Rabies Virus Vector Transgene Expression Level and Cytotoxicity Improvement Induced by Deletion of Glycoprotein Gene

The glycoprotein (G) of rabies virus (RV) is required for binding to neuronal receptors and for viral entry. G-deleted RV vector is a powerful tool for investigating the organization and function of the neural circuits. It gives the investigator the ability to genetically target initial infection to particular neurons and to control trans-synaptic propagation. In this study we have quantitatively evaluated the effect of G gene deletion on the cytotoxicity and transgene expression level of the RV vector. We compared the characteristics of the propagation-competent RV vector (rHEP5.0-CVSG-mRFP) and the G-deleted RV vector (rHEP5.0-ΔG-mRFP), both of which are based on the attenuated HEP-Flury strain and express monomeric red fluorescent protein (mRFP) as a transgene. rHEP5.0-ΔG-mRFP showed lower cytotoxicity than rHEP5.0-CVSG-mRFP, and within 16 days of infection we found no change in the basic electrophysiological properties of neurons infected with the rHEP5.0-ΔG-mRFP. The mRFP expression level of rHEP5.0-ΔG-mRFP was much higher than that of rHEP5.0-CVSG-mRFP, and 3 days after infection the retrogradely infected neurons were clearly visualized by the expressed fluorescent protein without any staining. This may be due to the low cytotoxicity and/or the presumed change in the polymerase gene (L) expression level of the G-deleted RV vector. Although the mechanisms remains to be clarified, the results of this study indicate that deletion of the G gene greatly improves the usability of the RV vector for studying the organization and function of the neural circuits by decreasing the cytotoxicity and increasing the transgene expression level.     

Effect of the E4 region on the persistence of transgene expression from adenovirus vectors.

The utility of adenovirus vectors for gene therapy is limited by the transience of expression that has been observed in various in vivo models. Immunological responses to viral targets can eliminate transduced cells and cause the loss of transgene expression. We previously described the characterization of an E4 modified adenovirus, Ad2E4ORF6, which is replication defective in cotton rats. We reasoned that gene transfer vectors based on Ad2E4ORF6 would have a reduced potential for viral gene expression in vivo which might be beneficial for achieving persistence of transgene expression. E1 replacement vectors expressing the cystic fibrosis transmembrane regulator or beta-galactosidase were constructed as series of vectors that differed with respect to the E4 region. Vectors containing a wild-type E4 region, E4 open reading frame 6, or a complete E4 deletion were compared in the lungs of BALB/c mice for persistence of expression. Results obtained with nude mice indicate that nonimmunological factors have a major influence on the longevity of transgene expression. Expression was transient from the E1a promoter with all vectors but persisted from the cytomegalovirus promoter only with a vector containing a wild-type E4 region. Transience of expression did not correlate with the disappearance of vector DNA, suggesting that promoter down-regulation may be involved. Coinfection studies indicate an E4 product(s) could be supplied in trans to allow persistent expression from the cytomegalovirus promoter. In summary, the choice of promoter is important for achieving persistence of expression; in addition, some promoters are highly influenced by the context of the vector backbone.     

Activation of transgene expression by early region 4 is responsible for a high level of persistent transgene expression from adenovirus vectors in vivo.

