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At least 685 records · Page 38Linked to original sources
PubMed2006

Efficient AAV1-AAV2 hybrid vector for gene therapy of hemophilia.

Adeno-associated virus (AAV) serotype 1 (AAV1) has been shown to be more effective than the well-studied AAV serotype 2 (AAV2) in muscle gene transfer. Replacement of amino acids 350 to 430 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector, termed AAV-221-IV, which behaved similarly to AAV1 in vitro and in vivo in muscle. Intramuscular injection of 1x10(11) vector particles per mouse of hybrid vector carrying a human FIX transgene in CD4 knockout mice resulted in an average level of human FIX in the plasma of 450 ng/ml, 4- to 10-fold higher than in mice injected with an AAV2 vector carrying the same transgene, and 80% of the transgene levels in animals treated with the same dose of AAV1. DNA analysis of injected muscle showed a 10-fold higher copy number after gene delivery by the hybrid vector compared with AAV2. A comparison of total DNA versus DNA from intact virus particles suggests a higher stability of hybrid virus particles. These results suggest that changes in the AAV capsid have an effect on virus-cell receptor interaction, and also influence trafficking and processing of the virus particle in the cell. This "hybrid vector" retains the heparin-binding sites of AAV2 and, therefore, can be purified by passage through a heparin-Sepharose column with the same efficiency as AAV2. When tested in vivo, either in CD4 knockout mice or in a hemophilic mouse model, the heparin-purified hybrid vector showed >10-fold higher activity than similarly purified AAV2. This demonstrates the utility of this hybrid vector in the performance of large-scale heparin column purification to generate a vector with a high expression profile for muscle-directed gene delivery. Initiation of clinical studies with this hybrid vector may be facilitated because it differs from AAV2 by only nine amino acids.

Animals↗
PubMed1996

Expression of hammerhead ribozymes by retroviral vectors to inhibit HIV-1 replication: comparison of RNA levels and viral inhibition.

We have analyzed expression of anti-HIV-1 hammerhead ribozymes in the context of retroviral vectors. To determine optimal vector designs for ribozyme expression, we compared three vectors, each of which contained the same pair of anti-HIV-1 hammerhead ribozymes in tandem. Despite the presence of vastly different amounts of vector-derived flanking sequences, the ribozymes produced by each vector had similar cleavage activity when assayed in vitro. The ribozyme vectors were packaged into amphotropic virion and used to transduce human CEM T lymphocytes. Analysis by Northern blot and RNAse protection assays demonstrated that the highest steady-state levels of ribozyme-containing transcripts were produced by a vector in which the ribozymes were expressed under transcriptional control of the vector MoMuLV LTR. Despite these differences in the levels of ribozyme transcripts achieved by the vectors, their ability to confer resistance to HIV-1 replication was similar. Therefore, other factors than the absolute levels of ribozymes play a role in determining the effectiveness of ribozyme vectors to inhibit HIV-1. These may include structural features of the transcripts that affect the antisense effects of the ribozyme constructs, the actual catalytic activity of the ribozymes, their RNA folding, the binding of proteins, and the intracellular localization. Greater understanding of these factors may permit more effective application of ribozymes to inhibit gene expression.

Base Sequence↗
PubMed2001

Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model.

