Gene therapy for malignant glioma using Sindbis vectors expressing a fusogenic membrane glycoprotein

BACKGROUND: Malignant glioma has a dismal prognosis. It was previously shown that glioma cells are efficiently killed when they express a gene coding for a hyperfusogenic mutant of the gibbon ape leukemia virus envelope glycoprotein (GALV.fus). However, production of viral vectors expressing GALV.fus has proven problematic because the transgene is toxic to vector-producing cells of human origin. We reasoned that Sindbis-virus-based vectors might be ideal for GALV.fus gene transfer because high-titer stocks can easily be generated in hamster cells and Sindbis virus efficiently infects human tumor cells through the high-affinity 67 kDa laminin receptor. In addition, Sindbis virus nonstructural proteins are potent inducers of apoptosis, and Sindbis vector RNAs expressing fusogenic viral proteins have been shown to spread from cell-to-cell in membrane-formed infectious particles. METHODS: Sindbis virus replicon-containing particles were generated by co-transfecting vector and helper RNAs into baby hamster kidney (BHK-21) cells. Packaged beta-galactosidase and GALV.fus expressing Sindbis vectors were used to infect glioma cell lines, which were then compared for syncytial cytopathic effect, cell killing, and release of infectious virus-like particles containing the vector genome. Finally, the efficacy of GALV.fus and beta-galactosidase Sindbis vectors was compared in an orthotopic intracerebral U87 glioma xenograft model in nude mice. RESULTS: High-titer stocks (>10(9) infectious units (iu)/ml) of the GALV.fus and beta-galactosidase vectors were obtained. Glioma cells infected with the GALV.fus vector formed large syncytia which died rapidly by apoptosis and released infectious membrane-formed particles that could transfer vector genomes to uninfected cells. The GALV.fus vector had significantly greater antitumor therapeutic potency than the beta-galactosidase vector in the U87 glioma xenograft model. CONCLUSIONS: Sindbis vectors expressing GALV.fus can be packaged into infectious viral particles to high titer, they exhibit potent bystander cytopathic potential and are active against U87 glioma xenografts. Sindbis-virus-based replicons appear to be efficient vector systems for delivery and expression of fusogenic membrane glycoproteins.     

Replicon vectors derived from Sindbis virus and Semliki forest virus that establish persistent replication in host cells

Alphavirus replicon vectors are well suited for applications where transient, high-level expression of a heterologous gene is required. Replicon vector expression in cells leads to inhibition of host macromolecular synthesis, culminating in eventual cell death by an apoptotic mechanism. For many applications, including gene expression studies in cultured cells, a longer duration of transgene expression without resulting cytopathic effects is useful. Recently, noncytopathic Sindbis virus (SIN) variants were isolated in BHK cells, and the mutations responsible were mapped to the protease domain of nonstructural protein 2 (nsP2). We report here the isolation of additional variants of both SIN and Semliki Forest virus (SFV) replicons encoding the neomycin resistance gene that can establish persistent replication in BHK cells. The SIN and SFV variant replicons resulted from previously undescribed mutations within one of three discrete regions of the nsP2 gene. Differences among the panel of variants were observed in processing of the nonstructural polyprotein and in the ratios of subgenomic to genomic RNAs. Importantly, high-level expression of a heterologous gene was retained with most replicons. Finally, in contrast to previous studies, efficient packaging was obtained with several of the variant replicons. This work expands the utility of noncytopathic replicons and the understanding of how alphavirus replicons establish persistent replication in cultured cells.     

Sindbis virus with a tricomponent genome

We established a system for propagation of Sindbis virus (SIN)-based replicons in tissue culture in the form of a tricomponent genome virus. Three RNA fragments containing complementing genetic information required for virus replication are packaged into separate viral particles, and each cell produces at least 1,000 packaged replicons and the number of packaged helpers sufficient to perform the next passage. This system can be used to generate large stocks of packaged replicons. The formation of infectious recombinant SIN virus was not detected in any experiments. These features make multicomponent genome SIN an attractive system for a variety of research and biotechnology applications.     

Comparison of the effects of Sindbis virus and Sindbis virus replicons on host cell protein synthesis and cytopathogenicity in BHK cells.

