Showing papers on "Vector (molecular biology) published in 2002"

Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy

Abstract: Tissues from rhesus monkeys were screened by PCR for the presence of sequences homologous to known adeno-associated virus (AAV) serotypes 1-6. DNA spanning entire rep-cap ORFs from two novel AAVs, called AAV7 and AAV8, were isolated. Sequence comparisons among these and previously described AAVs revealed the greatest divergence in capsid proteins. AAV7 and AAV8 were not neutralized by heterologous antisera raised to the other serotypes. Neutralizing antibodies to AAV7 and AAV8 were rare in human serum and, when present, were low in activity. Vectors formed with capsids from AAV7 and AAV8 were generated by using rep and inverted terminal repeats (ITRs) from AAV2 and were compared with similarly constructed vectors made from capsids of AAV1, AAV2, and AAV5. Murine models of skeletal muscle and liver-directed gene transfer were used to evaluate relative vector performance. AAV7 vectors demonstrated efficiencies of transgene expression in skeletal muscle equivalent to that observed with AAV1, the most efficient known serotype for this application. In liver, transgene expression was 10- to 100-fold higher with AAV8 than observed with other serotypes. This improved efficiency correlated with increased persistence of vector DNA and higher number of transduced hepatocytes. The efficiency of AAV8 vector for liver-directed gene transfer of factor IX was not impacted by preimmunization with the other AAV serotypes. Vectors based on these novel, nonhuman primate AAVs should be considered for human gene therapy because of low reactivity to antibodies directed to human AAVs and because gene transfer efficiency in muscle was similar to that obtained with the best known serotype, whereas, in liver, gene transfer was substantially higher than previously described.

MAKING AN ALLY FROM AN ENEMY: Plant Virology and the New Agriculture

Abstract: Historically, the study of plant viruses has contributed greatly to the elucidation of eukaryotic biology. Recently, concurrent with the development of viruses into expression vectors, the biotechnology industry has developed an increasing number of disease therapies utilizing recombinant proteins. Plant virus vectors are viewed as a viable option for recombinant protein production. Employing pathogens in the process of creating added value to agriculture is, in effect, making an ally from an enemy. This review discusses the development and use of viruses as expression vectors, with special emphasis on (+) strand RNA virus systems. Further, the use of virus expression vectors in large-scale agricultural settings to produce recombinant proteins is described, and the technical challenges that need to be addressed by agriculturists and molecular virologists to fully realize the potential of this latest evolution of plant science are outlined.

Geminivirus‐based vectors for gene silencing in Arabidopsis

Abstract: Gene silencing, or RNA interference, is a powerful tool for elucidating gene function in Caenorhabditis elegans and Drosophila melanogaster. The vast genetic, developmental and sequence information available for Arabidopsis thaliana makes this an attractive organism in which to develop reliable gene-silencing tools for the plant world. We have developed a system based on the bipartite geminivirus cabbage leaf curl virus (CbLCV) that allows silencing of endogenous genes singly or in combinations in Arabidopsis. Two vectors were tested: a gene-replacement vector derived from the A component; and an insertion vector derived from the B component. Extensive silencing was produced in new growth from the A component vectors, while only minimal silencing and symptoms were seen in the B component vector. Two endogenous genes were silenced simultaneously from the A component vector and silencing of the genes was maintained throughout new growth. Because the CbLCV vectors are DNA vectors they can be inoculated directly from plasmid DNA. Introduction of these vectors into intact plants bypasses transformation and extends the kinds of silencing studies that can be carried out in Arabidopsis.

Chromosomal effects of adeno-associated virus vector integration.

Abstract: Adeno-associated virus (AAV) vectors are currently being used in several clinical gene-therapy trials (see the NIH OBA Human Gene Transfer Clinical Trials Database); however, little is known about the chromosomal effects of vector integration. Here we report that integrated vector proviruses are associated with chromosomal deletions and other rearrangements and are frequently located on chromosome 19 (although not at the wildtype AAV integration site).

