Alphavirus Vectors for Gene Therapy Applications

Introduction

Expression vectors for several members of alphaviruses were developed already a decade ago. The most commonly used vectors are based on the Semliki Forest virus (SFV) (Liljeström and Garoff, 1991), Sindbis virus (SIN) (Xiong et al., 1989) and Venezuelan Equine Encephalitis virus (VEE) (Davis et al., 1989). In principle, three different types of vectors have been constructed. I Replication-deficient vectors: RNA molecules containing the viral nonstructural genes (nsP1-4) and the foreign gene of interest are packaged into alphavirus particles with the aid of a helper vector containing the viral structural genes. The generated recombinant alphavirus particles are capable of infection of host cells, but because no viral structural genes are accommodated, no further virus replication occurs. The obtained transgene expression is therefore of a transient nature. II Replication-competent vectors: In contrast to the suicide vectors described above, these vectors contain a second subgenomic promoter and the foreign gene of interest added to the full-length alphavirus genome. Infection of host cells with replication-competent particles will obviously lead to virus replication. III Layered DNA-vectors: An RNA polymerase II expression cassette is introduced to drive the transcription of a self-amplifying RNA (replicon) vector, which allows direct use of plasmid DNA for transfection and expression studies (Berglund et al., 1996, Dubensky et al., 1996).

The generally favorable features of alphavirus vectors are: rapid production of high-titer virus, broad host range (including a variety of mammalian cell lines and primary cell cultures), high RNA replication rate in the cytoplasm and extreme transgene expression levels obtained. Typical disadvantages for alphavirus vectors are their short-term expression mode and their strong cytotoxic effects on host cells. On the other hand, both these properties can be considered as advantageous under certain circumstances, for example for some cancer gene therapy applications as well as for vaccine production.

In this review the use of alphavirus vectors as potential vehicles in gene therapy applications are discussed. Obviously, over-expression of anti-tumor and immunostimulatory genes is the most straightforward approach. Alphavirus vectors for direct intra-tumoral injections as well as targeted vectors for systemic delivery have been developed. Prophylactic vaccination with RNA and DNA vectors is a potential approach. Furthermore, SFV vectors have been employed for the co-expression of retrovirus gene products and retroviral genomes, which has allowed the assembly of retrovirus-like particles with relatively high titers. Much effort has been spent on vector development for broadening the application range of alphavirus vectors. The infection and spread of alphavirus vectors in the central nervous system (CNS) is well characterized and stands as a good basis for engineering of gene delivery vectors.

Gene Delivery to CNS

A prerequisite for successful gene therapy applications is efficient gene delivery and expression in vivo. Considerable work has been done to clarify the route of SFV infection in the CNS by intransal and peripheral administration of replication-competent virus. Remarkable differ-ences were obtained between virulent and avirulent SFV strains. The virulent SFV4 strain caused lethal encephelalitis in mice of all ages, whereas the avirulent SFV A7 was asymptomatic in adult mice, but lethal in neonatal mice (Fazakerley et al., 1993). The most likely explanation for this age-related virulence is the capability of virus production only in propagating neurons and not in mature ones (Oliver et al., 1998). Determination of the molecular basis of SFV virulence has revealed a long untranslated 3 ‘end region with multiple repeats in the avirulent SFV A7 strain missing from the virulent SFV4 strain, and also multiple amino acid substitutions (Glasgow et al., 1994, Santagati et al., 1994). Chimeric constructs between SFV4 and A7 suggested that the virulence determinants are polygenic (Atkins et al., 1999). Three regions appeared to be important: the E2 gene, the nsP3 gene and the 5’ end noncoding region (Tarbatt et al., 1997, Santagati et al., 1998). Recent studies have confirmed that specifically the nsP3 gene containing an opal termination codon and some other amino acid substitutions are most important for the change from a virulent to an avirulent phenotype (Tuittila et al., 2000). Introduction of suitable point mutations in the nsP3 gene could therefore be useful for the development of novel less neurovirulent SFV vectors.

