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At least 1,153 records · Page 64Linked to original sources
PubMed2006

Enhanced gene transfer efficiency in the murine striatum and an orthotopic glioblastoma tumor model, using AAV-7- and AAV-8-pseudotyped vectors.

In this study, recombinant AAV vectors pseudotyped with viral capsids derived from AAV serotypes 7 and 8 were evaluated for gene transfer in the murine striatum relative to vectors pseudotyped with AAV serotypes 2, 5, and 6. In comparison with rAAV serotype 2, pseudotyped vectors derived from AAV-7 and AAV-8 have increased transduction efficiency in the murine CNS, with the rank order rAAV-7 > rAAV-8 > rAAV-5 > rAAV-2 = rAAV-6, with all vectors demonstrating a marked tropism for neuronal transduction. Pseudotyped rAAV vector gene transfer in the brain after preimplantation of a murine 4C8 glioblastoma tumor was also evaluated. Efficiency of gene transfer to the orthotopic tumor was increased when using AAV-6, -7, and -8 capsid proteins in comparison with serotype 2, with the order rAAV-8 = rAAV-7 > rAAV-6 > rAAV-2 > rAAV-5. The increased gene transfer efficiency of rAAV vectors pseudotyped with the rAAV-8 capsid also provided enhanced therapeutic efficacy in a mouse model of glioblastoma multiforme, using vectors encoding an inhibitor of the vascular endothelial growth factor pathway. These studies demonstrate that rAAV vectors pseudotyped with capsids derived from AAV serotypes 7 and 8 provide enhanced gene transfer in the murine CNS and may offer increased therapeutic efficacy in the treatment of neurological disease.

Animals↗
PubMed2002

Adenovirus gene transfer vector toxicity to mouse embryos: implications for human IVF.

BACKGROUND: The promulgation and diversification of micromanipulation procedures which open the zona pellucida of the oocyte or early embryo is steadily increasing the chance that zygotes will encounter infectious viral agents or gene transfer vectors derived from these agents. Such interactions could lead to toxic effects on the embryo or to insertion of foreign genes into the germ line. Adenovirus is a ubiquitous human viral pathogen that is commonly used as a gene therapy vector. However, the toxicity of this virus or its vector derivatives to embryos has not been extensively investigated. METHODS: In the present study, a mouse model was used to investigate the time of appearance of embryo toxicity, the manifestations of that toxicity, and the mechanism by which adenoviruses exert toxicity. The effects of exposure to adenovirus on in-vivo embryo development was also examined. RESULTS: These vectors exerted no deleterious effects until after the 2-cell stage, where they caused developmental delay and disorganized cleavage. Toxicity was associated with expression of a lacZ reporter gene cloned into the vectors, and with the proportion of infectious particles within the virus preparations: preparations with a high proportion of 'empty capsids', including a preparation of wild-type virus, were less toxic than a replication-defective vector with a high proportion of functional, infectious particles. Subzonal insertion of adenovirus vector followed by embryo transfer led to a dramatic reduction in the number of embryos which developed to term-findings which establish the physiological significance of the findings from in-vitro culture. CONCLUSIONS: These results indicate that adenoviruses and their vector derivatives are not likely to insert genetic material into the germ line, but they may pose a significant threat to the viability of human embryos undergoing opening of the zona pellucida during IVF.

Adenoviridae↗
PubMed1992

Japanese encephalitis virus in Bangkok: factors influencing vector infections in three suburban communities.

An unexpected outbreak of Japanese encephalitis (JE) in Bangkok in 1985 led us to investigate the vector ecology of urban JE from January 1986 to June 1987 at three suburban sites that displayed a wide range of factors imputed to influence JE transmission. Culex tritaeniorhynchus Giles and Cx. gelidus Theobald, suspected vectors, comprised 71-96% of all mosquitoes collected by CO2-baited CDC traps at the three sites. Mean of mosquito abundance per two trap-nights per month ranged from 28 to 5,728 mosquitoes at the sites of lowest and highest abundance, respectively. Cx. tritaeniorhynchus yielded more JE isolates (n = 16) than Cx. gelidus (n = 7), but the minimum infection rates of the two species (number of JE isolates per 1,000 mosquitoes tested; MIR, 0.17 and 0.47, respectively) were comparable and covaried with vector abundance. Moreover, the proportion of sentinel pigs that had JE antibodies generally increased proportionately with vector abundance at the sites. Vector abundance was high in monsoon (May-October), moderate in transition (March-April and November-December), and low in dry (January-February) seasons. Mosquitoes collected in monsoon seasons yielded 96% of the JE isolates, whereas 4 and 0% of the isolates were obtained from transition and dry season collections, respectively. More pigs seroconverted in monsoon and transition seasons than in dry seasons. Indices of JE transmission activity (vector abundance, pig seroconversions, and MIRs) increased proportionately with rainfall. Despite higher indices at the site of greatest vector abundance than elsewhere, the risk of human infection appeared greatest at the site with moderate vector abundance because of its greatest human population density.