The persistence of transgene expression has become a hallmark for adenovirus vector evaluation in vivo. Although not all therapeutic benefit in gene therapy is reliant on long-term transgene expression, it is assumed that the treatment of chronic diseases will require significant persistence of expression. To understand the mechanisms involved in transgene persistence, a number of adenovirus vectors were evaluated in vivo in different strains of mice. Interestingly, the rate of vector genome clearance was not altered by the complete deletion of early region 4 (E4) in our vectors. The GV11 (E1- E4-) vector genome cleared with a similar kinetic profile as the GV10 (E1-) vector genome in immunocompetent and immunocompromised mice. These results suggest that the majority of adenovirus vector genomes are eliminated from transduced tissue via a mechanism(s) independent of T-cell, B-cell, and NK cell immune mechanisms. While the levels of persistence of transgene expression in liver or lung transduced with GV10 and GV11 vectors expressing beta-galactosidase, cystic fibrosis transmembrane conductance regulator, or secretory alkaline phosphatase were similar in immunocompetent mice, a marked difference was observed in immunocompromised animals. Levels of transgene expression initially from both GV10 and GV11 vectors were the same. However, GV11 transgene expression correlated with loss of vector genome, while GV10 transgene expression persisted at a high level. Coadministration and readministration of GV10 vectors showed that E4 provided in trans could activate transgene expression from the GV11 vector genome. While transgene expression activity per genome from the GV10 vector is clearly activated, expression from a cytomegalovirus promoter expression cassette in a GV11 vector appeared to be further inactivated as a function of time. Understanding the molecular mechanisms underlying these expression effects will be important for developing persistent adenovirus vectors for chronic applications.     

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.     

Lentiviral vectors: optimization of packaging, transduction and gene expression

Gene transfer vectors based on retroviruses including oncogenic retroviruses and lentiviruses provide effective means for the delivery, integration and expression of exogenous genes in mammalian cells. Lentiviral (LV) vectors provide attractive gene delivery vehicles in the context of non-dividing cells. This review summarizes the different optimized LV genetic systems that have been developed to date. In all cases, the production of LV-derived vectors consists of a genetically split gene expression design. The viral elements that are specifically required are (i). the LV packaging helper proteins consisting of at least the gag-pol genes, (ii). the LV transfer vector RNA containing the transgene expression cassette, and (iii). an heterologous glycoprotein. While the genetic requirements and performances of the two former viral elements will be treated herein, the latter element relative to the envelope pseudotyping of LV vectors will not be further described (cf. review by Cosset in this issue).     

The histone deacetylase inhibitor sodium butyrate inhibits baculovirus-mediated transgene expression in Sf9 cells

Recent studies have indicated that histone deacetylase inhibitors (HDACis) could enhance and prolong expression of exogenous genes delivered by various viral vehicles in mammalian cells, including baculovirus vectors. In this study, the effects of HDACis on expression of a baculovirus-mediated eGFP reporter gene under control of baculovirus late promoter p10 in Sf9 cells were evaluated. It was found that sodium butyrate (NaBu) decreased the expression level of the target gene driven by p10 promoter by four to fivefold. Moreover, addition of NaBu increased DNaseI-sensitivity of transgene p10 promoter region and did not influence viral DNA replication. FACS assay has shown that both NaBu and fluorodeoxyuridine (FdUrd) blocked Sf9 cells at G1 phase and inhibited the target gene expression. Another HDACi, trichostatin, had little effects on both cell cycle and Ac-p10-eGFP expression, strongly suggesting that cell cycle arrest accounts for the mechanisms by which NaBu inhibits Ac-p10-eGFP expression. The inhibiting effects of NaBu on baculovirus transgene expression in Sf9 cells are promoter specific since the enhancement of NaBu on transgene expression in insect and mammalian cells are mediated by baculovirus harboring a murine cytomegalovirus (mCMV) immediate early promoter. This study was aimed at improving the productivity of the recombinant proteins and providing a better understanding of the epigenetic regulation of baculovirus gene expression.     

Chromosomal integration pattern of a helper-dependent minimal adenovirus vector with a selectable marker inserted into a 27.4-kilobase genomic stuffer

