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 systemic 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 extended 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 administration in the rodent brain. Injection of ogy in parallel in different mammalian cell lines. The SIN vectors expressing b-galactosidase into the mouse functional coupling of GPCRs to G proteins has been nucleus caudatus/putamen and nucleus accumbens demonstrated by measurement of intracellular Ca2+ septi resulted in neuron-specific reporter gene expres- release, inositol phosphate accumulation, cAMP stimula- sion.15 The expression was transient, with maximum tion and GTPgS binding.8 expression at 2 days postinjection and some minor Large-scale production of recombinant receptors has staining still observed at 14 days. Likewise, administra- also been established for SFV-infected mammalian tion of SFV-LacZ particles into the striatum and suspension cell cultures in bioreactors and spinner amygdala generated high local levels of b-galactosidase flasks.9 This approach has been successful in producing expression.16 In situ hybridizations demonstrated the up to 10 mg receptor protein per liter culture for presence of mRNA for at least 4 days post-injection. structural biology applications. Recently, the SFV system Minor b-galactosidase expression was still visible after 28 has been applied in a structural genomics program days, which most likely was due to the high stability of to overexpress 100 GPCRs with the aim of solubilization, the recombinant enzyme. Interestingly, the SFV particles purification and ultimately crystallization of GPCRs.10 caused no severe toxicity in the injected animals and when monitored for general health (food intake, body weight, body temperature), sensory motor function, Neuroscience exploratory behavior and muscle strength, no differences to control rats were detected. Administration of as few as The high rate of neuronal infection of alphaviruses has 105 infectious particles generated local highly neuron- made them attractive tools for applications in neuro- specific expression at the injection site, with no spread to science. Replication-deficient vectors have been shown to other brain regions. infect primary cultures of hippocampal and cortical neurons efficiently.11 Interestingly, cultures on a feeder layer of glial cells suggest that the expression is highly Gene therapy neuron-specific and very few glial cells show any transgene expression. Similarly, the b-galactosidase The first step towards gene therapy application was the expression pattern was highly neuron-specific for both demonstration of high transduction rates of various

Table 1 Applications of alphavirus vectors

Virus Application Objectives References

SFV Gene expression Cell lines Protein characterization 7,8 Large-scale production Drug screening, structural biology 9,10 Primary neurons Localization, electrophysiology 11 Hippocampal slices Localization, electrophysiology 12,13 In vivo, rat brain Localization, duration 16

Gene therapy Cancer vaccines Tumor protection, tumor regression 20 Tumor cell lines Transduction, cell killing 17,18 Intratumoral injection Tumor regression 23,24 Liposome-encapsulation Tumor-targeted gene delivery, therapy 28,29

SIN Gene expression Cell lines Expression of toxic proteins 31 Primary neurons Expression, localization 34 Hippocampal slices Localization 12 Mouse brain Localization, duration 15

Gene therapy Cancer vaccines Tumor protection, tumor regression 21 Envelope modiﬁcations Tumor targeting 26 Systemic delivery Tumor targeting 27

VEE Gene therapy Cancer vaccines Tumor protection, tumor regression 22

Gene Therapy Alphaviruses in gene therapy K Lundstrom S95 human prostate tumor cell lines with SFV vectors.17 This mice implanted with human lung tumors demonstrated study demonstrated not only infection rates of 70–95% that intratumoral injection of recombinant SFV-GFP in tumor cell lines, but also efficient b-galactosidase particles were responsible for a substantial tumor reporter gene expression in ex vivo transduced biopsies regression.24 Repeated injections were possible as these from prostate cancer patients. SFV-infected cells showed mice were immunodeficient. The best efficacy was a high degree of apoptosis. In another report, human obtained after SFV injections on three consecutive days, cancer cell lines were infected with relatively low followed by three additional injections a week later. concentrations of SFV-GFP and SFV-GFP/TK viruses to Additionally, the expression of the proapoptotic gene mimic an in vivo situation with reduced access to tumor Bax reduced the growth of AT3-Neo and AT3-Bcl2 cells.18 Clearly, the infection rate was modest, although tumors in nude mice.25 a bystander effect was observed for the therapeutic TK A major issue of concern has been the potential spread (thymidine kinase) after ganciclovir treatment. of virus into vital organs and especially into the central Another application of alphaviruses for cancer therapy nervous system after systemic delivery of alphaviruses. has been vaccination with tumor antigens.19 Application For this reason, several attempts have been made to of alphavirus vectors as RNA molecules, DNA plasmids target alphaviruses to specific cell types or tissue. As the and recombinant particles have all generated antibody RNA genome prevents any use of tissue-specific promo- responses in rodent and primate models. For instance, ters, the approach has been to introduce specific immunization with SFV-LacZ RNA led to tumor regres- targeting domains into the envelope structure of alpha- sion in mice.20 Antitumor activity and immune protec- viruses. Typically, introduction of IgG binding domains tion against melanomas was observed in mice after of protein A into the E2 envelope protein of SIN reduced administration of a SIN plasmid DNA construct carrying the infection rate of BHK and other host cells by a factor the tyr-related protein 1 gene.21 Moreover, when mice of 10 000.26 However, the chimeric SIN virus was able to were vaccinated with recombinant VEE particles expres- infect cells treated with a monoclonal antibody against sing the human papilloma virus (HPV), E7 gene a surface protein through the protein A domains. protection against tumor challenges was established.22 Surprisingly, a recent in vivo study suggested that SFV vectors have also been subjected to intratumoral conventional SIN vectors targeted tumors in several injections in animal tumor models. For instance, admin- mouse models.27 It was claimed that SIN-targeted istration of SFV particles expressing the interleukin-12 laminin receptors present at much higher concentrations (IL-12) subunits p40 and p35 to a B16 melanoma tumor on tumor cells than on normal cells. Most astonishingly, model resulted in significant tumor regression and peripheral fibrosarcomas located on the tail showed SIN- inhibition of tumor blood vessel formation measured based reporter gene expression after intraperitoneal by Doppler ultrasonography.23 Additional studies in administration.