Mice surviving infections with the avirulent SFV A7 strain show demyelination in special areas of the CNS, and provides therefore a model for human demyelating diseases such as multiple sclerosis (MS) (Atkins et al., 1994). Another demyelinating disease, experimental autoimmune encephalomyelitis (EAE) has also shown a correlation to SFV-infection (Mokhtarian and Swoveland, 1987). Using SFV as a model for disease will not only increase the understanding of the disease itself, but perhaps also help in designing gene therapy vectors for treatment.

Both SFV and SIN vectors have shown high reporter gene expression in rodent brain. Injection of replication-deficient SIN-LacZ virus resulted in transient local expression of b-galactosidase in mouse nucleus caudata/putamen and nucleus accumbens septi (Altman-Hamandzic et al., 1997). Likewise, injection of SFV-LacZ virus led to high reporter gene expression in rat striatum and amygdala (Lundstrom et al., 1999a). The transient nature of b-galactosidase expression became evident through histological examinations of stained brain slices as well as in situ hybridization. Maximal transgene expression was observed at 1-2 days post-injection followed by decrease with time. Interestingly, injection of 10⁵ infectious SFV-LacZ particles resulted in no behavioral changes compared to control animals when monitored for general health (food intake, body weight, body temperature), sensorimotor function, exploratory behavior and muscle strength. Only minor virus-induced inflammation was observed at the injection site. Typically, injections into rat brain resulted in highly neuron-specific expression, which was also the case with alphavirus injections into organotypic hippocampal slice cultures (Ehrengruber et al., 1999). No major cell toxicity could be seen after SFV-mediated neuronal gene transfer in vivo.

Tumor Cell Lines and Tumor Models in Animals

Due to their broad host range alphavirus vectors can naturally efficiently infect many different tumor cell lines. Recently, it was shown that several human prostate tumor cell lines demonstrated high levels of SFV-mediated b-galactosidase expression. Moreover, tumor cells infected with SFV-LacZ virus were subjected to a strong induction of apoptosis (Hardy et al., 2000). Additionally, infection of human prostate tissue ex vivo led to high expression of b-galactosidase, mainly in duct epithelial cells and could therefore be promising for treatment of prostate tumors.

SFV vectors have recently been tested in animal tumor models. The genes for the p40 and p35 subunits of interleukin-12 (IL-12) were introduced into the same SFV vector using two subgenomic 26S promoters in tandem, which resulted in high IL-12 activity in cell lines (Zhang et al., 1997). This SFV-IL-12 vector was then injected into mice with B16 melanoma tumors and monitored for tumor regression by Doppler ultrasonography (Asselin-Paturel et al., 1999). Significant tumor regression and inhibition of tumor blood vessel formation was detected already after a single intratumoral injection. Repeated injections resulted in improved anti-tumor effect. Most encouragingly, repeated injections showed no toxicity in the treated animals and did not elicit any SFV-related antibody response. In another study, nude mice with implanted human lung carcinomas were monitored for tumor shrinkage after intratumoral injections with SFV vectors (Murphy et al., 2000). It could be demonstrated that SFV-LacZ, SFV-GFP as well as empty SFV particles (containing only the SFV nonstructural genes and no foreign gene) induced p53-independent apoptosis. It was also evident that the best results for tumor regression were obtained after repeated viral injections according to a scheme of 3 injections on consecutive days followed by another set of 3 injections one week later. Again, no antiviral response was detected in the injected animals.