Construction and Evaluation of Plasmid Vectors Optimized for Constitutive and Regulated Gene Expression in Burkholderia cepacia Complex Isolates

Abstract: Genetic studies with Burkholderia cepacia complex isolates are hampered by the limited availability of cloning vectors and by the inherent resistance of these isolates to the most common antibiotics used for genetic selection. Also, some of the promoters widely employed for gene expression in Escherichia coli are inefficient in B. cepacia. In this study, we have utilized the backbone of the vector pME6000, a derivative of the pBBR1 plasmid that was originally isolated from Bordetella bronchiseptica, to construct a set of vectors useful for gene expression in B. cepacia. These vectors contain either the constitutive promoter of the S7 ribosomal protein gene from Burkholderia sp. strain LB400 or the arabinose-inducible PBAD promoter from E. coli. Promoter sequences were placed immediately upstream of multiple cloning sites in combination with the minimal sequence of pME6000 required for plasmid maintenance and mobilization. The functionality of both vectors was assessed by cloning the enhanced green fluorescent protein gene (e-gfp) and determining the levels of enhanced green fluorescent protein expression and fluorescence emission for a variety of clinical and environmental isolates of the B. cepacia complex. We also demonstrate that B. cepacia carrying these constructs can readily be detected intracellularly by fluorescence microscopy following the infection of Acanthamoeba polyphaga.

Noninvasive gene transfer to the lung for systemic delivery of therapeutic proteins

Abstract: This study evaluates the use of vectors based on adeno-associated viruses (AAVs) to noninvasively deliver genes to airway epithelial cells as a means for achieving systemic administration of therapeutic proteins. We intranasally delivered AAV vectors to mice in which the same AAV2 genome encoding a cellular marker was packaged in capsids from AAV1, 2, or 5 (AAV2/1, AAV2/2, or AAV2/5, respectively). Gene expression levels achieved in both airways and alveoli were higher with AAV2/5 than with AAV2/1 and were undetectable with AAV2/2. The same set of vectors encoding a secreted therapeutic protein, erythropoietin (Epo), under the control of a lung-specific promoter (CC10) was intranasally delivered to mice, resulting in polycythemia with the highest levels of serum Epo obtained with AAV2/5 vectors. After a single intranasal administration of this vector, secretion of Epo was documented for 150 days. Similarly, intranasal administration of an AAV2/5-CC10-factor IX vector resulted in secretion of functional recombinant protein in the bloodstream of hemophiliac, factor IX-deficient mice. In addition, we demonstrate successful readministration of AAV2/5 to the lung 5 months after the first delivery of the same vector. In conclusion, we show that intranasal administration of AAV vectors results in efficient gene transfer to the lung only when the vector contains the AAV5 capsid and that this noninvasive route of administration results in sustained secretion of therapeutic proteins in the bloodstream.

RNA 3' readthrough of oncoretrovirus and lentivirus: implications for vector safety and efficacy.

Abstract: The expression of reporter genes driven by the same human elongation factor 1α (EF1α) promoter in murine leukemia virus (MLV)- and human immunodeficiency virus type 1 (HIV-1)-based vectors was studied in either transfected or virally transduced cells. The HIV-1 vectors consistently expressed 3 to 10 times higher activity than the MLV vectors at both the RNA and protein levels. The difference was not attributable to transcriptional interference, alternative enhancer/silencer, or differential EF1α intron splicing. Based on nuclear run-on assays, both vectors exhibited similar EF1α transcriptional activity. The reduced RNA levels of MLV vectors could not be explained by the decrease in RNA half-lives. Southern analysis of proviral DNA indicated that both HIV-1 and MLV vectors efficiently propagated the EF1α intron in the transduced cells. To decipher the discrepancy in transgene expression between MLV and HIV-1 vectors, the role of RNA 3′-end processing was examined using a sensitive Cre/lox reporter assay. The results showed that MLV vectors, but not HIV-1 vectors, displayed high frequencies of readthrough of the 3′ polyadenylation signal. Interestingly, the polyadenylation signal of a self-inactivating (SIN) HIV-1 vector was as leaky as that of the MLV vectors, suggesting a potential risk of oncogene activation by the lentiviral SIN vectors. Together, our results suggest that an efficient polyadenylation signal would improve both the efficacy and the safety of these vectors.