Infection of BHK cells by Sindbis virus leads to rapid inhibition of host cell protein synthesis and cytopathic effects (CPE). We have been studying these events to determine whether the expression of a specific viral gene is required and, in the present study, have focused our attention on the role of the structural proteins--the capsid protein and the two membrane glycoproteins. We tested a variety of Sindbis viruses and Sindbis virus replicons (virus particles containing an RNA that is self-replicating but with some or all of the viral structural protein genes deleted) for their abilities to inhibit host cell protein synthesis and cause CPE in infected BHK cells. Our results show that shutoff of host cell protein synthesis occurred in infected BHK cells when no viral structural proteins were synthesized and also under conditions in which the level of the viral subgenomic RNA was too low to be detected. These results support the conclusion that the early steps in viral gene expression are the ones required for the inhibition of host cell protein synthesis in BHK cells. In contrast, the Sindbis viruses and Sindbis virus replicons were clearly distinguished by the time at which CPE became evident. Viruses that synthesized high levels of the two membrane glycoproteins on the surface of the infected cells caused a rapid (12 to 16 h postinfection) appearance of CPE, and those that did not synthesize the glycoprotein spikes showed delayed (30 to 40 h) CPE.     

Rapid, specific detection of alphaviruses from tissue cultures using a replicon-defective reporter gene assay

We established a rapid, specific technique for detecting alphaviruses using a replicon-defective reporter gene assay derived from the Sindbis virus XJ-160. The pVaXJ expression vector containing the XJ-160 genome was engineered to form the expression vectors pVaXJ-EGFP expressing enhanced green fluorescence protein (EGFP) or pVaXJ-GLuc expressing Gaussia luciferase (GLuc). The replicon-defective reporter plasmids pVaXJ-EGFPΔnsp4 and pVaXJ-GLucΔnsp4 were constructed by deleting 1139 bp in the non-structural protein 4 (nsP4) gene. The deletion in the nsP4 gene prevented the defective replicons from replicating and expressing reporter genes in transfected BHK-21 cells. However, when these transfected cells were infected with an alphavirus, the non-structural proteins expressed by the alphavirus could act on the defective replicons in trans and induce the expression of the reporter genes. The replicon-defective plasmids were used to visualize the presence of alphavirus qualitatively or detect it quantitatively. Specificity tests showed that this assay could detect a variety of alphaviruses from tissue cultures, while other RNA viruses, such as Japanese encephalitis virus and Tahyna virus, gave negative results with this system. Sensitivity tests showed that the limit of detection (LOD) of this replicon-defective assay is between 1 and 10 PFU for Sindbis viruses. These results indicate that, with the help of the replicon-defective alphavirus detection technique, we can specifically, sensitively, and rapidly detect alphaviruses in tissue cultures. The detection technique constructed here may be well suited for use in clinical examination and epidemiological surveillance, as well as for rapid screening of potential viral biological warfare agents.     

Packaging signals in alphaviruses.

Alphaviruses synthesize large amounts of both genomic and subgenomic RNA in infected cells, but usually only the genomic RNA is packaged. This implies the existence of an encapsidation or packaging signal which would be responsible for selectivity. Previously, we had identified a region of the Sindbis virus genome that interacts specifically with the viral capsid protein. This 132-nucleotide (nt) fragment lies within the coding region of the nsP1 gene (nt 945 to 1076). We proposed that the 132-mer is important for capsid recognition and initiates the formation of the viral nucleocapsid. To study the encapsidation of Sindbis virus RNAs in infected cells, we designed a new assay that uses the self-replicating Sindbis virus genomes (replicons) which lack the viral structural protein genes and contain heterologous sequences under the control of the subgenomic RNA promoter. These replicons can be packaged into viral particles by using defective helper RNAs that contain the structural protein genes (P. Bredenbeek, I. Frolov, C. M. Rice, and S. Schlesinger, J. Virol. 67:6439-6446, 1993). Insertion of the 132-mer into the subgenomic RNA significantly increased the packaging of this RNA into viral particles. We have used this assay and defective helpers that contain the structural protein genes of Ross River virus (RRV) to investigate the location of the encapsidation signal in the RRV genome. Our results show that there are several fragments that could act as packaging signals. They are all located in a different region of the genome than the signal for the Sindbis virus genome. For RRV, the strongest packaging signal lies between nt 2761 and 3062 in the nsP2 gene. This is the same region that was proposed to contain the packaging signal for Semliki Forest virus genomic RNA.     