Established technologies Clinical Trials

tumor in vivo regression gene delivery

intratumoral vector injections recombinant improve- protein liposome-encapsulation ment expression

systemic delivery improved targeting

large-scale production

anti- sense immunization ribo- tumor zyme regression production RNAi of retrovirus- virus or like particles replication- tumor competent challenge cell-specific targeting vectors

Figure 3 Schematic presentation of alphavirus applications. Recombinant SFV particles can be applied for applications in gene expression, vaccine production, intratumoral and systemic administration.

Gene Therapy Alphaviruses in gene therapy K Lundstrom S96 Another approach to obtain targeted delivery of viral therapy (Figure 3). The simple and rapid production of vectors has been to encapsulate SFV particles in high-titer virus has made alphaviruses attractive for gene liposomes that can specifically target tumors.28 This expression studies. Large-scale production especially strategy has several advantages. The delivery is targeted of membrane proteins in mammalian suspension cell to tumors, which allows systemic administration and cultures has made structural genomics initiatives possi- causes reduced damage to normal tissue. The liposome ble. Improved expression vectors and their targeting encapsulated particles stay for a prolonged time in in vivo have also facilitated gene therapy applications. circulation and thereby provide extended time of gene However, there are still avenues to explore to further delivery and expression. The cell membrane permeabil- reduce cytotoxicity for certain applications. On the other ity of liposomes improves the viral delivery in tumor hand, for cancer therapy, vectors with increased toxicity tissue. Most importantly, liposome-encapsulation pro- might be superior. Furthermore, studies on replication- vides a protection against detection by the host immune competent and persistent vectors might be advantageous system, which makes repeated injections feasible. In- to achieve better tissue-distribution of the transgene traperitoneal administration of encapsulated SFV-LacZ expression. However, in this context, targeting is of particles in SCID mice implanted with human LNCaP crucial importance. As additional large-scale virus tumors resulted in highly tumor-specific b-galactosidase production is requested for structural biology studies expression. Moreover, a single intraperitoneal injection of and further clinical trials are initiated, issues related to encapsulated SFV-IL-12 particles resulted in a significant reduced costs and improved quality (GMP grade) virus tumor growth inhibition in a pancreatic tumor mouse production need to be addressed. Only future develop- model.29 Preliminary clinical data from a phase I study ment can ensure the efficient and safe use of viral with encapsulated SFV-IL-12 particles on kidney carci- vectors, which will contribute to gene therapy as the noma and melanoma patients suggested that there was medicine of the future. no liposome- or SFV-related toxicity or any other adverse events.28 The IL-12 levels in the serum of patients were transiently increased five-fold with maximal expression References after 2 days, with a steady decrease until base levels were reached after 5 days. Repeated administration of 1 Strauss JH, Strauss EG. The alphaviruses: gene expression, encapsulated SFV particles generated no detectable replication, and evolution. Microbiol Rev 1994; 58: 491–562. immune response. 2 Mathiot CC et al. An outbreak of human Semliki Forest virus infections in Central African Republic. Am J Trop Med Hyg 1990; 4: 386–393. 3 Liljestro¨m P, Garoff H. A new generation of animal cell Vector development expression vectors based on the Semliki Forest virus replicon. Although alphaviruses have been proven suitable for a Bio/Technology 1991; 9: 1356–1361. wide range of applications, the induction of apoptosis, 4 Xiong C et al. Sindbis virus: an efficient, broad host range vector for gene expression in animal cells. Science 1989; 243: 1189–1191. the cytotoxicity in infected cells and the relatively short- 5 Davis NL, Brown KW, Johnston RE. In vitro synthesis of term transgene expression has restricted their use. 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Expression of heterologous proteins in which showed a temperature-dependent expression cultured rat hippocampal neurons using the Semliki Forest virus 35 pattern in hippocampal slice cultures.33 At 371C, the vector. J Neurosci Res 1993; : 445–451. 12 Ehrengruber MU et al. Recombinant Semliki Forest virus and GFP expression pattern was mostly glial specific, 1 Sindbis virus efficiently infect neurons in hippocampal slice whereas at 31 C, the majority of GFP-positive cells were cultures. Proc Natl Acad Sci USA 1999; 96: 7041–7046. of neuronal origin. 13 Ehrengruber MU et al. Gene transfer into neurons from hippocampal slices: comparison of recombinant Semliki Forest Virus, adenovirus, adeno-associated virus, lentivirus, and Conclusion and future aspects measles virus. Mol Cell Neurosci 2001; 17: 855–871. 14 Hennou S et al. Homer-1a/Vesl-1S enhances hippocampal Alphavirus vectors have found a large number of synaptic transmission. 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