Recombinant vectors based on adeno-associated virus (AAV) or human immunodeficiency 1 (lentivirus) are promising tools for long term in vivo gene delivery. Their design allows the exchange of capsids or envelopes, respectively, theoretically providing the opportunity to transduce a range of cell types. We constructed AAV vectors encoding enhanced green fluorescent protein (EGFP) within an AAV serotype 2 (AAV2) genome contained in an AAV2, five or one capsid (called AAV2/2, AAV2/5 and AAV2/1, respectively). Similarly we produced lentiviral vectors, encoding the same expression cassette present in the AAV vectors, pseudotyped with proteins from vesicular stomatitis virus glycoprotein (VSVG) or Mokola envelopes. Transduction characteristics of these vectors were evaluated in the murine retina following subretinal or intravitreal administration. The time of onset of transgene expression and the targeted cell types differed between the various recombinants. Onset of transgene expression was 3-4 days for lentiviral vectors and AAV2/1. In contrast, onset was at 2-4 weeks for AAV2/5 and AAV2/2, respectively. After subretinal injection, both lenti-VSVG and AAV2/5 transduced the retinal pigment epithelium (RPE) and photoreceptors efficiently whereas transgene expression was restricted to RPE cells using lenti with the Mokola envelope or AAV2/1. After intravitreal administration, only AAV2/2 and lenti-VSVG transduced the inner retina. Vector-mediated fluorescence was detected in the retina for over 12 weeks for all of the vectors. We conclude that pseudotyping provides a useful means to manipulate viral vector cell targeting specificity as well as retinal transduction characteristics of vectors containing the same genome.

Animals↗
PubMed1989

Stability and expression of bacterial genes in replicating geminivirus vectors in plants.

Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.

Chromosome Deletion↗
PubMed2004

Spatial distribution of bluetongue virus and its Culicoides vectors in Sicily.

During the recent Mediterranean epizootic of bluetongue, an extensive programme of serological and vector (Culicoides biting midges (Diptera: Ceratopogonidae)) surveillance was carried out across Sicily. This paper presents the analysis of 911 light trap catches collected at the times of peak Culicoides abundance (summer to autumn 2000-2002) in 269 sites, in order to produce detailed maps of the spatial distribution of the main European vector, Culicoides imicola Kieffer and that of potential novel vectors. Whereas C. imicola was found at only 12% of sites, potential novel vectors, Culicoides obsoletus group Meigen, Culicoides pulicaris Linnaeus and Culicoides newsteadi Austen were present at over 50% of sites. However, the spatial distribution of C. imicola showed the closest correspondence to that of the 2000 and 2001 bluetongue (BT) outbreaks and its presence and abundance were significant predictors of the probability of an outbreak, suggesting that it was the main vector during these years. Although C. imicola may have played a role in transmission in several sites near Paternó, it was absent from the majority of sites at which outbreaks occurred in 2002 and from all sites in the province of Messina. All three potential novel vectors were widespread across sites at which outbreaks occurred during 2002. Of these, C. newsteadi was an unlikely candidate, as it was significantly less prevalent in outbreak vs. non-outbreak sites in Messina. It is hypothesized that the yearly distribution and intensity of outbreaks is directly attributable to the distribution and abundance of the vectors involved in transmission during each year. When C. imicola operated as the main vector in 2000 and 2001, outbreaks were few in number and were restricted to coastal regions due to low abundance and prevalence of this species. In 2002, it is hypothesized that BTV transmission was handed over to more prevalent and abundant novel vector species, leading to numerous and widespread outbreaks and probably to overwintering of the virus between 2001 and 2002. Based on catch ranges in outbreak vs. non-outbreak sites, it is tentatively suggested that nightly catches of 400 or more C. obsoletus and 150 or more C. pulicaris allow BTV transmission at a site, and provide a strategy for a fuller examination of the relationship between BTV transmission and the abundance and distribution of different vector species.

Animals↗
PubMed2005

Development of food-grade cloning and expression vectors for Lactococcus lactis.

AIMS: To develop food-grade cloning and expression vectors for use in genetic modification of Lactococcus lactis. METHODS AND RESULTS: Two plasmid replicons and three dominant selection markers were isolated from L. lactis and used to construct five food-grade cloning vectors. These vectors were composed of DNA only from L. lactis and contained no antibiotic resistance markers. Three of the vectors (pND632, pND648 and pND969) were based on the same plasmid replicon and carried, either alone or in combination, the three different selectable markers encoding resistance to nisin, cadmium and/or copper. The other two (pND965DJ and pND965RS) were derived from a cadmium resistance plasmid, and carried a constitutive promoter and a copper-inducible promoter, respectively, immediately upstream of a multicloning site. All vectors were stable in L. lactis LM0230 for at least 40 generations without selection pressure. The two groups of vectors were compatible in L. lactis LM0230. The vectors pND648 and pND965RS, as representatives of the two groups, were transferred successfully by electroporation into and maintained in an industrial strain of L. lactis. The usefulness of the vectors was further demonstrated by expressing a phage resistance gene (abiI) in another industrial strain of L. lactis. CONCLUSIONS: The five food-grade vectors constructed are potentially useful for industrial strains of L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: These vectors represent a new set of molecular tools useful for food-grade modifications of L. lactis.