Regulated Expression of a Sindbis Virus Replicon by Herpesvirus Promoters

We describe the use of herpesvirus promoters to regulate the expression of a Sindbis virus replicon (SINrep/LacZ). We isolated cell lines that contain the cDNA of SINrep/LacZ under the control of a promoter from a herpesvirus early gene which requires regulatory proteins encoded by immediate-early genes for expression. Wild-type Sindbis virus and replicons derived from this virus cause death of most vertebrate cells, but the cells discussed here grew normally and expressed the replicon and β-galactosidase only after infection with a herpesvirus. Vero cell lines in which the expression of SINrep/LacZ was regulated by the herpes simplex virus type 1 (HSV-1) infected-cell protein 8 promoter were generated. One Vero cell line (V3-45N) contained, in addition to the SINrep/LacZ cDNA, a Sindbis virus-defective helper cDNA which provides the structural proteins for packaging the replicon. Infection of V3-45N cells with HSV-1 resulted in the production of packaged SINrep/LacZ replicons. HSV-1 induction of the Sindbis virus replicon and packaging and spread of the replicon led to enhanced expression of the reporter gene, suggesting that this type of cell could be used to develop sensitive assays to detect herpesviruses. We also isolated a mink lung cell line that was transformed with SINrep/LacZ cDNA under the control of the promoter from the human cytomegalovirus (HCMV) early gene UL45. HCMV carries out an abortive infection in mink lung cells, but it was able to induce the SINrep/LacZ replicon. These results, and those obtained with an HSV-1 mutant, demonstrate that this type of signal amplification system could be valuable for detecting herpesviruses for which a permissive cell culture system is not available.     

An alphavirus replicon particle chimera derived from venezuelan equine encephalitis and sindbis viruses is a potent gene-based vaccine delivery vector

Alphavirus replicon particle-based vaccine vectors derived from Sindbis virus (SIN), Semliki Forest virus, and Venezuelan equine encephalitis virus (VEE) have been shown to induce robust antigen-specific cellular, humoral, and mucosal immune responses in many animal models of infectious disease and cancer. However, since little is known about the relative potencies among these different vectors, we compared the immunogenicity of replicon particle vectors derived from two very different parental alphaviruses, VEE and SIN, expressing a human immunodeficiency virus type 1 p55(Gag) antigen. Moreover, to explore the potential benefits of combining elements from different alphaviruses, we generated replicon particle chimeras of SIN and VEE. Two distinct strategies were used to produce particles with VEE-p55(gag) replicon RNA packaged within SIN envelope glycoproteins and SIN-p55(gag) replicon RNA within VEE envelope glycoproteins. Each replicon particle configuration induced Gag-specific CD8(+) T-cell responses in murine models when administered alone or after priming with DNA. However, Gag-specific responses varied dramatically, with the strongest responses to this particular antigen correlating with the VEE replicon RNA, irrespective of the source of envelope glycoproteins. Comparing the replicons with respect to heterologous gene expression levels and sensitivity to alpha/beta interferon in cultured cells indicated that each might contribute to potency differences. This work shows that combining desirable elements from VEE and SIN into a replicon particle chimera may be a valuable approach toward the goal of developing vaccine vectors with optimal in vivo potency, ease of production, and safety.     

Development of improved Sindbis virus-based DNA expression vector

We have constructed an improved DNA expression vector based on the Sindbis virus. Several DNA-based Sindbis virus vectors were constructed to investigate the efficiency of transgene expression. These vectors, when transfected into mammalian cells, have been used to express heterologous genes. A recombinant genome of Sindbis plasmid DNA, in which the structural genes were replaced by a polylinker cassette to allow for insertion of heterologous genes, was placed under the control of a simian virus (SV 40) promoter with a hepatitis delta virus (HDV) antigenomic ribozyme and a polyadenylation signal. Transfection of mammalian cells with this Sindbis-based plasmid vector, pSin-SV40-HDV-SV40pA, resulted in transient high-level expression of the beta-galactosidase reporter gene. The expression level of beta-galactosidase from pSin-SV40-HDV-SV40pA was more than 16-fold higher than that of pSin-Lux originally reported by Herweijer et al. In vivo expression was also detected after injection of plasmid DNA into mouse quadriceps. In vivo expression was transient and undetectable after day 14. Furthermore, we demonstrate that the transfection of cells with this Sindbis virus vector results in apoptotic death on glioma cells. We have demonstrated a high-level expression of the exogenous beta-galactosidase gene from the pSin-SV40-HDV-SV40pA construct using a Sindbis replication system.     

