idealized viral vector. 

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Viruses are infectious particles formed by nucleic acid, proteins, and in some cases lipids. As viruses (for example, retro- and adenoviruses) transfer viral genes into cells with high efficiency, modified forms are sometimes used as vectors for gene transfer. However, procedures using virus-based vectors are often significantly more complicated and time-consuming than other transfection methods. In addition, viral vectors are potentially hazardous, and biological safety issues need to be considered carefully. Therefore, techniques that combine... 

Because HIV-1 is a human pathogen, there is some concern about the use of HIV-1-based lentiviral vectors. The current HIV-1 lentiviral vector system is deprived of all accessory proteins (except rev) and viral sequences in the vectors have been minimized therefore, replication of these vectors is highly disabled and the possibility of homologous recombination is minimized. In addition, codon-optimization of the packaging construct further decreases the risk of homologous recombination. Furthermore, changes in the codons makes the production of the structural proteins independent of rev, and therefore additional viral sequences (RRE) can be eliminated from the packaging construct (82). 

Vaccine adjuvants Plasmid vaccines that can successfully elicit humoral and cellular immune response have the potential to provide safer alternatives than live viral or heat-killed bacterial vaccines. Therapeutically relevant immune response is attainable with adjuvants such as cholera toxin and lipid A delivered using cationic nanoparticles by topical and subcutaneous administration, respectively [45]. Additionally, protein-based vaccines may be encapsulated in pH sensitive polymers, where low pH in the phagosomes of antigen-presenting cells disrupt the vectors to release vaccines [ 37]. 

RNAi technology has obvious therapeutic potential as an antisense agent, and initial therapeutic targets of RNAi include viral infection, neurological diseases and cancer therapy. The synthesis of dsRNA displaying the desired nucleotide sequence is straightforward. However, as in the case of additional nucleic-acid-based therapeutic approaches, major technical hurdles remain to be overcome before RNAi becomes a therapeutic reality. Naked unmodified siRNAs for example display a serum half-life of less than 1 min, due to serum nuclease degradation. Approaches to improve the RNAi pharmacokinetic profile include chemical modification of the nucleotide backbone, to render it nuclease resistant, and the use of viral or non-viral vectors, to achieve safe product delivery to cells. As such, the jury remains out in terms of the development and approval of RNAi-based medicines, in the short to medium term at least. 

This transfection-based platform allows to produce proteins in a plant host at a cost of US 1 to 10 per gram of crude protein. The platform is essentially free from limitations (gene insert size limit, inability to express more than one gene) of current viral vector-based platforms. The expression levels reach 5 g per kilogram of fresh leaf tissue (or some 50% of total cellular protein ) in 5 to 14 days after inoculation. Since the virus process (in addition to superhigh production of its own proteins, including the protein of interest) leads to the shut-off of the other... 

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