Targeted Alphavirus Vectors

To further improve the alphavirus vectors for cancer therapy, the question of the broad host range needs to be addressed. Additionally, the high preference of infection of neurons in hippocampal slice cultures (Ehrengruber et al., 1999) as well as in rat brain (Lundstrom et al., 1999a) is of a major concern for applications of these vectors to the CNS. One approach to target alphavirus vectors has been the introduction of IgG binding domains of protein A into the SIN envelope protein E2. This resulted in a substantially modified host range of the chimeric SIN virus (Ohno et al., 1997). For instance, the infection rate of BHK cells was reduced 10⁵-fold. At the same time, it was demonstrated that treatment of cells with monoclonal antibodies (mAbs) directed against surface receptors/markers allowed binding of chimeric SIN particles through the protein A domains. Targeted infection of host cells could also be achieved by the introduction of a- and b-hCG gene sequences into the SIN envelope, where no infection of BHK cells nor human cancer cells lacking LH/CG receptors occurred, while choriocarcinoma cells showed high infection rates (Sawai and Meruelo, 1998). SIN vectors have also been used to in vitro transcribe biotinylated and self-replicating SIN genomic RNA with streptavidin-protein A fusion protein and mAbs and could in the presence of cationic liposomes result in specific transfection of cancer cells in a mAb dose-dependent manner (Sawai et al., 1998).

Vaccination with RNA/DNA Vectors

The use of alphavirus vectors for vaccine production has been reviewed in detail elsewhere (Leitner et al., 1999, Lundstrom, 2001) and is therefore only briefly summarized here. Several studies conducted with alphavirus particles have shown that efficient immune responses could be elicited. Specifically, intravenous injection of SFV particles expressing influenza nucleoprotein (NP) into BALB/c mice resulted in high antibody responses (Zhou et al., 1994). Another study demonstrated that as few as 100 SFV-NP particles were sufficient to induce a strong cytotoxic T cell (CTL) response (Zhou et al., 1995). To avoid the use of infectious, albeit replication-deficient virus particles, naked RNA molecules as well as layered DNA vectors have been applied successfully for vaccination approaches (reviewed by Leitner et al., 1999, Lundstrom, 2001). Injection of naked SFV RNA containing the influenza NP gene into the quadriceps muscle of mice led to strong humoral responses (Zhou et al., 1994). More related to gene therapy applications, it was demonstrated that a single intramuscular injection of SFV-LacZ RNA prolonged the survival time of mice with established tumors and even protected mice from tumor challenge (Ying et al., 1999). SFV-mediated expression of the P1A gene led to P185 tumor immunity (Colmenero et al., 1999) and immunization with SFV particles expressing the human papillomavirus E6 and E7 early genes protected mice from cervical cancer challenge (Daemen et al., 2000).

The sensitivity of self-replicating, layered DNA vectors indicated that to achieve antigene-specific responses 1,000-fold lower DNA concentrations were required compared to conventional DNA vectors (Berglund et al., 1998). For instance, significant immune responses against herpes simplex virus glycoprotein B were achieved with a single dose of 10 ng SIN-HSVgB plasmid (Hariharan et al., 1998).

Generation of Retrovirus Particles from SFV Vectors

Indirect approaches in the context of gene therapy have been to employ SFV vectors for the production of retrovirus-like particles. This was achieved by co-transfection of gag-pol, env and LTR-y⁺-neo-LTR constructs for Moloney murine leukemia virus (MMLV) from three separate SFV vectors into BHK cells. The generated extracellular virus-like particles showed relatively high titers and possessed reverse-transcriptase activity (Li and Garoff, 1996). Furthermore, this retrovirus packaging technology allows the use of constructs containing intron sequences (Li and Garoff, 1998). The advantage of this method is the rapid and simple helper-free virus production procedure, which should allow efficient production of virus with amphotropic as well as ecotropic envelopes.