Base Sequence↗
PubMed1995

Salivary apyrase in New World blackflies (Diptera: Simuliidae) and its relationship to onchocerciasis vector status.

Salivary gland apyrase is believed to be critical to blood-feeding in arthropod vectors. This enzyme was measured in six New World blackflies representing three taxonomic pairs of non-vectors and vectors of Onchocerca volvulus. In Simulium (Psilopelmia) ochraceum, a highly anthropophilic vector in Mexico and Guatemala, apyrase exhibited maximum activity between pH 8.0 and 9.0, mean 39.8 +/- 4.7 milliUnits/pair of gland equivalents (mU), and was enhanced when ATP was used as a substrate. In the zoophilic non-vector Simulium (Psilopelmia) bivittatum maximum activity was significantly less (5.1 +/- 0.7 mU) under all conditions examined. Preference for ADP or ATP as substrate was a function of the pH of the reaction for this species. Apyrase activity in Simulium (Simulium) metallicum Bellardi (29.5 +/- 11.5 mU), a zoophilic secondary vector in Mexico and Guatemala, resembled that of S. (Ps.) ochraceum (24.8 +/- 13.7 mU at pH 8.5) with ADP as substrate, but showed reduced activity with ATP. Both these Central American vectors had higher apyrase activity than found in Simulium (Notolepria) exiguum, a vector of O. volvulus in Ecuador and Colombia. However, maximum apyrase activity, measured at pH 8.0 with ADP as substrate, was greater in S. (N.) exiguum (10.9 +/- 0.6 mU) than in Simulium (Notolepria) gonzalezi (5.9 +/- 1.9 mU), a non-vector species widespread in Central America. Therefore, for the consubgeneric species pairs examined, a positive association was detected between higher concentrations of apyrase activity and their vector status for O.volvulus.

Adenosine Diphosphate↗
PubMed2005

Behavioural heterogeneity of Anopheles species in ecologically different localities in Southeast Asia: a challenge for vector control.

In Southeast Asia the biodiversity of Anopheles species in the domestic environment is very high. Only few species are considered major vectors throughout the region, whereas the vector status of other species varies from area to area. Often it is difficult to identify an Anopheles species as a malaria vector in areas with low malaria incidence. The behaviour of Anopheles species largely determines their vector status, and insights into their behaviour are essential to evaluate the appropriateness of vector control measures. This study was conducted in six ecologically different localities in Southeast Asia to rank the different Anopheles species in terms of anthropophily and endophagy in order to estimate their current epidemiological importance. Concurrently, the biting and resting behaviour of the vectors was analysed to evaluate the appropriateness of insecticide-impregnated bed nets and residual house spraying in vector control. Anopheles dirus A was highly anthropophilic at all sites where it occurred. By contrast, the degree of anthropophily exhibited by An. minimus A depended on availability of cattle. Anopheles campestris, An. nimpe, An. sinensis, An. maculatus, An. aconitus showed a high degree of anthropophily in certain villages, indicating their potential of participating in malaria transmission, although the actual incidence of malaria in the study villages can be fully explained by transmission of the major vectors (An. dirus A, An. minimus A and An. sundaicus). Late biting of An. minimus A and biting activity throughout the night of An. sundaicus favour bed nets as a control method for these species, whilst exophilic and outdoor biting in combination with early feeding behaviour of An. dirus A will make both insecticide-impregnated bed nets and indoor residual spraying less suitable for controlling this species. Spatial variation in biting and resting behaviour was observed within almost all Anopheles species. These heterogeneities may result in the differences in epidemiological importance and in response to vector control of Anopheles species in different areas. Moreover, environmental changes and changes in human practice are expected to influence the behaviour, hence the role of the different species in malaria transmission. The effect of environmental changes on vector behaviour should be followed up carefully.

