Introduction

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Alphaviruses belong to the Togaviruses and harbor a single-stranded RNA genome surrounded by a capsid structure and envelope spike proteins embedded in a lipid bilayer (1). The genome consists of four nonstructural genes (nsP1-4) and the capsid protein and two or three envelope proteins (E1-E3). The function of nsP1 is to initiate the minus-strand RNA synthesis and capping of viral RNAs, whereas nsP2 possesses protease and helicase activities and nsP4 contains the catalytic subunit of viral RNA polymerase (1). Although the precise function of nsP3 is not known, it is described to

From: Cancer Drug Discovery and Development: Gene Therapy for Cancer Edited by: K. K. Hunt, S. A. Vorburger, and S. G. Swisher © Humana Press Inc., Totowa, NJ

be a phosphoprotein involved in RNA replication (2). The host range for alphaviruses is broad and includes insect, amphibian, reptile, avian, and mammalian cells. The infection occurs through the recognition of cell-surface receptors and, although these have not been definitely identified for all alphaviruses, it is suggested that common receptors for many cell types, such as laminin receptors, are the targets for alphaviruses (3). After the initial cell recognition step, the virus particles are brought into the cytoplasm either through fusion of the viral envelope structures to the host cell or by endocytosis depending on the cell type. Next a minus-strand copy is generated from the plus-strand genome as a template for generation of extreme numbers of new plus strand copies. Both full-length 42S RNA and the subgenomic 26S RNA encoding the structural genes are synthesized in the cells. The capsid protein forms the nucleocapsid structure together with the single-stranded viral RNA and nucleocapsids are transported to the plasma membrane. Simultaneously, the envelope proteins are processed through the rough endo-plasmatic reticulum and Golgi to the plasma membrane encapsulating the nucleocapsid, which results in release of mature virus particles by budding. The process is highly efficient generating virus particles with titers of 109-1010 particles/mL within 24 h.

To apply alphaviruses for heterologous gene expression, basically three types of vectors have been engineered (see Fig. 1).

1. Replication-deficient vectors. For this application, the alphavirus nonstructural and structural genes have been split on separate plasmid vectors. The nonstructural genes and the subgenomic 26S promoter followed by a multilinker cloning region have been introduced into the expression vector downstream of a prokaryotic T7 or SP6 RNA polymerase promoter for in vitro transcription of RNA (4). Likewise the structural genes are transcribed from helper vectors in trans as a safety precautious to prevent the generation of replication competent particles. Furthermore, to eliminate homologous recombination between RNA generated from expression and helper vectors, the capsid and envelope genes have been split on separate helper vectors (5). Once RNA molecules have been transcribed from the two vectors, these are introduced into BHK-21 (baby hamster kidney) cells by either electroporation or applying transfection reagents. Rapid RNA replication generates up to 200,000 RNA copies/cell, which results in efficient production of recombinant virus particles. As the packaging signal is located on the recombinant RNA only, this species of RNA will be packaged into the nucleocap-sid leading to generation of replication-deficient particles. However, these particles can be applied for only one round of infection of various host cells to generate high transient expression levels of recombinant proteins as described below.

2. Replication-competent vectors. Especially for in vivo applications, where an extended expression pattern and spread of infection is advantageous, vectors with ability to replicate could be attractive. In this context, a second subgenomic promoter with a down stream multilinker cloning region was engineered into vectors with a full-length alphavirus genome (6). Although different constructs have been engineered the most common site for the insertion of the second subgenomic promoter has been downstream of the E1 gene in the 3' nontranslated region (6). Generally, the replication-competent vectors have been less frequently used than the replication-deficient ones.

3. DNA-based vectors. Replacement of the T7 or SP6 RNA polymerase promoter with a cytomegalo virus (CMV) or RSV promoter has allowed the use of alphavirus vectors directly in the form of plasmid DNA (7). In this form, plasmid DNA can conveniently be directly introduced into mammalian cells for transient heterologous gene expression. However, using this approach the advantage of the broad range of alphavirus

Fig. 1. Schematic presentation of alphavirus vectors. (A) Replication-deficient RNA-based expression vector. (B) RNA-based helper vector. (C) Replication-proficient RNA-based expression vector. (D) DNA-based expression vector. Gol, Gene of interest; Rep, Replicase signal.

infection is lost as the gene delivery success relies on DNA transfection methods. Introduction of a type II promoter in the helper vector, DNA-based vectors have also been applied for virus production although the titers have generally been 100-1000 fold lower than observed from RNA-based vectors (8).

Several alphaviruses have been subjected to vector development. The most frequently used alphaviruses are Semliki Forest virus (SFV) (4), Sindbis virus (SIN) (9), and Venezuelan equine encephalitis virus (VEE) (16). SFV, SIN, and VEE have shown very similar features concerning host range, cytotoxic effect on infected host cells and transgene expression. Both SFV and SIN have been frequently used for recombinant protein

Fig. 2. Broad host range of alphaviruses. (A) SFV-LacZ infection of BHK cells. (B) SFV-GFP infection of rat primary hippocampal neurons. (C) SFV-LacZ infection of human prostate tumor cell line DU-145. (D) Ex vivo SFV-LacZ infection of prostate biopsy. (E) SFV-LacZ infection of rat hippocampal slice culture. (F) Stereotactic injection of SFV-LacZ virus into rat brain. (G) Systemic delivery of liposome-encapsulated SFV-LacZ virus in SCID mice with human LnCaP xenografts.

Fig. 2. Broad host range of alphaviruses. (A) SFV-LacZ infection of BHK cells. (B) SFV-GFP infection of rat primary hippocampal neurons. (C) SFV-LacZ infection of human prostate tumor cell line DU-145. (D) Ex vivo SFV-LacZ infection of prostate biopsy. (E) SFV-LacZ infection of rat hippocampal slice culture. (F) Stereotactic injection of SFV-LacZ virus into rat brain. (G) Systemic delivery of liposome-encapsulated SFV-LacZ virus in SCID mice with human LnCaP xenografts.

expression in mammalian cell lines (11,12), in primary neurons (13), in hippocampal slice cultures (14), and in vivo (15,16) (Fig 2). SFV in particular has been applied for successful high-level expression of integral membrane proteins such as G protein-coupled receptors and ligand-gated ion channels, generally known to be difficult to express (17). This has allowed studies on pharmacology and cell biology of many therapeuti-cally important receptors and has provided material for drug screening programs and structural biology. Alphavirus vectors have been frequently used for expression of tumor antigens (18) and viral antigens (19) in approaches to develop vaccines. Moreover, alphavirus vectors have been used as gene delivery tools in neuroscience and also as vectors in experimental cancer gene therapy.

In this chapter, the applications of alphavirus vectors for cancer vaccines and also as delivery vehicles for therapeutic genes in cancer are described. Much attention is paid to vector development to improve the expression properties treatment and to modify the toxi-city of the alphaviruses themselves. Safety issues related to the use of viral vectors are also discussed. Finally, the possibility of targeting systemic delivery of alphaviruses is presented.

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