Separating fact from fiction: assessing the potential of modified adenovirus vectors for use in human gene therapy.

Abstract: One of the major hurdles to successful gene therapy of genetic and/or acquired disease is the ability to efficiently introduce a foreign gene into the tissue of interest and, in the case of some genetic diseases, achieve long-term expression of the transgene. Due to their ability to transduce a wide variety of cell types in a cell-cycle independent fashion, adenovirus (Ad)-based vectors have received considerable attention in recent years as delivery vehicles for multiple gene therapy applications. Effective use of early "first-generation" versions of these vectors was hampered by not only the induction of strong immune responses in the host to the Ad vector and transduced cells, but also to direct acute and chronic toxicity caused by the vector itself. Furthermore, transgene expression was typically transient, lasting only a few weeks. Despite these limitations, these vectors have been used in a number of human clinical trials, eliciting both interesting as well as controversial results, some of which are summarized herein. Because of these limitations, a number of advances in adenovirus "vectorology", manifested primarily as the development of multiply attenuated Ads and vectors deleted of all viral protein coding sequences, has resulted in vectors which retain all of the advantages of Ad vectors and, in addition, do not exhibit the deleterious characteristics associated with [E1-]deleted Ads. This review focuses on the current state of the art regarding the potential for human use of Ad-based vectors, and how the use of this vector continues to offer the potential for successful use as a gene delivery tool for the treatment of a great number of human genetic and non-genetic diseases.

Safety of local delivery of low- and intermediate-dose adenovirus gene transfer vectors to individuals with a spectrum of morbid conditions.

Abstract: To help define the safety profile of the use of adenovirus (Ad) gene transfer vectors in humans, this report summarizes our experience since April 1993 of the local administration of E1-/E3- Ad vectors to humans using low (<109 particle units) or intermediate (109-1011 particle units) doses. Included in the study are 90 individuals and 12 controls, with diverse comorbid conditions, including cystic fibrosis, colon cancer metastatic to liver, severe coronary artery disease, and peripheral vascular disease, as well as normals. These individuals received 140 different administrations of vector, with up to seven administrations to a single individual. The vectors used include three different transgenes (human cystic fibrosis transmembrane conductance regulator cDNA, E. coli cytosine deaminase gene, and the human vascular endothelial growth factor 121 cDNA) administered by six different routes (nasal epithelium, bronchial epithelium, percutaneous to solid tumor, intradermal, epicardial injection of the myocard...

Comparison of the efficiency and safety of non-viral vector-mediated gene transfer into a wide range of human cells.

Abstract: Non-viral gene transfer into a wide range of human cells was examined in order to clarify the factors that affect the efficiency and safety of non-viral vectors and to optimize the conditions so that high efficiency and low toxicity could be achieved. Six non-viral vectors (Lipofectin, LipofectAMINE PLUS, SuperFect, Effectene, DMRIE-C and DOTAP) were used to transfect a mammalian expression plasmid pCMVβ into 16 types of human primary cells and cultured cell lines. Transfection efficiency was quantified using a galactosidase assay. Cytotoxic effects were measured by lactate dehydrogenase (LDH) assay and WST-8 assay. In serum-free conditions, LipofectAMINE PLUS, Effectene and SuperFect, on average, transfected DNA more successfully than Lipofectin, DMRIE-C, and DOTAP, although the levels of gene expression with these vectors varied remarkably in different cells. The most effective vector also differed depending on the cell type. Serum was found to inhibit gene transfer and reduce the cytotoxicity of all of these vectors except Effectene. The efficiency and toxicity of the non-viral vectors used depended on the type of vector, the DNA/vector ratio, the type of cell, and the presence of serum. These results provided useful information for the optimization of transfer conditions of these non-viral vectors.

