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PubMed1996

Plasmoviruses: nonviral/viral vectors for gene therapy.

We have generated a chimeric gene transfer vector that combines the simplicity of plasmids with the infectivity and long-term expression of retroviruses. We replaced the env gene of a Moloney murine leukemia virus-derived provirus by a foreign gene, generating a plasmid that upon transfer to tumor cells generates noninfectious retroviral particles carrying the transgene. We added to this plasmid an independent expression cassette comprising a cytomegalovirus promoter, an amphotropic retroviral envelope, and a polyadenylylation signal from simian virus 40. These constructs were designed to minimize the risk of recombination generating replication-competent retroviruses. Their only region of homology is a 157-bp sequence with 53% identity. We show that the sole transfection of this plasmid in various cell lines generates infectious but defective retroviral particles capable of efficiently infecting and expressing the transgene. The formation of infectious particles allows the transgene propagation in vitro. Eight days after transfection in vitro, the proportion of cells expressing the transgene is increased by 10-60 times. There was no evidence of replication-competent retrovirus generation in these experiments. The intratumoral injection of this plasmid, but not of the control vector lacking the env gene, led to foci of transgene-expressing cells, suggesting that the transgene had propagated in situ. Altogether, these "plasmoviruses" combine advantages of viral and non-viral vectors. They should be easy to produce in large quantity as clinical grade materials and should allow efficient and safe in situ targeting of tumor cells.

3T3 Cells↗
PubMed2006

Mutations in the palm region of a plus-strand RNA virus polymerase result in attenuated phenotype.

The three-dimensional structure of RNA-dependent RNA polymerases (RdRps) is highly conserved among RNA viruses. In a previous study, a unique set of mutant strains of Bovine viral diarrhea virus was obtained, encompassing either a genomic deletion of six codons or duplications of between 1 and 45 codons; these mutations affect different parts of the palm region, the most conserved part of RdRps containing the catalytic centre. In the present study, a detailed characterization of the RdRp mutant viruses was performed, demonstrating different degrees of a small-plaque phenotype in cell culture, correlating with significantly reduced viral RNA synthesis and delayed virus replication. Taken together, the results of this study demonstrate a surprising flexibility within the palm region of a plus-strand RNA virus RdRp, resulting in viral attenuation in vitro. This interesting insight into an essential viral protein may have implications for the development of vaccines and attenuated viral vectors.

Animals↗
PubMed2004

Design and expression of chimeric U1/ribozyme transgenes.

The U1snRNA/ribozyme/antisense construct (designated U1/ribozyme) is a chimeric transgene that has proven to be very useful for inhibiting the expression of targeted genes in vitro and in vivo. It consists of a combination of hammerhead ribozyme flanked by target-specific antisense sequences that are in turn flanked by the loops and promoter of U1 snRNA. To construct U1/ribozymes, antisense/ribozyme sequences are first designed corresponding to ribozyme-cleavage consensus "GUC" sequences that are present in the targeted mRNA. Antisense/ribozyme sequences are then inserted between the U1 snRNA loops, and the conceptual secondary structure of the encoded regulatory RNA is analyzed to ensure proper folding. Appropriate antisense/ribozymes are then synthesized as oligonucleotides, annealed, and ligated into the pU1 vector containing the U1 snRNA promoter and loops to yield the pU1/ribozyme expression vector. Constructs can then be transiently or stably expressed in vitro and in vivo to inhibit the expression of target genes. U1/ribozymes can also be expressed in viral vectors for more efficient transfection, or complexed to liposomes for systemic delivery.

Animals↗
PubMed2003

[Gene therapy of pain].

Possible practical applications of gene therapy for pain are at the beginning. Very intensive research on animal models has been effectuated and some partial results of clinical applications in men already exist. The article describes different possibilities of these applications, namely the application of viral vectors and antisense nucleotides as well as the first experimental results of possible gene therapy. BDNF (brain derived nervous factor), herpes simplex virus, cytokine-interleukin 10, viral mediated transfer of the decarboxylase of the glumatic acid and down--regulated genes are recently used. Experimation of beta endorphin is also possible. In the article describes also possible molecular and gene therapy of pain and the molecular neurosurgical approach to the treatment of pain, which is called the molecular neurosurgery. From all these results it is possible to conclude, that gene therapy will be definitely used for the treatment of pain and that only the intensive research in this field may bring some important results.