Animals↗
PubMed1991

A novel BK virus-based episomal vector for expression of foreign genes in mammalian cells.

A composite mammalian cell-E. coli shuttle vector was developed based on the human papova virus BK and pSV-neo. The vector contains a dioxin-responsive enhancer (DRE) controlling a mouse mammary tumor virus (MMTV) promoter for the inducible expression of inserted genes. In human cells the vector replicates episomally, presumably utilizing the BKV rather than the SV40 origin, and expresses the BK T/t antigens. A deletion in the late BK region precludes the expression of the core/capsid proteins VP1, VP2, and VP3, thereby preventing the infectious lytic cycle. HeLa cells which were transfected with this vector and selected for resistance to the antibiotic G418 maintained the construct primarily in episomal form during more than one year of continuous culture, with little or no integration into the host genome. Transformed cells cultured in higher concentrations of G418 contained higher copy numbers of the vector. This permits one to vary the dosage of an inserted gene easily and reversibly without the need of conventional amplification techniques and clonal analysis. Using a chloramphenicol acetyl transferase (CAT) reporter gene inserted downstream of the MMTV promoter, we found that CAT expression was greater in clones with higher vector copy number. CAT expression was inducible with 2,3,7,8-tetrachlorodibenzo-p-dioxin, but inducibility was found to be inversely proportional to the copy number. Transformation of bacteria with plasmid molecules retrieved from the mammalian host was efficient, making this vector well adapted for the screening of cDNA libraries for the ability to express a phenotype in mammalian cells. Moreover, DNA sequences were stable during long-term passage in mammalian cells; vector passaged continuously for more than one year retained fully functional bacterial genes for resistance to chloramphenicol and ampicillin.

BK Virus↗
PubMed1996

High-titer bicistronic retroviral vectors employing foot-and-mouth disease virus internal ribosome entry site.

Bicistronic retroviral vectors were constructed containing the foot-and-mouth disease virus (FMDV) internal ribosome entry site (IRES) followed by the coding region of beta-galactosidase (beta-gal) or therapeutic genes, with the selectable neomycin phosphotransferase gene under the control of the viral long terminal repeat (LTR) promoter. LNFX, a vector with a multiple cloning site 3' to foot-and-mouth disease virus IRES, was used to construct vectors encoding rat erythropoietin (EP), rat granulocyte colony-stimulating factor (G-CSF), human adenosine deaminase (ADA) and beta-gal. In transduced primary rat vascular smooth muscle cells the cytokines were expressed at high levels, similar to those obtained from vectors employing the viral LTR promoter. LNFZ, a vector encoding beta-gal, had a 10-fold increase in titer over that of LNPoZ, a comparable vector containing the poliovirus (Po) internal ribosome entry site. Primary canine vascular smooth muscle cells infected with LNFZ and LNPoZ expressed similar activities of beta-gal and neomycin phosphotransferase (NPT). Overall, these vectors had titers between 10(6) and 2 x 10(7) c.f.u./ml, indicating that foot-and-mouth disease virus IRES provides high-titer bicistronic vectors with high-level two gene expression.

3T3 Cells↗
PubMed1995

Retroviruses as vectors.