Animals↗
PubMed1996

Genetic variation in arthropod vectors of disease-causing organisms: obstacles and opportunities.

An overview of the genetic variation in arthropods that transmit pathogens to vertebrates is presented, emphasizing the genetics of vector-pathogen relationships and the biochemical genetics of vectors. Vector-pathogen interactions are reviewed briefly as a prelude to a discussion of the genetics of susceptibility and refractoriness in vectors. Susceptibility to pathogens is controlled by maternally inherited factors, sex-linked dominant alleles, and dominant and recessive autosomal genes. There is widespread interpopulation (including intercolony) and temporal variation in susceptibility to pathogens. The amount of biochemical genetic variation in vectors is similar to that found in other invertebrates. However, the amount varies widely among species, among populations within species, and temporally within populations. Biochemical genetic studies show that there is considerable genetic structuring of many vectors at the local, regional, and global levels. It is argued that genetic variation in vectors is critical in understanding vector-pathogen interactions and that genetic variation in vectors creates both obstacles to and opportunities for application of genetic techniques to the control of vectors.

Animals↗
PubMed1988

Comparison of expression in hemopoietic cells by retroviral vectors carrying two genes.

In order to identify factors that influence expression by retroviral vectors in hemopoietic cells, we have compared viral RNA levels in cells infected with several different recombinant viruses. All of the vectors tested carry the neomycin resistance gene and provide for the insertion of a second gene which, in these studies, comprised sequences from the myc or myb oncogenes or the gene encoding granulocyte-macrophage colony-stimulating factor. The vectors utilize two different strategies for the coexpression of the two genes: alternate splicing and the use of a separate internal promoter. We found that expression in hemopoietic cells could be increased by substituting sequences from the myeloproliferative sarcoma virus long terminal repeat for those of the Moloney murine leukemia virus long terminal repeat. However, none of the vectors examined was able to express a second gene at levels equivalent to those achieved by the parental vectors carrying only the neomycin resistance gene. The reasons for this varied with the different vectors and included inefficient splicing and/or a reduction in the level of unspliced transcripts upon insertion of a second gene. Although the basis of the latter phenomenon is not clear, it is probably related to the position--near the 5' long terminal repeat--at which the second gene was inserted, since insertion of the same genes near the 3' end of another vector had no effect on viral RNA levels. In an attempt to circumvent some of these problems, we constructed a vector that employs an internal beta-actin promoter. Although this vector could express granulocyte-macrophage colony-stimulating factor sequences in a responsive hemopoietic cell line, the level of granulocyte-macrophage colony-stimulating factor produced was disappointingly low. The results from these studies suggest approaches to the design of improved vectors for effective expression of genes in hemopoietic cells.

Animals↗
PubMed1994

Analysis in human immunodeficiency virus type 1 vectors of cis-acting sequences that affect gene transfer into human lymphocytes.

Human immunodeficiency virus type 1 (HIV-1) can be used to generate recombinant viral vectors for delivery of heterologous genes to human CD4-positive lymphocytes. To define the cis-acting sequences required for efficient gene transfer, a number of HIV-1 vectors containing a previously identified packaging signal, long terminal repeats, and additional gag, pol, and env viral sequences were designed. By providing the viral proteins in trans, recombinant viruses were generated and analyzed for their abilities to transfer genes into human T lymphocytes. Inclusion of up to 653 nucleotides derived from the 5' end of the gag gene in the vector improved the efficiency of gene transfer, but inclusion of additional gag or pol sequences did not further improve this efficiency. The increased efficiency of gene transfer associated with the inclusion of 5' gag sequences in the vector arose, at least in part, from an increase in the packaging of vector RNA. The presence of the Rev-responsive element (RRE) increased the efficiency of transfer of vectors containing significant lengths of gag sequence, as expected from the Rev requirement for nucleus-to-cytoplasm transport of unspliced vector RNA containing intact packaging signals. However, the presence of a RRE did not affect the transfer efficiency of smaller vectors lacking significant lengths of gag sequences, arguing against a specific role for the RRE in packaging or vector transfer. These results contribute to an understanding of the minimal cis-acting sequences that operate in the context of HIV-1 vectors for delivering genes into human lymphocytes.