Animals↗
PubMed2003

Improved gene transfer selectivity to hepatocarcinoma cells by retrovirus vector displaying single-chain variable fragment antibody against c-Met.

Engineered retroviruses are widely used vectors for cancer gene therapy approaches. However, the ability to target cells of therapeutic interest while controlling the expression of the transferred genes would improve both the efficiency and the safety of viral vectors. In this study, we investigated the ability of a retroviral amphotropic envelope displaying single-chain variable-fragment (scFv) directed against the c-Met receptor, to target the entry of recombinant retroviruses to human hepatocarcinoma cells. Four single-chain antibody fragments directed against the c-Met receptor were generated and inserted into the viral envelope protein as an N-terminal fusion. The modified envelopes were incorporated into virus particles and one of the chimeric viruses, 3D6-Env, transduced preferentially human hepatoma cells rather than proliferating human hepatocytes. In another construct, the urokinase cleavage site was inserted between the scFv moiety and the envelope. Chimeric scFv-urokinase-Env viruses transduced hepatoma cells with a similar efficiency to that of the control virus and their infectivity in human hepatocytes remained low. These results indicate that amphotropic retroviruses with engineered envelopes to display scFv directed against the c-Met receptor can efficiently and selectively deliver genes into hepatoma cells.

Antibodies↗
PubMed2003

Distribution of adenoviral vector in brain after intravenous administration.

The delivery of transgenes to the central nervous system (CNS) can be a valuable tool to treat CNS diseases. Various systems for the delivery to the CNS have been developed; vascular delivery of viral vectors being most recent. Here, we investigated gene transfer to the CNS by intravenous injection of recombinant adenoviral vectors, containing green fluorescence protein (GFP) as a reporter gene. Expression of GFP was first observed 6 days after the gene transfer, peaked at 14 days, and almost diminished after 28 days. The observed expression of GFP in the CNS was highly localized to hippocampal CA regions of cerebral neocortex, inferior colliculus of midbrain, and granular cell and Purkinje cell layers of cerebellum. It is concluded that intravenous delivery of adenoviral vectors can be used for gene delivery to the CNS, and hence the technique could be beneficial to gene therapy.

Adenoviruses, Human↗
PubMed2003

Large-scale production of pseudotyped lentiviral vectors using baculovirus GP64.

Unlike oncoretroviruses, lentiviral vectors can insert large genes and can target both dividing and nondividing cells; thus they hold unique promise as gene transfer agents. To enhance target range, the native lentiviral envelope glycoprotein is replaced (pseudotyped) with vesicular stomatitis virus G (VSVG), and the genes of interest are packaged in nonreplicating vectors by transient transfection with three plasmids. However, because of cytotoxic effects of VSVG expression in producer cells (293T cells) it has been difficult to generate a packaging cell line, required for even modest scale-up of vector production. Here we introduce a pseudotyped lentivirus vector using the baculovirus GP64 envelope glycoprotein. Compared with VSVG, GP64 vectors exhibited a similar broad tropism and similar native titers. GP64-pseudotyped vectors were usually highly concentrated without much loss of titer. Because, unlike VSVG, GP64 expression does not kill cells, we generated 293T-based cell lines constitutively expressing GP64. Our results demonstrate that the baculovirus GP64 protein is an attractive alternative to VSVG for viral vectors used in the large-scale production of high-titer virus required for clinical and commercial applications.

Animals↗
PubMed2004

The potential of virus-induced gene silencing for speeding up functional characterization of plant genes.

Virus-induced gene silencing (VIGS) has been shown to be of great potential in plant reverse genetics. Advantages of VIGS over other approaches, such as T-DNA or transposon tagging, include the circumvention of plant transformation, methodological simplicity and robustness, and speedy results. These features make VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is already well established in Nicotiana benthamiana; however, efforts are being made to improve VIGS in other species, including monocots. Current research is focussed on unravelling the mechanisms of post-transcriptional gene silencing and VIGS, as well as on finding novel viral vectors in order to broaden the host species spectrum. We examined how VIGS has been used to assess gene functions in plants, including molecular mechanisms involved in the process, available methodological elements, such as vectors and inoculation procedures, and we looked for examples in which the system has been applied successfully to characterize gene function in plants.

DNA, Viral↗
PubMed2000

Conditionally replicative adenoviral vectors for cancer gene therapy.