Gene therapy of muscular dystrophy

Abstract: Development of gene therapy for the muscular dystrophies represents a daunting challenge requiring significant advances in our knowledge of the defective genes, muscle promoters, viral vectors, immune system surveillance and methods for systemic delivery of vectors. However, tremendous progress has been made in developing improved viral vectors and avoiding immune reactions against gene transfer. This review summarizes recent progress and highlights problems that must be solved before an effective treatment is available.

Immunological aspects of recombinant adeno-associated virus delivery to the mammalian brain

Abstract: Recombinant adeno-associated viruses (rAAV) are highly efficient vectors for gene delivery into the central nervous system (CNS). However, host inflammatory and immune responses may play a critical role in limiting the use of rAAV vectors for gene therapy and functional genomic studies in vivo. Here, we evaluated the effect of repeated injections of five rAAV vectors expressing different genetic sequences (coding or noncoding) in a range of combinations into the rat brain. Specifically, we wished to determine whether a specific immune or inflammatory response appeared in response to the vector and/or the transgene protein after repeated injections under conditions of mannitol coinjection. We show that readministration of the same rAAV to the CNS is possible if the interval between the first and second injection is more than 4 weeks. Furthermore, our data demonstrate that rAAV vectors carrying different genetic sequences can be administered at intervals of 2 weeks. Our data therefore suggest that the AAV capsid structure is altered by the vector genetic sequence, such that secondary structures of the single-stranded genome have an impact on the antigenicity of the virus. This study provides guidelines for more rational design of gene transfer studies in the rodent brain and, in addition, suggests the use of repeated administration of rAAV as a viable form of therapy for the treatment of chronic diseases.

Lentiviruses in gene therapy clinical research

Abstract: Gene therapy vectors derived from lentiviruses offer many potentially unique advantages over more conventional retroviral gene delivery systems. Principal amongst these is their ability to provide long-term and stable gene expression and to infect non-dividing cells, such as neurons. However, the use of lentiviral-based vectors in the clinic also raises specific safety and ethical issues. Concerns include the possible generation of replication competent lentiviruses during vector production, mobilisation of the vector by endogenous retroviruses in the genomes of patients, insertional mutagenesis leading to cancer, germline alteration resulting in trans-generational effects and dissemination of new viruses from gene therapy patients. Investigators proposing to conduct this type of research should take due account of the potential risks for interaction of lentiviral gene therapy vectors with other retroviral elements in human subjects, such as Human Immunodeficiency Virus. In addition, strict quality control for replication competent lentiviruses and suitable measurements of lentiviral infectious particle number will be required before these types of viral vector can proceed to the clinic.

Improved Primate Foamy Virus Vectors and Packaging Constructs

Abstract: Foamy virus (FV) vectors that have minimal cis-acting sequences and are devoid of residual viral gene expression were constructed and analyzed by using a packaging system based on transient cotransfection of vector and different packaging plasmids. Previous studies indicated (i) that FV gag gene expression requires the presence of the R region of the long terminal repeat and (ii) that RNA from packaging constructs is efficiently incorporated into vector particles. Mutants with changes in major 5′ splice donor (SD) site located in the R region identified this sequence element as responsible for regulating gag gene expression by an unidentified mechanism. Replacement of the FV 5′ SD with heterologous splice sites enabled expression of the gag and pol genes. The incorporation of nonvector RNA into vector particles could be reduced to barely detectable levels with constructs in which the human immunodeficiency virus 5′ SD or an unrelated intron sequence was substituted for the FV 5′ untranslated region and in which gag expression and pol expression were separated on two different plasmids. By this strategy, efficient vector transfer was achieved with constructs that have minimal genetic overlap.