CD4-Positive T-Lymphocytes↗
PubMed1996

Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

A novel recombinant adenovirus vector, Av3nBg, was constructed with deletions in adenovirus E1, E2a, and E3 regions and expressing a beta-galactosidase reporter gene. Av3nBg can be propagated at a high titer in a corresponding A549-derived cell line, AE1-2a, which contains the adenovirus E1 and E2a region genes inducibly expressed from separate glucocorticoid-responsive promoters. Av3nBg demonstrated gene transfer and expression comparable to that of Av1nBg, a first-generation adenovirus vector with deletions in E1 and E3. Several lines of evidence suggest that this vector is significantly more attenuated than E1 and E3 deletion vectors. Metabolic DNA labeling studies showed no detectable de novo vector DNA synthesis or accumulation, and metabolic protein labeling demonstrated no detectable de novo hexon protein synthesis for Av3nBg in naive A549 cells even at a multiplicity of infection of up to 3,000 PFU per cell. Additionally, naive A549 cells infected by Av3nBg did not accumulate infectious virions. In contrast, both Av1nBg and Av2Lu vectors showed DNA replication and hexon protein synthesis at multiplicities of infection of 500 PFU per cell. Av2Lu has a deletion in E1 and also carries a temperature-sensitive mutation in E2a. Thus, molecular characterization has demonstrated that the Av3nBg vector is improved with respect to the potential for vector DNA replication and hexon protein expression compared with both first-generation (Av1nBg) and second-generation (Av2Lu) adenoviral vectors. These observations may have important implications for potential use of adenovirus vectors in human gene therapy.

Adenoviridae↗
PubMed1997

A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging.

Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with a double-stranded linear genome of 36 kb. The icosohedral virion has been shown to accommodate up to 105% of the wild-type genome length, and genomes larger than this size are either unpackageable or extremely unstable, frequently undergoing DNA rearrangement. Here we show that the Ad virion also has a lower packaging limit of approximately 75% of the wild-type genome length. We have constructed a series of vectors with sizes ranging from 15.1 to 33.6 kb and used these to show that in our Cre/loxP helper-dependent system (R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham, Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes greater than or equal to 27.7 kb are packaged with equal efficiencies, whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb vector, approximately half the size of the wild-type adenovirus genome, was packaged with an efficiency intermediate between that of the small (21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector DNA after amplification in helper virus-infected cells showed that vectors below 75% of the Ad genome had undergone DNA rearrangements, whereas larger vectors were unaltered. The 15.1-kb vector was recovered primarily as a mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30 kb. We conclude that the Ad virion can efficiently accommodate viral DNA of greater than 75% of the viral genome but that smaller viral genomes tend to undergo rearrangement, resulting in a final size of greater than approximately 27 kb before they can be efficiently packaged. Knowledge of the lower limit to Ad DNA packaging should allow for the design of better and more stable vectors.

Adenoviruses, Human↗
PubMed1998

A third-generation lentivirus vector with a conditional packaging system.

