Gene Therapy (2005) 12, S92–S97 & 2005 Nature Publishing Group All rights reserved 0969-7128/05 $30.00 www.nature.com/gt CONFERENCE PAPER Biology and application of alphaviruses in gene therapy K Lundstrom Regulon Inc./BioXtal, Epalinges, Switzerland The broad host cell range and high expression levels of lenges against lethal viruses and tumors in rodent and transgenes are features that have made alphaviruses primate models. Intratumoral injections of alphavirus vectors attractive for gene expression studies and gene therapy expressing reporter and immunostimulatory genes have led applications. Particularly, Semliki Forest virus vectors have to significant tumor regression in mouse models. Modifica- been applied for large-scale production of recombinant tions of the viral envelope structure have generated targeted membrane proteins for drug screening purposes and Sindbis virus vectors. Astonishingly, conventional Sindbis structural biology studies. The high preference of expression vectors have demonstrated tumor-specific targeting in animal in neuronal cells has led to many applications of alphavirus models due to the high density of laminin receptors on vectors in neuroscience. Studies on localization and trans- cancer cells. Moreover, encaspulation of Semliki Forest virus port of recombinant proteins as well as electrophysiological vectors in liposomes has provided a means of achieving recording have become feasible in primary cultures of tumor targeting and protection against the host immune neurons and hippocampal slice cultures. Alphaviruses have response. Much attention has also been given to the frequently been used as vaccine vectors for expression engineering of novel mutant alphavirus vectors with proper- of antigens against viruses and tumors. Administration ties such as reduced cytotoxicity, prolonged duration of of recombinant viral particles, DNA plasmids or in vitro transgene expression and improved survival of host cells. transcribed RNA has resulted in protection against chal- Gene Therapy (2005) 12, S92–S97. doi:10.1038/sj.gt.3302620 Keywords: alphavirus; gene expression; gene delivery, neuron-specific expression; tumor targeting; cancer therapy Introduction taneously, the envelope proteins are processed through the Golgi apparatus and the endoplasmic reticulum to The family of Togaviruses are single-stranded RNA the plasma membrane, where they surround nucleocap- viruses with an envelope structure. The genus Alphavir- sids and finally mature virus particles are released by idae, alphaviruses, has 26 members, all with a genome budding. of approximately 12 kb and are commonly residing in Expression systems have been engineered for several many species such as mosquitoes, birds and rodents and alphaviruses. The most commonly used alphavirus other mammals.1 Some of the members of alphaviruses vectors are based on Semliki Forest virus (SFV)3 (Figure are mild pathogens in humans and some epidemics with 2), Sindbis virus4 and Venezuelan equine encephalitis flu-like symptoms have been described in central Africa.2 virus (VEE).5 In principle, three types of vector systems The life cycle of alphaviruses includes infection through have been engineered. I. Replication-deficient viral particles: recognition of cell surface receptors such as heparin In this approach, an expression vector carrying the sulfate and laminin receptors and uptake of viral nonstructural genes and the gene of interest and a helper particles through either fusion with the cell membrane vector with the structural genes are subjected to in vitro or endocytosis (Figure 1). After the release of the RNA preparation followed by cotransfection into baby positive-strand RNA genome in the cytoplasm, immedi- hamster kidney (BHK) cells where recombinant virus ate translation of the viral nonstructural proteins (nsP1-4) particles are assembled. The generated particles are occurs leading to the formation of the replicase complex capable of one round of infection, but the lack of any responsible for highly efficient RNA replication. A full- structural genes prevents any further virus production. length minus strand copy is made as a template and the As the half-life of the RNA and the replicase complex replication generates approximately 200 000 new plus (replicon) are limited, the transgene expression is strand copies per cell. Also copies of a subgenomic RNA transient. II. Replication-competent viral particles: These containing the structural genes (capsid, envelope pro- vectors contain the full-length alphavirus genome and an teins) are generated. Assembly of RNA and capsid additional subgenomic promoter upstream of the trans- protein into nucleocapsids occurs in the cytoplasm gene of interest. The produced recombinant particles are followed by