Translation Driven by Picornavirus IRES Is Hampered from Sindbis Virus Replicons: Rescue by Poliovirus 2A Protease

Alphavirus replicons are very useful for analyzing different aspects of viral molecular biology. They are also useful tools in the development of new vaccines and highly efficient expression of heterologous genes. We have investigated the translatability of Sindbis virus (SV) subgenomic mRNA bearing different 5′-untranslated regions, including several viral internal ribosome entry sites (IRESs) from picornaviruses, hepatitis C virus, and cricket paralysis virus. Our findings indicate that all these IRES-containing mRNAs are initially translated in culture cells transfected with the corresponding SV replicon but their translation is inhibited in the late phase of SV replication. Notably, co-expression of different poliovirus (PV) non-structural genes reveals that the protease 2A (2A^pro) is able to increase translation of subgenomic mRNAs containing the PV or encephalomyocarditis virus IRESs but not of those of hepatitis C virus or cricket paralysis virus. A PV 2A^pro variant deficient in eukaryotic initiation factor (eIF) 4GI cleavage or PV protease 3C, neither of which cleaves eIF4GI, does not increase picornavirus IRES-driven translation, whereas L protease from foot-and-mouth disease virus also rescues translation. These findings suggest that the replicative foci of SV-infected cells where translation takes place are deficient in components necessary to translate IRES-containing mRNAs. In the case of picornavirus IRESs, cleavage of eIF4GI accomplished by PV 2A^pro or foot-and-mouth disease virus protease L rescues this inhibition. eIF4GI co-localizes with ribosomes both in cells electroporated with SV replicons bearing the picornavirus IRES and in cells co-electroporated with replicons that express PV 2A^pro. These findings support the idea that eIF4GI cleavage is necessary to rescue the translation driven by picornavirus IRESs in baby hamster kidney cells that express SV replicons.     

Rubella virus capsid protein modulates viral genome replication and virus infectivity

The structural proteins (SP) of the Togaviridae can be deleted in defective interfering RNAs. The dispensability of viral SP has allowed construction of noninfectious viral expression vectors and replicons from viruses of the Alphavirus and Rubivirus genera. Nevertheless, in this study, we found that the SP of rubella virus (RUB) could enhance expression of reporter genes from RUB replicons in trans. SP enhancement required capsid protein (CP) expression and was not due to RNA-RNA recombination. Accumulation of minus- and plus-strand RNAs from replicons was observed in the presence of SP, suggesting that SP specifically affects RNA synthesis. By using replicons containing an antibiotic resistance gene, we found 2- to 50-fold increases in the number of cells surviving selection in the presence of SP. The increases depended significantly on the amount of transfected RNA. Small amounts of RNA or templates that replicated inefficiently showed more enhancement. The infectivity of infectious RNA was increased by at least 10-fold in cells expressing CP. Moreover, virus infectivity was greatly enhanced in such cells. In other cells that expressed higher levels of CP, RNA replication of replicons was inhibited. Thus, depending on conditions, CP can markedly enhance or inhibit RUB RNA replication.     

Antibody-directed targeting of retroviral vectors via cell surface antigens

Targeted stable transduction of specific cells is a highly desirable goal for gene therapy applications. We report an efficient and broadly applicable approach for targeting retroviral vectors to specific cells. We find that the envelope of the alphavirus Sindbis virus can pseudotype human immunodeficiency virus type 1- and murine leukemia virus-based retroviral vectors. When modified to contain the Fc-binding domain of protein A, this envelope gives a significant enhancement in specificity in combination with antibodies specific for HLA and CD4 relative to that without antibody. Unlike previous targeting strategies for retroviral transduction, the virus titers are relatively high and stable and can be further increased by ultracentrifugation. This study provides proof of principle for a targeting strategy that would be generally useful for many gene therapy applications.     