Polycistronic Viral Vectors

Abstract: Traditionally, vectors for gene transfer/therapy experiments were mono- or bicistronic. In the latter case, vectors express the gene of interest coupled with a marker gene. An increasing demand for more complex polycistronic vectors has arisen in recent years to obtain complex gene transfer/therapy effects. In particular, this demand is stimulated by the hope of a more powerful effect from combined gene therapy than from single gene therapy in a process whose parallels lie in the multi-drug combined therapies for cancer or AIDS. In the 1980's we had only splicing signals and internal promoters to construct such vectors: now a new set of biotechnological tools enables us to design new and more reliable bicistronic and polycistronic vectors. This article focuses on the description and comparison of the strategies for co-expression of two genes in bicistronic vectors, from the oldest to the more recently described: internal promoters, splicing, reinitiation, IRES, self-processing peptides (e.g. foot-and-mouth disease virus 2A), proteolytic cleavable sites (e.g. fusagen) and fusion of genes. I propose a classification of these strategies based upon either the use of multiple transcripts (with transcriptional mechanisms), or single transcripts (using translational/post-translational mechanisms). I also examine the different attempts to utilize these strategies in the construction of polycistronic vectors and the main problems encountered. Several potential uses of these polycistronic vectors, both in basic research and in therapy-focused applications, are discussed. The importance of the study of viral gene expression strategies and the need to transfer this knowledge to vector design is highlighted.

Plasmid vectors encoding cholera toxin or the heat-labile enterotoxin from Escherichia coli are strong adjuvants for DNA vaccines.

Abstract: Two plasmid vectors encoding the A and B subunits of cholera toxin (CT) and two additional vectors encoding the A and B subunits of the Escherichia coli heat-labile enterotoxin (LT) were evaluated for their ability to serve as genetic adjuvants for particle-mediated DNA vaccines administered to the epidermis of laboratory animals. Both the CT and the LT vectors strongly augmented Th1 cytokine responses (gamma interferon [IFN-γ]) to multiple viral antigens when codelivered with DNA vaccines. In addition, Th2 cytokine responses (interleukin 4 [IL-4]) were also augmented by both sets of vectors, with the effects of the LT vectors on IL-4 responses being more antigen dependent. The activities of both sets of vectors on antibody responses were antigen dependent and ranged from no effect to sharp reductions in the immunoglobulin G1 (IgG1)-to-IgG2a ratios. Overall, the LT vectors exhibited stronger adjuvant effects in terms of T-cell responses than did the CT vectors, and this was correlated with the induction of greater levels of cyclic AMP by the LT vectors following vector transfection into cultured cells. The adjuvant effects observed in vivo were due to the biological effects of the encoded proteins and not due to CpG motifs in the bacterial genes. Interestingly, the individual LT A and B subunit vectors exhibited partial adjuvant activity that was strongly influenced by the presence or absence of signal peptide coding sequences directing the encoded subunit to either intracellular or extracellular locations. Particle-mediated delivery of either the CT or LT adjuvant vectors in rodents and domestic pigs was well tolerated, suggesting that bacterial toxin-based genetic adjuvants may be a safe and effective strategy to enhance the potency of both prophylactic and therapeutic DNA vaccines for the induction of strong cellular immunity.

Development of a replication-selective, oncolytic poxvirus for the treatment of human cancers.

Abstract: Tumor directed gene therapy for the purpose of destroying cancer cells through replicative "oncolysis" or by intratumoral expression of toxic or immunostimulatory genes requires an efficient, tumor targeted vector. Vectors are limited by inefficient replication in vivo, inefficient tumor targeting, and safety concerns. As a unique approach to addressing these limitations, our laboratory has studied poxviruses as tumor selective replicating vectors. The best in vivo antitumor results achieved to date have been with a mutated WR strain of vaccinia virus. The unique advantage of this strain of vaccinia over other vectors currently being explored for this purpose is the efficiency of in vivo replication. Intradermal injection of 10(6) pfu of the wild type (non-mutated) vaccinia in non-human primates leads to a 108 cm(2) zone of necrosis in 8 days - directly related to cellular destruction from viral replication. We have mutated the virus through insertional deletion of both the thymidine kinase (TK) gene and vaccinia growth factor (VGF) gene. The mutant virus no longer causes destruction of normal tissue, but has completely preserved replication efficiency in tumor tissue and can safely be delivered systematically to successfully treat subcutaneous tumors in mice. Plans are now underway for clinical trials.