In another approach, SFV-mediated cyto-plasmic synthesis of retrovirus vector RNA was applied. Retrovirus virion RNA was cloned downstream of the SFV 26S subgenomic promoter, full-length chimeric SFV-retrovirus RNA was transcribed in vitro and introduced into retrovirus packaging cell lines by electroporation or SFV infection to generate fully functional viral particles (Wahlfors et al., 1997). These retroviral particles could transduce target cells, demonstrated reverse transcriptase activity and had the capacity to integrate into the host cell genome. Furthermore, minigene-containing retroviral vectors were produced using an alphavirus/retrovirus hybrid vector system (Wahlfors and Morgan, 1999). When the Phoenix retroviral packaging cell line was infected with alphavirus/retrovirus particles, cytoplasmically produced factor IX minigene-containing retroviral vectors were generated. Transduction of TE671 cells resulted in stable transfer of the minigene and factor IX expression. In another approach, the envelope protein genes of SFV were replaced with the env gene from murine leukemia virus (MuLV), which led to packaging of minimal virus particles capable of specifically infecting cells carrying MuLV receptors (Lebedeva, et al., 1997).

Vector Development

Although alphavirus vectors have turned out to be efficient for many gene therapy applications as described above, there has naturally been some limitations, particularly related to host cell toxicity and the transient nature of expression. To address these questions, novel non-cytopathogenic vectors have been constructed for both SIN (Agapov et al., 1998) and SFV (Lundstrom et al., 1999b). In the case of the SIN vector it was demonstrated that a single point mutation in the nsP2 gene (Pro726Ser) was responsible for the non-cytopathogenic phenotype. However, the RNA replication of this vector was significantly impaired and the phenotype restricted only to few cell lines. Molecular analysis of a less virulent SFV strain pointed to a single amino acid substitution in the nuclear localization signal (NLS) of the nsP2 gene at position 649, which retained the nsP2 protein in the cytoplasm and caused less damage to host cell DNA synthesis (Rikkonen, 1996). Introduction of this mutation (Arg649Asp) into the SFV expression vector resulted in a less cytotoxic phenotype. However, mutagenesis of the adjacent amino acid residue (Arg650Asp) in combination with another point mutation in the nsP2 gene (Ser259Pro) generated a vector with dramatically increased transgene expression and substantially prolonged survival of host cells compared to the conventional SFV vector (Lundstrom et al., 1999b). The non-cytopathogenic phenotype was not restricted to a limited number of cell lines, but present in all cell lines tested (BHK, CHO, HeLa, HEK293 cells), so far, as well as in primary hippocampal neurons in culture. The titers of recombinant SFV particles generated from the double-mutant vector were approximately 10-fold lower than for the conventional SFV vector, but lowering the virus production temperature to 31^oC resulted in only slightly lower titers.

Another point recently addressed for both SIN and SFV vectors was the possibility to prolong the duration of expression. Constructs containing randomly mutagenized replicon (nsP1-4 genes) and the neo gene were transfected into BHK cells followed by selection of G418-resistant clones with prolonged expression. Characterization of the isolated clones revealed SIN and SFV vectors with either point mutations or deletions in the nsP2 gene (Perri et al., 2000). These novel vectors demonstrated a persistent replication in host cells and should therefore be valuable tools for establishment of prolonged expression.

Interestingly, both the puromycin selection that led to a non-cytopathogenic Sindbis mutant (Agapov et al., 1998) and the G418 selection from which several replication-persistent SFV and Sindbis vectors were isolated (Perri et al., 2000) all were located in the nsP2 gene. Logically, it would make sense to carry out mutagenesis studies on the nsP3 gene, especially based on the differences seen in virulence between SFV4 and A7 strains that correlate with amino acid differences in the nsP3 gene (Tuittila et al., 2000).

An area of significant interest with potential relevance for future gene therapy applications is the development of temperature-sensitive alphavirus vectors. A tightly temperature-regulated SIN vector demonstrated expression of the highly toxic apoptosis inducing death domain of the receptor interacting protein (RipDD) only after induction at 29^oC, with no expression and high cell viability at the non-permissive temperature of 37^oC (Boorsma et al, 2000). In a similar way, temperature-sensitive SFV vectors have been developed (Lundstrom et al., 2001). Introduction of two additional point mutations to the ones described for the non-cytopathogenic SFV vector above, resulted in a novel SFV vector with both a non-cytopathogenic and temperature-sensitive phenotype. This vector resulted in further increased reporter gene expression levels.