PKR-dependent and -independent mechanisms are involved in translational shutoff during Sindbis virus infection

The replication of Sindbis virus (SIN) profoundly affects the metabolism of infected vertebrate cells. One of the main events during SIN infection is the strong inhibition of translation of cellular mRNAs. In this study, we used a combination of approaches, including the study of SIN replication in PKR(-/-) mouse embryo fibroblasts or in the presence of an excess of catalytically inactive PKR. We show that the PKR-dependent inhibition of translation is not the only and most likely not the major pathway mediating translational shutoff during SIN infection. The PKR-independent mechanism strongly affects the translation of cellular templates, whereas translation of SIN subgenomic RNA is resistant to inhibition, and this leads to a benefit for viral replication. Our findings suggest that both PKR-dependent and non-PKR-dependent mechanisms of SIN-induced translational shutoff can be manipulated by using SIN replicons expressing mutated SIN nsP2 or kinase-defective PKR. Specifically, we show that expression of heterologous genes from SIN-based and most likely other alphavirus-based replicons can be increased by downregulating both the PKR-dependent and PKR-independent translational shutoffs.     

Development of dengue virus replicons expressing HIV-1 gp120 and other heterologous genes: a potential future tool for dual vaccination against dengue virus and HIV

Background  

Toward the goals of providing an additional vector to add to the armamentarium available to HIV vaccinologists and of creating a bivalent vaccine effective against dengue virus and HIV, we have attempted to create vectors which express dengue virus non-structural proteins and HIV immunogens. Previously we reported the successful construction of dengue virus replicons which lack structural genes necessary for virion release and spreading infection in culture but which can replicate intracellularly and abundantly produce dengue non-structural proteins. Here we attempted to express heterologous genetic material from these replicons.     

Release of virus-like particles from cells infected with poliovirus replicons which express human immunodeficiency virus type 1 Gag.

The effectiveness of attenuated poliovirus vaccines when given orally to induce both systemic and mucosal immune responses against poliovirus has resulted in an effort to develop poliovirus-based vectors to express foreign proteins. We have previously described the construction of poliovirus genomes (referred to as replicons) in which the complete human immunodeficiency virus type 1 (HIV-1) gag gene was substituted for the capsid gene (P1) (D.C. Porter, D.C. Ansardi, and C.D. Morrow, J. Virol. 69:1548-1555, 1995). Infection of cells with encapsidated replicons resulted in the expression of a 55-kDa protein. To further characterize the biological features of the HIV-1 Gag proteins expressed in cells infected with encapsidated replicons, we utilized biochemical analysis and electron microscopy. Expression of the 55-kDa protein in cells infected with encapsidated replicons resulted in myristylation of the Pr55gag protein. The Gag precursor protein was released from infected cells; analysis on sucrose density gradients revealed that the precursor sedimented at a density consistent with that of an HIV-1 virus-like particle. Analysis of replicon-infected cells by electron microscopy demonstrated the presence of condensed structures at the plasma membrane and the release of virus-like particles. These studies demonstrate that poliovirus-based vectors can be used to express foreign proteins which require posttranslational modifications, such as myristylation, and assemble into higher-order structures, providing a foundation for the future use of poliovirus replicons as vaccine vectors.     

Translation of Sindbis virus mRNA: analysis of sequences downstream of the initiating AUG codon that enhance translation.

Alphaviruses, particularly Sinbis virus and Semliki Forest virus, are proving to be useful vectors for the expression of heterologous genes. In infected cells, these self-replicating vectors (replicons) transcribe a subgenomic mRNA that codes for a heterologous protein instead of the structural proteins. We reported recently that translation of the reporter gene lacZ is enhanced 10-fold when the coding sequences of this gene are fused downstream of and in frame with the 5' half of the capsid gene (I. Frolov and S. Schlesinger, J. Virol. 68:8111-8117, 1994). The enhancing sequences, located downstream of the AUG codon that initiates translation of the capsid protein, have a predicted hairpin structure. We have mutated this region by making changes in the codons which do not affect the protein sequence but should destabilize the putative hairpin structure. These changes caused a decrease in the accumulation of the capsid-beta-galactosidase fusion protein. When these alterations were inserted into the capsid gene in the context of the intact Sindbis virus genome, they led to a decrease in the rate of virus formation but did not affect the final yield. We also altered the original sequence to one that has 12 contiguous G.C base pairs and should form a stable hairpin. The new sequence was essentially as effective as the original had been in enhancement of translation and in the rate of virus formation. The position of the predicted hairpin structure is important for its function; an insertion of 9 nucleotides or a deletion of 9 nucleotides decreased the level of translation. The insertion of a hairpin structure at a particular location downstream of the initiating AUG appears to be a way that alphaviruses have evolved to enhance translation of their mRNA, and, as a consequence, they produce high levels of the structural proteins which are needed for virus assembly. This high level of translation requires an intracellular environment in which host cell protein synthesis is inhibited.     