During the past century, many attempts have been made to exploit the ability of some viruses to infect and destroy cancer cells. Crippled, non-replicative viruses have been used as vectors to transfer genes into tumours. Both strategies have serious limitations. The time is now ripe, however, for full convergence of these two research tracks. On the one hand, the intratumoral propagation of replicative viruses would overcome the low levels of gene transfer achieved by current viral vectors. On the other hand, the versatility provided by vectors encoding foreign genes, which are limited in their uses only by our ingenuity, would overcome the physiological barriers to robust propagation of the viral progeny in the tumour. This empowering synthesis will provide truly new opportunities that might realise the promises of gene transfer for the therapy of cancer.

Adenoviridae↗
PubMedDate not supplied

Ca2+-sensitive cytosolic nucleases prevent efficient delivery to the nucleus of injected plasmids.

BACKGROUND: Efficient gene delivery by synthetic vectors is a major challenge in gene therapy. However, inefficient nuclear delivery of cDNA is thought to be a major limiting step in gene transfer using non-viral vectors. It is commonly thought that, in the cytosol, cDNA has to be released from its vector before importation to the nucleus. The stability of naked cDNA in the cytoplasm is not well established. METHODS: cDNA plasmids, either free or complexed with poly(ethyleneimine) (PEI), were microinjected into the cytoplasm of mammalian cells and their turnover was assessed by fluorescence in situ hybridization (FISH). Incubations of cDNA plasmids in cytosolic extracts were also performed. RESULTS: FISH experiments showed that naked cDNA rapidly fade with time when injected into the cytosol. Fading was not observed when naked cDNA plasmids were injected into the nucleus. Incubation of naked cDNA in a cytosolic fraction isolated from mammalian cells reproduced cDNA degradation as observed in microinjection experiments. Nuclease inhibitors, including aurin tricarboxylic acid or Zn2+, prevented in vitro cDNA degradation. The cytosolic nuclease activity was optimal at physiological pH and physiological Ca2+ concentration. By contrast, it was insensitive to Mg2+ or Na+ concentrations. Finally, cDNA complexation with PEI or addition of oligonucleotides prevented in vitro cDNA degradation. CONCLUSION: Altogether, these experiments suggest that cDNA digestion by cytosolic nucleases occur when the decomplexed transgene is present in the cytosol. We propose that the inefficient transfer of cDNA into the nucleus during transfection with synthetic vectors may result from rapid digestion of naked cDNA by a Ca2+-sensitive cytosolic nuclease.

Animals↗
PubMed2005

In vivo application of RNA interference: from functional genomics to therapeutics.

RNAi has rapidly become a powerful tool for drug target discovery and validation in cell culture, and now has largely displaced efforts with antisense and ribozymes. Consequently, interest is rapidly growing for extension of its application to in vivo systems, such as animal disease models and human therapeutics. Studies on RNAi have resulted in two basic methods for its use for gene selective inhibition: 1) cytoplasmic delivery of short dsRNA oligonucleotides (siRNA), which mimics an active intermediate of an endogenous RNAi mechanism and 2) nuclear delivery of gene expression cassettes that express a short hairpin RNA (shRNA), which mimics the micro interfering RNA (miRNA) active intermediate of a different endogenous RNAi mechanism. Non-viral gene delivery systems are a diverse collection of technologies that are applicable to both of these forms of RNAi. Importantly, unlike antisense and ribozyme systems, a remarkable trait of siRNA is a lack of dependence on chemical modifications blocking enzymatic degradation, although chemical protection methods developed for the earlier systems are being incorporated into siRNA and are generally compatible with non-viral delivery systems. The use of siRNA is emerging more rapidly than for shRNA, in part due to the increased effort required to construct shRNA expression systems before selection of active sequences and verification of biological activity are obtained. In contrast, screens of many siRNA sequences can be accomplished rapidly using synthetic oligos. It is not surprising that the use of siRNA in vivo is also emerging first. Initial in vivo studies have been reported for both viral and non-viral delivery but viral delivery is limited to shRNA. This review describes the emerging in vivo application of non-viral delivery systems for RNAi for functional genomics, which will provide a foundation for further development of RNAi therapeutics. Of interest is the rapid adaptation of ligand-targeted plasmid-based nanoparticles for RNAi agents. These systems are growing in capabilities and beginning to pose a serious rival to viral vector based gene delivery. The activity of siRNA in the cytoplasm may lower the hurdle and thereby accelerate the successful development of therapeutics based on targeted non-viral delivery systems.