Comparison to other Viral Vectors

Many viral vectors have been subjected to gene therapy applications in animal models and even clinical trials. The comparison between gene delivery efficacy and therapeutic success from the different vectors would require a review of its own. Only a brief overview of some applications of other viral vectors and a comparison to alphavirus vectors is presented here. Needless to say, many viral delivery systems, like retrovirus and adenovirus vectors, have been the targets for intensive research for years, whereas alphaviruses must be considered as newcomers in the field.

Concerning the capacity to accommodate foreign genes, alphavirus vectors are relatively good compared to lentivirus and adeno-associated virus (AAV) with a size restriction of approximately 4 kb. It is possible to introduce at least 7 kb inserts, which means that several genes, either under separate subgenomic promoters (Zhang et al., 1998) or Internal Ribosomal Entry Site (IRES) sequences, can be inserted. The generation of recombinant SFV particles is extremely rapid. High-titer virus stocks (10⁹-10¹⁰ infectious particles / ml) can be produced within two days and no further purification or concentration is required. This is an advantage compared to the more time consuming and labor-intensive methods and the relatively low yields obtained for retrovirus, lentivirus and AAV vectors.

For most viral vectors high efficacy has been achieved in different gene therapy approaches. A replication-defective adenovirus vector was used to express the herpes simplex virus thymidine kinase gene (HSV-tk) in combination with ganciclovir (GCV) treatment in an orthopic murine bladder cancer model (Sutton et al., 2000). Direct transvesical injection of 5 x 10⁸ pfu showed a good distribution of the marker protein b-galactosidase and a 2-fold reduction in tumor growth. In another study intratumoral injection of an adenovirus vector expressing IL-2 in a rat CC531 model for hepatic metastases of colorectal cancer led to cessation of tumor growth in 80% of the injected tumors (Geutskens et al., 2000). Immunoreactivity problems encountered for adenovirus vectors were reported in a study where high doses of adenovirus expressing the HSV-tk gene injected into the cerebrospinal fluid (CSF) of rats and non-human primates showed signs of viral meningitis (Driesse et al., 2000). AAV vectors were tested for in vitro and in vivo gene delivery to healthy normal mouse knee or arthritic knee in transgenic mice overexpressing TNFa (Goater et al., 2000). The transduction efficiency was very low in normal knee, whereas in diseased animals the expression was 10-fold higher and correlated with the joint damage. In a murine model for hereditary tyrosinemia type 1 (HTI) AAV expressing fumarylacetoacetate hydrolase was transduced into a limited number of hepatocytes (Chen et al., 2000). The AAV containing hepatocytes could be expanded in vivo by withdrawal of the drug that prevented the accumulation of toxic metabolites, which resulted in repopulation of hepatocytes with a functional and apparently integrated AAV provirus. This type of long-term gene delivery is obviously not possible with alphavirus vectors, at least in their present form. Herpes simplex virus (HSV) vectors were applied to a nude mouse model of human glioblastoma in combination with gamma-knife surgery (Niranjan et al., 2000). Intratumoral injections of 2 x 10⁶ infectious HSV particles expressing HSV-tk and TNFa combined with GCV treatment and radiosurgery showed survival of 8 out of 9 animals for 75 days. Pseudotyped Moloney leukemia murine leukemia virus expressing HSV-tk showed an improved transduction frequency and a 5- to 10-fold higher sensitivity to GCV (Howard et al., 2000).

Lentiviruses have shown great promise also in other gene therapy applications than tumor targeting. Injection of lentivirus expressing GDNF into striatum and substantia nigra resulted in GDNF augmented dopaminergic function in aged rhesus monkeys (Kordower et al., 2000). In MPTP-treated animals functional deficits were reversed and the nigrostriatal degradation completely prevented. Long-term GDNF expression (8 months) was achieved in rhesus monkeys. In another study lentivirus-based GDNF expression promoted regeneration and functional recovery in 6-OHDA-lesioned rats and MPTP-lesioned monkeys (Bjorklund et al., 2000). Recently, a non-primate lentivirus vector, unable to replicate in human cells, was developed as an alternative to using HIV-based lentivirus vectors (Mitrophanous, et al., 1999). The expression vector based on the equine infectious anaemia virus (EIAV) showed comparable transduction efficiency to HIV-based vectors in the rat CNS.