transport to the plasma membrane. Simul- infectious and able to produce progeny virus in host cells. III. DNA-based vectors: To allow direct application of Correspondence: Dr K Lundstrom, Regulon Inc.,/BioXtal, Chemin des plasmid DNA the SP6 RNA polymerase promoter has Croisettes 22, CH-1066 Epalinges, Switzerland. been replaced by a CMV promoter. In this case, transient Alphaviruses in gene therapy K Lundstrom S93 transfection of plasmid DNA will result in high expres- advantages are the rapid production of high titer (109– sion levels of the transgene of interest. It is also possible 1010 infectious particles/ml) particles that require no to cotransfect a DNA-based helper vector and to obtain further concentration or purification. Moreover, the recombinant particles.6 However, the titers are signifi- broad cell host range and high transgene expression cantly lower than for RNA-based particles. levels are positive features. The induction of apoptosis Most applications of alphavirus vectors have so far can also be seen as a plus for applications in cancer been for replication-deficient particles. The obvious therapy. Among the disadvantages of alphaviruses is the short-term transgene expression, which lasts in vivo for 5–7 days. Another concern is the strong cytotoxicity upon Alphavirus Life-cycle host cells, resulting in a dramatic shutdown on endo- -strand RNA template genous gene expression, which can seriously affect BHK-21 cells signal-transduction events and gene expression kinetics. +strand RNA replication In this review, the applications of alphavirus vectors Packaging signal for recombinant protein expression, localization and infection functional studies in neurons and gene therapy approaches including intratumoral injections and sys- temic delivery are described (Table 1). Moreover, the targeting of alphaviruses to tumor tissue specifically as nucleocapsid an important improvement of efficacy and safety are generation discussed. Finally, the development of novel vectors with mature virus particles lower cytotoxicity properties and improved and ex- tended expression profiles is described. virus budding Gene expression Figure 1 Schematic presentation of the life cycle of alphaviruses. Infection Recombinant protein expression has in general been of BHK-21 (baby hamster kidney) cells with alphaviruses lead to an early onset of production of nonstructural proteins nsP1–4, which will form relatively successful for soluble proteins in different the replicase complex responsible for RNA replication. Expression of expression systems such as bacteria, yeast, insect and alphaviral structural proteins results in nucleocapsid formation followed mammalian cells. However, to obtain high yields of by maturation of virus progeny by budding. integral transmembrane proteins in their native form has Replication-deficient SFV system SP6 DNA In vitro transcription SFV RNA foreign Expression Vector SFV 26S MCS Packaging signal SFV nonstructural genes + SP6 SFV 26S SFV SFV 26S SFV 26S Electroporation Helper foreign Vector SFV structural genes Capsid, p62, E1 BHK-21 cells Recombinant SFV particles Expression of recombinant protein Infection of host cells Figure 2 Schematic presentation of generation of replication-deficient SFV particles. In vitro transcribed RNAs from the expression and helper vectors are transfected into BHK-21 cells, where recombinant SFV particles are generated. Due to the presence of the packaging signal only in the expression vector the generated particles contain only the nonstructural genes and the gene of interest and are therefore replication-deficient. BHK-21, baby hamster kidney cells; E1, SFV envelope E1 protein; p62, precursor protein p62 for SFV E2 and E3 envelope proteins; SFV26S, SFV subgenomic promoter; SP6, SP6 RNA polymerase promoter. Gene Therapy Alphaviruses in gene therapy K Lundstrom S94 been extremely difficult. In this context, SFV vectors have SIN and SFV vectors injected into hippocampal slices been employed for overexpression of a large number of cultured in vivo.12 SFV-based expression indicated that G protein-coupled receptors (GPCRs) and several ligand- more than 90% of the GFP-positive cells were of neuronal gated ion channels.7 The high expression levels, in the origin.13 Transduction of primary neurons and hippo- range of 20–200 pmol receptor per milligram protein, campal slice cultures has allowed studies on protein suggesting receptor densities of up to 10 million transport and localization as well as functional activity, receptors per cell, has made the SFV system applicable typically by electrophysiological recordings.14 for drug screening programs. Additionally, the broad Moreover, alphavirus vectors have been applied for host range has allowed studies on receptor pharmacol- in vivo