Vectors derived from human immunodeficiency virus (HIV) are highly efficient vehicles for in vivo gene delivery. However, their biosafety is of major concern. Here we exploit the complexity of the HIV genome to provide lentivirus vectors with novel biosafety features. In addition to the structural genes, HIV contains two regulatory genes, tat and rev, that are essential for HIV replication, and four accessory genes that encode critical virulence factors. We previously reported that the HIV type 1 accessory open reading frames are dispensable for efficient gene transduction by a lentivirus vector. We now demonstrate that the requirement for the tat gene can be offset by placing constitutive promoters upstream of the vector transcript. Vectors generated from constructs containing such a chimeric long terminal repeat (LTR) transduced neurons in vivo at very high efficiency, whether or not they were produced in the presence of Tat. When the rev gene was also deleted from the packaging construct, expression of gag and pol was strictly dependent on Rev complementation in trans. By the combined use of a separate nonoverlapping Rev expression plasmid and a 5' LTR chimeric transfer construct, we achieved optimal yields of vector of high transducing efficiency (up to 10(7) transducing units [TU]/ml and 10(4) TU/ng of p24). This third-generation lentivirus vector uses only a fractional set of HIV genes: gag, pol, and rev. Moreover, the HIV-derived constructs, and any recombinant between them, are contingent on upstream elements and trans complementation for expression and thus are nonfunctional outside of the vector producer cells. This split-genome, conditional packaging system is based on existing viral sequences and acts as a built-in device against the generation of productive recombinants. While the actual biosafety of the vector will ultimately be proven in vivo, the improved design presented here should facilitate testing of lentivirus vectors.

Animals↗
PubMed1998

Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery.

In vivo transduction of nondividing cells by human immunodeficiency virus type 1 (HIV-1)-based vectors results in transgene expression that is stable over several months. However, the use of HIV-1 vectors raises concerns about their safety. Here we describe a self-inactivating HIV-1 vector with a 400-nucleotide deletion in the 3' long terminal repeat (LTR). The deletion, which includes the TATA box, abolished the LTR promoter activity but did not affect vector titers or transgene expression in vitro. The self-inactivating vector transduced neurons in vivo as efficiently as a vector with full-length LTRs. The inactivation design achieved in this work improves significantly the biosafety of HIV-derived vectors, as it reduces the likelihood that replication-competent retroviruses will originate in the vector producer and target cells, and hampers recombination with wild-type HIV in an infected host. Moreover, it improves the potential performance of the vector by removing LTR sequences previously associated with transcriptional interference and suppression in vivo and by allowing the construction of more-stringent tissue-specific or regulatable vectors.

3T3 Cells↗
PubMed1998

Transient inhibition of CD28 and CD40 ligand interactions prolongs adenovirus-mediated transgene expression in the lung and facilitates expression after secondary vector administration.

Recombinant adenovirus vectors have been used to transfer genes to the lungs in animal models, but the extent and duration of primary transgene expression and the ability to achieve expression after repeated vector administration have been limited by the development of antigen-specific immunity to the vector and, in some cases, to vector-transduced foreign proteins. To determine if focused modulation of the immune response could overcome some of these limitations, costimulatory interactions between T cells and B cells/antigen-presenting cells were transiently blocked around the time of vector administration. Systemic treatment at the time of primary-vector administration with a monoclonal antibody (MR1) against murine CD40 ligand, combined with recombinant murine CTLA4Ig and intratracheal coadministration of an adenovirus vector transducing the expression of murine CTLA4Ig, prolonged adenovirus-transduced beta-galactosidase expression in the airways for up to 28 days and resulted in persistent alveolar expression for >90 days (the duration of the experiment). Consistent with these results, this treatment regimen reduced local inflammation and markedly reduced the T-cell and T-cell-dependent antibody response to the vector. A secondary adenovirus vector, administered >90 days after the last systemic dose of MR1 and muCTLA4Ig, resulted in alkaline phosphatase expression at levels comparable to those seen with primary-vector administration. Expression of the secondary transgene persisted in the alveoli (but not in the airways) for up to 24 days (the longest period of observation) at levels similar to those observed on days 3 to 4. These results indicate that transient inhibition of costimulatory molecule interactions substantially enhanced gene transfer to the alveoli but was much less effective in the airways. This suggests that there are differences in the efficiency or nature of mechanisms limiting transgene expression in the airways and in the alveoli.

Abatacept↗
PubMed1999

Isolation of recombinant adeno-associated virus vector-cellular DNA junctions from mouse liver.