Helper-dependent minimal adenovirus vectors are promising tools for gene transfer and therapy because of their high capacity and the absence of immunostimulatory or cytotoxic viral genes. In order to characterize this new vector system with respect to its integrative properties, the integration pattern of a minimal adenovirus vector with a neo(r) gene inserted centrally into a noncoding 27.4-kb genomic stuffer element derived from the human X chromosome after infection of a sex chromosome aneuploid (X0) human glioblastoma cell line was studied. Our results indicate that even extensive homologies and abundant chromosomal repeat elements present in the vector did not lead to integration of the vector via homologous or homology-mediated mechanisms. Instead, integration occurred primarily by insertion of a monomer with no or little loss of sequences at the vector ends, apparently at random sites, which is very similar to E1 deletion adenovirus vectors. It is therefore unlikely that the incorporation of stuffer elements derived from human genomic DNA, which were shown to allow long-term transgene expression in vivo in a number of studies, leads to an enhanced risk of insertional mutagenesis. Furthermore, our findings indicate that the potential of minimal adenovirus vectors as tools for targeted insertion and gene targeting is limited despite the possibility of incorporating long stretches of homologous sequences. However, we found an enhanced efficiency of stable neo(r) transduction of the minimal adenovirus vector compared to an E1 deletion adenovirus vector, possibly caused by the absence of potential growth-inhibitory viral genes. Complete integration of the vector and tolerance of the integrated vector sequences by the cell might indicate a potential use of these vectors as tools for stable transfer of (large) genes.     

Hybrid vector designs to control the delivery, fate and expression of transgenes

One of the greatest challenges to gene therapy is the targetting of gene delivery selectively to the sites of disease and regulation of transgene expression without adverse effects. Ultimately, the successful realization of these goals is dependent upon improvements in vector design. Over the years, viral vector design has progressed from various types of replication-defective viral mutants to replication-conditioned viruses and, more recently, to 'gutted' and hybrid vectors, which have, respectively, eliminated expression of non-relevant or toxic viral genes and incorporated desired elements of different viruses so as to increase the efficacy of gene delivery in vivo. This review will focus on the different viral and cellular elements which have been incorporated into virus vectors to: improve transduction efficiencies; alter the entry specificity of virions; control the fate of transgenes in the host cells; and regulate transgene expression.     

Improved HSV-1 amplicon packaging using virion host shutoff mutants lacking mRNAse activity

Given their generous transgene capacity and inherent neurotropism, herpes simplex virus (HSV-1)-based viral vectors are promising tools for gene delivery to the central nervous system. Despite their widespread pre-clinical use, vector toxicity remains a concern with regard to the use of herpes vectors in humans. One potential source of toxicity stems from the tegument-associated virion host shutoff protein (vhs), which induces translational arrest in the host cell through non-specific mRNAse activity. In the current study we utilized a series of HSV-1 viruses containing a deletion in the U(L)41 open reading frame to investigate: (1) the requirement of intact vhs function in amplicon packaging and (2) whether vhs influences the post-transduction survival of dissociated cortical neurons. Our results demonstrate that while amplicon yield was reduced an order of magnitude, U(L)41 deletion was associated with reduced vector toxicity. Furthermore, partial reconstitution of vhs function using mRNAse-inactive point mutants improved amplicon titers without imparting the toxicity observed with wild-type controls. These findings offer a novel approach to improving the titer and toxicity profiles of HSV-based viral vectors.     

Tumor-specific gene expression in hepatic metastases by a replication-activated adenovirus vector

Clinical applications of tumor gene therapy require tumor-specific delivery or expression of therapeutic genes in order to maximize the oncolytic index and minimize side effects. This study demonstrates activation of transgene expression exclusively in hepatic metastases after systemic application of a modified first-generation (E1A/E1B-deleted) adenovirus vector (AdE1-) in mouse tumor models. The discrimination between tumors and normal liver tissue is based on selective DNA replication of AdE1- vectors in tumor cells. This new AdE1- based vector system uses homologous recombination between inverted repeats to mediate precise rearrangements within the viral genome. As a result of these rearrangements, a promoter is brought into conjunction with a reporter gene creating a functional expression cassette. Genomic rearrangements are dependent upon viral DNA replication, which in turn occurs specifically in tumor cells. In a mouse tumor model with liver metastases derived from human tumor cells, a single systemic administration of replication activated AdE1- vectors achieved transgene expression in every metastasis, whereas no extra-tumoral transgene induction was observed. Here we provide a new concept for tumor-specific gene expression that is also applicable for other conditionally replicating adenovirus vectors.     

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.