Viruses as vectors for the expression of foreign sequences in plants.

Abstract: General abbreviations: Ac, transposon activator unit; bp, base pair~ BMS. Black Mexican sweetcorn; CAT, chloramphenicol acetyl transfer\"se; Cd, cadnlium~ ChsA, chalcone synthase A; CP. coat protein; CR. corllinon region; DHFR, dihydrofolate reductase: Dr RNA. defective interfering RNA; dRNA. defectjve RNA; ds, double-stranded; Ds, dissociation clcJncnt; f}gal, ~-galactosida8e; GFP. green fluorescent proteill~ kbp~ kilobase pair; GR. glutathione reductase~ OS, glutatninc synthase; GUS, P~glucuronidase;He-Pro, helper cotllponcnt-protcinase; HPT, hygronlycin phosphotraJ1sferase~ IFN-aD, interrcron aD: IFN-y. interferon y; LIR, large intel'genic rcagion; Inc, luciferase: MP, movelnenl pl'otein; MTIL Inetallothioncin II: nos. nopaline synthase; NPT-II. neomycin phosphotransferase type II: OAS. origin of asselllbly: oes, octopine synthase; ORF. open reading Craine; ori, origin of replication; PAT, phosphinothricin acetyl tl'ansferase~PDS~ phytoene desuturase: PSY t phylocne synLhase; PTGS, post-transcriptional genesilencing; Rz, ribozylne; ss. single-stranded;N. labacum.. Nicot;(I'UlltlbtlcUnl; N. betllhamill1lllt JViCO/;llIUI helltIUlIn;(IJl(I: RBS, ribulose biphosphate carboxylase; ScFv. singlc·chain variable fragnlcnt; sg, subgcnomic: SIR, snu,H ilnergenic region: TGB~ triple gene block~ VIGS. virus-induced gene silencing; VLPs. virus-like particles. Note: In the text nanlCS of genes are given in lower case italics. nanlC~ of the con·csponding gene products in upper case. Abbreviations for virus mUllCS: ACMV, Africnn cassava 1110saic vil\"us~ AIMV. alfalfa mosaic virus; BaMV, balltboo Iltosaic vinL~; BMV, bronlc Illosaic virus; BSMV, bilrley stripe [nosuic virus; BYV, beet yel1ow~ vinls; CaMV I cauliflower nl0s,~icvirus: CCMV. cowpea chlorotic rnottlc virus: CJYVV. clover yellow vein virus: CMV. cucunlbcr mosnic vil1Js: CPMVt co\\vpea nlosaic virus; CyRSY.. cymbidiuJll ringspot vinls; FMDV. foot~and-]nouthdisease virus; HIV-I. hUEnnn inullllnodeficiency vii-us I: MiSV, rlliscanthus streak virus; MSV. nlaize streak vinls~ ORSV. odontoglo~sUJllringspot virus; PEBV. peH early browning virll~: PepRSV, pepper ringspOl viru~; PMMoV.. pepper nlild 1110ttle vinls~ PPV, pllUn pox virl1s~ PVV, potato virus V; PYX, potato virus X: RABV, nlbies virus: RHDV. rabbit haeJllOrrhagic disease virus: SHI\\1V. SUDn hemp Illosaic virus: sTRSV, satellite of tob,lCCO ringspot vinls; TAV. tOJnato aspenny virlls: TBSV, tonlato bushy stunt virus: TEV, tobacco etch virus~ ToMV, tonlato lnosaic vinls; TMV. tobacco Dl0saic virus~ TYMV, turnip yellow mosaic vinls~TYDV . tobacco yello\\\\' dwarFvirus: TGMV.10nlato golden 1l1osaic vinls; TRV, tob,lCCO rattle virus; TSWV. 10nlato spotted wilt virus; TCV. turnip crinkle virus: WDV. wheat dwarf virus; WSMV, wheat streak J110saic vinls.

Virus-based gene delivery systems.