A Novel System for the Launch of Alphavirus RNA Synthesis Reveals a Role for the Imd Pathway in Arthropod Antiviral Response

Alphaviruses are RNA viruses transmitted between vertebrate hosts by arthropod vectors, primarily mosquitoes. How arthropods counteract alphaviruses or viruses per se is not very well understood. Drosophila melanogaster is a powerful model system for studying innate immunity against bacterial and fungal infections. In this study we report the use of a novel system to analyze replication of Sindbis virus (type species of the alphavirus genus) RNA following expression of a Sindbis virus replicon RNA from the fly genome. We demonstrate deficits in the immune deficiency (Imd) pathway enhance viral replication while mutations in the Toll pathway fail to affect replication. Similar results were observed with intrathoracic injections of whole virus and confirmed in cultured mosquito cells. These findings show that the Imd pathway mediates an antiviral response to Sindbis virus replication. To our knowledge, this is the first demonstration of an antiviral role for the Imd pathway in insects.     

Noncytopathic replication of Venezuelan equine encephalitis virus and eastern equine encephalitis virus replicons in Mammalian cells

Venezuelan equine encephalitis (VEE) and eastern equine encephalitis (EEE) viruses are important, naturally emerging zoonotic viruses. They are significant human and equine pathogens which still pose a serious public health threat. Both VEE and EEE cause chronic infection in mosquitoes and persistent or chronic infection in mosquito-derived cell lines. In contrast, vertebrate hosts infected with either virus develop an acute infection with high-titer viremia and encephalitis, followed by host death or virus clearance by the immune system. Accordingly, EEE and VEE infection in vertebrate cell lines is highly cytopathic. To further understand the pathogenesis of alphaviruses on molecular and cellular levels, we designed EEE- and VEE-based replicons and investigated their replication and their ability to generate cytopathic effect (CPE) and to interfere with other viral infections. VEE and EEE replicons appeared to be less cytopathic than Sindbis virus-based constructs that we designed in our previous research and readily established persistent replication in BHK-21 cells. VEE replicons required additional mutations in the 5' untranslated region and nsP2 or nsP3 genes to further reduce cytopathicity and to become capable of persisting in cells with no defects in alpha/beta interferon production or signaling. The results indicated that alphaviruses strongly differ in virus-host cell interactions, and the ability to cause CPE in tissue culture does not necessarily correlate with pathogenesis and strongly depends on the sequence of viral nonstructural proteins.     

Enhancement of sindbis virus self-replicating RNA vaccine potency by linkage of herpes simplex virus type 1 VP22 protein to antigen

Recently, self-replicating and self-limiting RNA vaccines (RNA replicons) have emerged as an important form of nucleic acid vaccines. Self-replicating RNA eventually causes lysis of transfected cells and does not raise the concern associated with naked DNA vaccines of integration into the host genome. This is particularly important for development of vaccines targeting proteins that are potentially oncogenic. However, the potency of RNA replicons is significantly limited by their lack of intrinsic ability to spread in vivo. The herpes simplex virus type 1 protein VP22 has demonstrated the remarkable property of intercellular transport and provides the opportunity to enhance RNA replicon vaccine potency. We therefore created a novel fusion of VP22 with a model tumor antigen, human papillomavirus type 16 E7, in a Sindbis virus RNA replicon vector. The linkage of VP22 with E7 resulted in a significant enhancement of E7-specific CD8+ T-cell activities in vaccinated mice and converted a less effective RNA replicon vaccine into one with significant potency against E7-expressing tumors. These results indicate that fusion of VP22 to an antigen gene may greatly enhance the potency of RNA replicon vaccines.