Animals↗
PubMed2003

Chromosome-based vectors for gene therapy.

Currently used vectors in human gene therapy suffer from a number of limitations with respect to safety and reproducibility. There is increasing agreement that the ideal vector for gene therapy should be completely based on chromosomal elements and behave as an independent functional unit after integration into the genome or when retained as an episome. In this review we will first discuss the chromosomal elements, such as enhancers, locus control regions, boundary elements, insulators and scaffold- or matrix-attachment regions, involved in the hierarchic regulation of mammalian gene expression and replication. These elements have been used to design vectors that behave as artificial domains when integrating into the genome. We then discuss recent progress in the use of mammalian artificial chromosomes and small circular non-viral vectors for their use as expression systems in mammalian cells.

Animals↗
PubMed2000

A replication-deficient rSV40 mediates liver-directed gene transfer and a long-term amelioration of jaundice in gunn rats.

BACKGROUND & AIMS: In the quest for a recombinant viral vector for liver-directed gene therapy that would permit both prolonged and efficient transgene expression in quiescent hepatocytes in vivo and repeated administration, we evaluated a recombinant simian virus 40 (rSV40). METHODS: The rSV40 was generated through replacement of the DNA encoding for the T antigens (Tag) by the coding region of human bilirubin-uridine 5'-diphosphate-glucuronosyl-transferase (BUGT) complementary DNA (SV-hBUGT). Helper-free rSV40 units were generated at infectious titers of 5 x 10(9) to 1 x 10(10) infectious units (IU)/mL in a Tag-producing packaging cell line (COS-7 cells). RESULTS: After 1, 3, or 7 daily infusions of 3 x 10(9) IU of SV-hBUGT through an indwelling portal vein catheter in bilirubin-UGT-deficient jaundiced Gunn rats, mean serum bilirubin concentrations decreased by 40%, 60% and 70%, respectively, in 3 weeks and remained at those levels throughout the duration of the study (40 days). Results of liver biopsies from SV-hBUGT-treated Gunn rats, but not from controls, were positive for human BUGT DNA, messenger RNA, and protein. Bilirubin-UGT activity in liver homogenates was 8%-12% of normal, and bilirubin glucuronides were excreted in bile. Immunostaining showed that >50%-60% of hepatocytes stably expressed the transgene. Portal vein infusion of an rSV40 expressing hepatitis B surface antigen (HBsAg) in a naive Gunn rat and a Gunn rat that had received 7 injections of SV-BUGT resulted in approximately equal levels of hepatic expression of HBsAg, indicating that multiple inoculations of SV-BUGT did not elicit neutralizing antibodies. Plasma alanine aminotransferase levels and liver histology remained normal despite repeated injections of rSV40. CONCLUSIONS: rSV40 vectors may represent a significant advance toward gene therapy for metabolic diseases.

Animals↗
PubMed2003

Ectopic bone formation by human bone morphogenetic protein-2 gene transfer to skeletal muscle using transcutaneous electroporation.

Therapy using recombinant human bone morphogenetic protein-2 (rhBMP-2) is expected to promote bone healing and regeneration. Previous studies using protein or virus vectors for direct clinical application had problems, including a lack of efficiency, safety, and simplicity of the delivery system, and required an expensive protein, carrier matrix, or antigenic viral vector. In vivo gene transfer by electroporation is a simple and inexpensive method that only requires a plasmid and an electroporation device. Here, we created a plasmid-based human BMP-2 construct (pCAGGS-BMP-2) and examined the induction of bone in the skeletal muscle of rats after transferring different doses of this plasmid (25 microg, 100 microg, and 400 microg) by transcutaneous electroporation (8 electrical pulses of 100 V and 50 msec, in 1 to 5 sessions). First, we verified the gene transfer by transcutaneous electroporation using pCAGGS-lacZ. Next, the BMP-2 gene transfer and the production and localization of BMP-2 were identified by reverse transcription-polymerase chain reaction (RT-PCR), Western blots, and immunohistochemistry. Ectopic bone formation was verified by radiography, histologic and immunohistochemical analyses, and quantitative examination. Ectopic bone formation, consisting of active osteoblasts and osteoclasts, was observed in all rats treated with electroporation. Thus, transcutaneous electroporation with pCAGGS-BMP-2 induced ectopic bone formation in the skeletal muscle of rats. This supports the possibility of applying human BMP-2 gene transfer using transcutaneous electroporation clinically.