Abstract: Within the past decade, gene therapy strategies have come to the forefront of novel therapeutics Tremendous advances in vector technology along with deeper understandings of vector biology and the molecular mechanisms of disease have significantly advanced the field of human gene therapy This manuscript will discuss the viral-based subset of current gene transfer vectors In particular, the most established viral vectors to date, including parvovirus, adenovirus, retrovirus, lentivirus, and herpesvirus-based vectors, are described, as well as the current innovative improvements being made to each From past experience, it has become evident that in addition to optimising the vectors in terms of transgene expression, minimising vector-related immunology, and vector production, methods of vector delivery resulting in optimum vector transduction of target cells need to be established This review will also illustrate several current improved physical delivery systems for optimal vector administration

Evaluation of E1B gene-attenuated replicating adenoviruses for cancer gene therapy.

Abstract: Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated adenoviral vectors with different combinations of E1B genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent adenoviruses. We show here that the cytolytic potency of each gene-attenuated replicating adenovirus differed significantly depending on the presence or deletion of E1B 55 kDa and E1B 19 kDa function. More specifically, among the constructed vectors (Ad-deltaE1B19, Ad-deltaE1B55, Ad-deltaE1B19/55, and Ad-wt), E1B 19 kDa-inactivated adenovirus (Ad-deltaE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. Further, cells infected with either Ad-deltaE1B19 or E1B19/55 kDa-deleted adenovirus (Ad-deltaE1B19/55) showed complete cell lysis with disintegrated cellular structure, whereas cells infected with Ad-wt maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL and DNA fragmentation assay also revealed that the Ad-deltaE1B19 or Ad-deltaE1B19/55 adenovirus-infected cells showed more profound induction of apoptosis in comparison to wild-type adenovirus-infected cells. The presence of E1B 55 kDa gene was required for efficient viral replication and deletion of E1B 19 kDa function in replicating adenovirus-induced apoptosis, leading to increased cytopathic effects. Moreover, Ad-deltaE1B19 adenovirus showed a better antitumor effect than other E1B-attenuated adenoviruses. Taken together, the replicating adenoviruses deleted in E1B 19 kDa function may serve as an improved vector for anticancer gene therapy in combination with apoptosis-inducing modalities such as chemotherapeutic agents and radiation therapy.

Stress responses and replication of plasmids in bacterial cells

Abstract: Plasmids, DNA (or rarely RNA) molecules which replicate in cells autonomously (independently of chromosomes) as non-essential genetic elements, play important roles for microbes grown under specific environmental conditions as well as in scientific laboratories and in biotechnology. For example, bacterial plasmids are excellent models in studies on regulation of DNA replication, and their derivatives are the most commonly used vectors in genetic engineering. Detailed mechanisms of replication initiation, which is the crucial process for efficient maintenance of plasmids in cells, have been elucidated for several plasmids. However, to understand plasmid biology, it is necessary to understand regulation of plasmid DNA replication in response to different environmental conditions in which host cells exist. Knowledge of such regulatory processes is also very important for those who use plasmids as expression vectors to produce large amounts of recombinant proteins. Variable conditions in large-scale fermentations must influence replication of plasmid DNA in cells, thus affecting the efficiency of recombinant gene expression significantly. Contrary to extensively investigated biochemistry of plasmid replication, molecular mechanisms of regulation of plasmid DNA replication in response to various environmental stress conditions are relatively poorly understood. There are, however, recently published studies that add significant data to our knowledge on relations between cellular stress responses and control of plasmid DNA replication. In this review we focus on plasmids derived from bacteriophage λ that are among the best investigated replicons. Nevertheless, recent results of studies on other plasmids are also discussed shortly.

Adenovirus as an integrating vector.