In conclusion, alphavirus vectors fair favorably in relation to ease and quantity of virus production, insertion capacity, host range and gene delivery. However, in their present form, alphavirus vectors demonstrate a highly transient expression pattern, which does not allow for long-term expression. On the other hand, independent studies have indicated that immunogenic responses against SFV are not generated after repeated injections.

Discussion and Future Aspects

As described above, there are clear indications that alphavirus vector are potentially useful for various gene therapy applications (Table 1). Promising results, monitored as tumor regression, have already been obtained in two independent studies in tumor animal models. The absence of immune response against SFV even after repeated injections is highly encouraging. The vaccination approach with alphavirus particles, naked RNA or layered DNA vectors is also interesting both from a therapeutic and a prophylactic point of view. Additionally, employing SFV vectors for generation of retrovirus-like particles is an attractive approach to facilitate vector production.

The use of viral vectors for clinical trials and future drug development requires naturally assurance of achieving the highest possible biosafety standards. This has been possible by using attenuated alphavirus strains, in most cases replication-deficient vectors and for vaccine applications either naked RNA or layered DNA vectors. In the case of SFV vectors, the introduction of three point mutations in the p62 precursor (for E2 and E3 envelope protein genes) in the second-generation helper vector led to production of only conditionally infectious recombinant particles (Berglund et al., 1993). This modification prevented the amplification of replication proficient viruses (RPVs) generated through homologous recombination between recombinant RNA and helper RNA in virus producing cells. To further increase the biosafety level of SFV vectors a split two-helper vector system was developed (Smerdou and Liljeström, 1999). By splitting the SFV capsid and envelope protein genes on two separate helper vectors the recombination events became negligible. In order to further reduce the probability of generating RPVs a point mutation was introduced into the capsid sequence (Ser219Ala) to prevent the self-cleaving activity. Combining the split two-helper vector system with this point mutation resulted in a packaging system with a theoretical RPV generation frequency of less than 4 x 10^-17. An important development that will significantly facilitate virus production under GMP conditions and the use of alphavirus vectors in future clinical trials is the construction of a packaging cell line. BHK cells stably expressing the SIN structural proteins can be transfected or infected with both SIN and SFV vectors for the production of recombinant alphavirus particles (Polo et al., 1999). So far, the titers have been at least one magnitude lower than what can routinely be achieved from packaging with helper vectors, but further development should resolve this problem.

Based on recent achievements it looks like the novel non-cytopathogenic alphavirus vectors have efficiently addressed the problem with host cell cytotoxicity. These modifications have also opened up completely new avenues for applications of alphavirus vectors. It is now possible to start applying these vectors for antisense and ribozyme technologies. Infection with conventional alphavirus vectors led to a rapid shut down of host cell protein synthesis and made it impossible to distingusih between this phenomenon and an antisense effect. Due to the rapid cytoplasmic RNA replication, these novel vectors should therefore be attractive for experiments targeted to specifically inhibit mRNA translation.

The transient nature of alphavirus-mediated gene expression has often been considered as a disadvantage. Indeed, this is a problem if long-term expression is desired. It needs to be analyzed, whether the novel replication-persistent SIN and SFV vectors can substantially extend the duration of expression and allow the use of alphavirus vectors also for long-term expression. On the other hand, it is under certain circumstances advan-tageous to obtain transient expression. In some therapeutic areas a short-term expression is enough to reach efficacy. Additionally, application of the vector in the form of RNA with relatively short half-life and no capacity of chromosomal integration increases the biosafety standards significantly.

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