Animals↗
PubMed2000

Transcriptional targeting to anterior pituitary lactotrophic cells using recombinant adenovirus vectors in vitro and in vivo in normal and estrogen/sulpiride-induced hyperplastic anterior pituitaries.

The use of pituitary cell type-specific promoters is a powerful molecular tool to achieve pituitary cell type-specific transcriptional targeting of transgenes encoded by viral vectors. It has recently been proposed that transcriptional targeting of therapeutic genes could be harnessed as a gene therapy strategy for the treatment of pituitary disease. We describe the successful use of the human PRL promoter (hPrl) encoded within recombinant adenovirus vectors to target transgene expression of Herpes Simplex Virus Type 1-Thymidine Kinase (HSV1-TK) or beta-galactosidase to lactotrophic cells in vitro and in vivo. Functionally, the restriction of expression of HSV1-TK to lactotrophic tumor cells, using the hPrl promoter, resulted in the cell type-specific induction of apoptosis in the lactotrophic GH3 tumor cell line, in the presence of ganciclovir (GCV). In the corticotrophic AtT20 cell line, we detected neither HSV1-TK expression, nor apoptosis in the presence of GCV. The hPrl promoter encoded within a recombinant adenoviral vector also restricted transgene expression to lactotrophic cells in primary anterior pituitary (AP) cultures, and importantly, within the anterior pituitary gland in vivo. When the HSV1-TK driven by hPrl promoter was used in an in vivo model ofestrogen/sulpiride lactotroph induced hyperplasia within the AP in situ, the treatment was not effective in either reducing the weight of the gland, the number of lactotrophic cells within the transduced area in vivo, or the circulating PRL levels. This is in contrast to the human cytomegalovirus promoter (hCMV) driving expression of HSV1-TK in the same experimental paradigm, which was effective in reducing pituitary weight and circulating PRL levels. Our results have important implications in the design of gene therapy strategies for pituitary tumors. We demonstrate that both the choice of the in vivo animal model, i.e. adenoma in the AP gland in situ, and the particular gene therapy strategy chosen, i.e. use of strong ubiquitous promoters vs. weaker but cell type-specific promoters, determine the experimental therapeutic outcome.

Adenoviridae↗
PubMed2003

In vivo high-efficiency transcoronary gene delivery and Cre-LoxP gene switching in the adult mouse heart.

High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.

Adenoviridae↗
PubMed2001

Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells.

We have investigated the possible usefulness of recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and are able to process and present, the Ag coded by the viral vector. One consistent feature of infection by ALVAC is that these viruses induce apoptosis, and we show cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor Ag in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation is evidenced in the 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests. Overall, our data indicate that DC infected with recombinant canarypox viruses may represent an efficient presentation platform for tumor Ags, which can be exploited in clinical studies.

Antigen Presentation↗
PubMed2002

Comparison of cis and trans tat gene expression in HIV LTR-based amplifier vectors.

The long terminal repeat (LTR) of the human immunodeficiency virus (HIV) drives highly efficient gene expression in the presence of the transactivator, Tat. Thus, tat-containing vectors may be very useful tools in gene therapy. However information about the optimal way of delivering the tat gene is limited. In this study, we compared the effects of cis and trans expressions of the tat gene and its effects on HIV LTR-driven gene expression in different cell lines using non-viral vectors. The human interleukin-2 (IL-2) gene was used as a reporter gene under the control of the HIV2 LTR (pHIV2-IL-2). The tat gene, driven by a cytomegalovirus (CMV) promoter, was either co-transfected separately (pCMV-Tat) or inserted downstream of the IL-2 gene (pHIV2-IL-2-neo-C-Tat). Our results showed that HIV2 LTR-Tat-based vectors were much more potent than CMV promoter-based vectors in transient expression. The co-transfection of both plasmids was comparable to a single transfection of pHIV-IL-2-neo-C-Tat in both high and low transfection efficiency cells. In conclusion, the co-placement of HIV2 LTR and tat genes on a single plasmid allows for gene expression as efficiently as a two-plasmid system, suggesting that HIV2 LTR-Tat-based vectors may be attractive tools for gene therapy.

Animals↗