Abstract: Recombinant adenoviral vectors have served as one of the most efficient gene delivery vehicles in vivo thus far. Multiply attenuated or completely gutless adenoviral vectors have been developed to achieve long-term gene expression in animal models by overcoming cellular immunity against de novo synthesized adenoviral proteins. However, since adenovirus lacks native integration machinery, the goal of gene therapy obtaining permanent expression cannot be realized with current adenoviral vector systems. Recent studies have shown that replication-incompetent adenoviral vectors randomly integrate into host chromosomes at frequencies of 0.001-1% of infected cells. To improve the integration frequencies of adenoviral vectors, a variety of hybrid vectors combining the highly efficient DNA delivery of adenovirus with the integrating machinery of retroviruses, adeno-associated viruses, and transposons, have been emerging. These hybrid vectors have shown promise, at least in in vitro systems. Furthermore, adenoviral vectors have shown potential as gene targeting vectors. These developments should eventually lead to more effective gene therapy vectors that can transduce a myriad of cell types stably in vivo.

Enzyme-activated Prodrug Therapy Enhances Tumor-specific Replication of Adenovirus Vectors

Abstract: Adenoviruses (Ads) that selectively replicate in tumor cells have shown promising preliminary results in clinical trials, especially in combination with chemotherapy. Here, we describe a system that combines the antitumor synergy of Ads and chemotherapeutic agents with the benefits of enzyme-activated prodrug therapy. In this system, a functional transgene expression cassette is created by homologous recombination during adenoviral DNA replication. Transgene expression is strictly dependent on viral DNA replication, which in turn is tumor specific. We constructed replication-activated Ad vectors to express a secreted form of beta-glucuronidase and a cytosine deaminase/uracil phosphoribosyltransferase, which activate the prodrugs 9-aminocamptothecin glucuronide to 9-aminocamptothecin and 5-fluorocytosine to 5-fluorouracil (5-FU) and further to 5-fluoro-UMP, respectively. We demonstrated replication-dependent transgene expression, prodrug activation, and induction of tumor cell toxicity by secreted beta-glucuronidase and cytosine deaminase/uracil phosphoribosyltransferase. Furthermore, exposure of cells to activated prodrug or drug at subtoxic concentrations enhanced viral DNA replication. Characteristically, these agents induced changes in the cell cycle status of exposed cells (G(2) arrest), which closely resembled the effect of wild-type Ad infection, and are thought to be favorable for viral replication. We tested a number of cytostatic drugs (camptothecin, etoposide, daunorubicin, cisplatin, 5-fluorouracil, hydroxyurea, Taxol, and actinomycin D) for their effect on viral DNA replication and found considerable differences between individual agents. Finally, we show that the combination of viral and prodrug therapy enhances viral replication and spread in liver metastases derived from human colon carcinoma or cervical carcinoma in a mouse model. Our data indicate that specific vector/drug combinations tailored to be synergistic may have the potential to improve the potency of either therapeutic approach. These data also provide a new rationale for expressing prodrug-activating enzymes from conditionally replicating Ads.

Viruses as gene delivery vectors: Application to gene function, target validation, and assay development

Abstract: A Biochemical Pharmacology Discussion Group Conference, was held at the headquarters of the New York Academy of Sciences on December 4, 2001 as part of an ongoing series designed to highlight and review areas important to modern drug development (Figure 1). Briefly introduced by Tom Kost (GlaxoSmithKline) and Michael Lotze (University of Pittsburgh), the focus was on the intersection of genomics, proteomics, and now “viromics.” The latter term refers to the use of viruses and viral gene transfer to explore the complexity arising from the vast array of new targets available from the human and murine genomes. Indeed, access to large numbers of genes using viral vectors is a key tool for drug discovery and drug delivery. With 38,000 genes identified within the human genome, only 5000 are considered readily druggable. Generating tools such as these to validate targets represents a major part of the armamentarium of the postgenomic scientist. During the last 12 years alone, there have been over 26,000 publications on virus vectors. Many of them have been found useful in target validation, assay development, and evaluation in in vivo models and gene therapy. Thus, there is now an extensive knowledge base for several viral vectors, with unique attributes within each of them providing versatility, efficiency, and ease of use. The individual scientists presenting at the meeting illustrated many of the unique and useful characteristics of such vector systems including retrovirus, adenovirus, herpes virus, simbis virus, and baculovirus.