Recombinant adeno-associated virus (rAAV) vectors allow for sustained expression of transgene products from mouse liver following a single portal vein administration. Here a rAAV vector expressing human coagulation factor F.IX (hF.IX), AAV-EF1alpha-F.IX (hF.IX expression was controlled by the human elongation factor 1alpha [EF1alpha] enhancer-promoter) was injected into mice via the portal vein or tail vein, or directly into the liver parenchyma, and the forms of rAAV vector DNA extracted from the liver were analyzed. Southern blot analyses suggested that rAAV vector integrated into the host genome, forming mainly head-to-tail concatemers with occasional deletions of the inverted terminal repeats (ITRs) and their flanking sequences. To further confirm vector integration, we developed a shuttle vector system and isolated and sequenced rAAV vector-cellular DNA junctions from transduced mouse livers. Analysis of 18 junctions revealed various rearrangements, including ITR deletions and amplifications of the vector and cellular DNA sequences. The breakpoints of the vector were mostly located within the ITRs, and cellular DNA sequences were recombined with the vector genome in a nonhomologous manner. Two rAAV-targeted DNA sequences were identified as the mouse rRNA gene and the alpha1 collagen gene. These observations serve as direct evidence of rAAV integration into the host genome of mouse liver and allow us to begin to elucidate the mechanisms involved in rAAV integration into tissues in vivo.

Animals↗
PubMed2001

ICP0, ICP4, or VP16 expressed from adenovirus vectors induces reactivation of latent herpes simplex virus type 1 in primary cultures of latently infected trigeminal ganglion cells.

In a previous study, we demonstrated that infected-cell polypeptide 0 (ICP0) is necessary for the efficient reactivation of herpes simplex virus type 1 (HSV-1) in primary cultures of latently infected trigeminal ganglion (TG) cells (W. P. Halford and P. A. Schaffer, J. Virol. 75:3240-3249, 2001). The present study was undertaken to determine whether ICP0 is sufficient to trigger HSV-1 reactivation in latently infected TG cells. To test this hypothesis, replication-defective adenovirus vectors that express wild-type and mutant forms of ICP0 under the control of a tetracycline response element (TRE) promoter were constructed. Similar adenovirus vectors encoding wild-type ICP4, wild-type and mutant forms of the HSV-1 origin-binding protein (OBP), and wild-type and mutant forms of VP16 were also constructed. The TRE promoter was induced by coinfection of Vero cells with the test vector and an adenovirus vector that expresses the reverse tetracycline-regulated transactivator in the presence of doxycycline. Northern blot analysis demonstrated that transcription of the OBP gene in the adenovirus expression vector increased as a function of doxycycline concentration over a range of 0.1 to 10 microM. Likewise, Western blot analysis demonstrated that addition of 3 microM doxycycline to adenovirus vector-infected Vero cells resulted in a 100-fold increase in OBP expression. Wild-type forms of ICP0, ICP4, OBP, and VP16 expressed from adenovirus vectors were functional based on their ability to complement plaque formation in Vero cells by replication-defective HSV-1 strains with mutations in these genes. Adenovirus vectors that express wild-type forms of ICP0, ICP4, or VP16 induced reactivation of HSV-1 in 86% +/- 5%, 86% +/- 5%, and 97% +/- 5% of TG cell cultures, respectively (means +/- standard deviations). In contrast, vectors that express wild-type OBP or mutant forms of ICP0, OBP, or VP16 induced reactivation in 5% +/- 5%, 8% +/- 0%, 0% +/- 0%, and 13% +/- 6% of TG cell cultures, respectively. In control infections, an adenovirus vector expressed green fluorescent protein efficiently in TG neurons but did not induce HSV-1 reactivation. Therefore, expression of ICP0, ICP4, or VP16 is sufficient to induce HSV-1 reactivation in latently infected TG cell cultures. We conclude that this system provides a powerful tool for determining which cellular and viral proteins are sufficient to induce HSV-1 reactivation from neuronal latency.

Adenoviridae↗