Recombinant retroviruses have long been used to deliver heterologous genes to mammalian cells. Convenient packaging cell lines and vector plasmids have been distributed widely and 'home-made' retroviral vectors have now become a useful research tool in many laboratories. Compared to more traditional methods of gene transfer, retroviral vectors are extraordinarily efficient gene delivery vehicles which cause no detectable harm as they enter their target cells. In the nucleus the retroviral necleic acid becomes integrated into chromosomal DNA, ensuring its long-term persistence and stable transmission to all future progeny of the transduced cell. Up to 8 kilobases of foreign gene sequence can be packaged in a retroviral vector and this is more than enough for most gene therapy applications. Retroviral vectors can also be manufactured in large quantities to meet very stringent safety specifications. They have therefore been selected as the vectors of choice in 80% of the clinical gene therapy trials that have been approved to date. So far there have been no reported short- or long-term toxicity problems associated with their use in human gene therapy trials, now dating back to 1989. However, despite this impressive record, there is still great scope (and need) for the development of new, improved retroviral vectors and packaging systems to fuel further advances in the field of human gene therapy. In the following discussion, existing retroviral vectors are reviewed and current areas of technological development are emphasised.

Gene Expression Regulation, Viral↗
PubMed1997

Herpes simplex viral vectors expressing Bcl-2 are neuroprotective when delivered after a stroke.

Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system for the purpose of decreasing necrotic neuronal injury. To that end, we have previously shown that a herpes simplex virus (HSV) vector expressing Bcl-2 could protect neurons from ischemia. In that study, vector was delivered before the ischemia. However, for such gene therapy to be of clinical use, vectors must be protective even if delivered after the onset of the insult. In the present study, we show that an HSV vector expressing Bcl-2 protects striatal neurons when delivered after focal ischemia. Rats were exposed to middle cerebral artery occlusion for 1 hour, followed by reperfusion, and damage was assessed 48 hours later. Delivery of the Bcl-2 vector 30 minutes after reperfusion (i.e., 1.5 hours after ischemia onset) prevented any significant loss of virally-targeted neurons in the striatum. In contrast, in rats microinfused with a vector only expressing a reporter gene, a highly significant loss of neurons occurred. By 4 hours into the reperfusion period (5 hours after ischemia onset), delivery of the Bcl-2 vector was no longer protective. These data show the efficacy of postinsult gene therapy strategies for the brain, underline the finite length of this temporal therapeutic window, and support the growing evidence attesting to the neuroprotective potential of Bcl-2.

Animals↗
PubMed2006

Gene transfer to the gastrointestinal tract after peroral administration of recombinant adeno-associated virus type 2 vectors.

OBJECTIVES: The transfer of exogenous genetic material to cells within the gastrointestinal (GI) tract has many potential therapeutic applications. An attractive feature of the GI tract for gene transfer is its accessibility through the orogastric route. In this study, we evaluated the stability of recombinant adeno-associated virus type 2 (rAAV2) vectors within the GI tract and whether rAAV2-mediated gene transfer could be increased through manipulation of the intraluminal environment. METHODS: The stability of rAAV2 vectors carrying beta-galactosidase and enhanced green fluorescence protein transgenes was determined in the presence of hydrochloric acid, pepsin, trypsin, chymotrypsin gastric fluid and intestinal fluid and after in vivo administration. For in vivo experiments, the rAAV2 vector carrying the beta-galactosidase transgene was administered perorally to FVB/NJ mice. Groups of mice received the vector alone or in combination with sodium bicarbonate and aprotinin. Gene transfer to the stomach and small intestine was evaluated by polymerase chain reaction and histochemical assays. RESULTS: The stability of rAAV2 was reduced by hydrochloric acid, trypsin, chymotrypsin, gastric fluid and intestinal fluid. The vector was not stable within the lumen of the GI tract. Gastric acid neutralization with sodium bicarbonate and protease inhibition with aprotinin increased the in vivo stability of the vector and the level of gene transfer to the stomach and all regions of the small bowel. In both groups of mice (vector alone and vector plus sodium bicarbonate and aprotinin), transgene-derived protein expression (beta-galactosidase) was below the level of detection of the histochemical assay. CONCLUSIONS: Recombinant AAV2 are adversely affected by physiological conditions within the proximal GI tract. Gastric acid neutralization and inhibition of intestinal protease activity improved rAAV2 stability and increased the level of gene transfer within the GI tract. Despite these changes, transduction of the GI tract after peroral rAAV2 administration remained low.

Animals↗
PubMed2006

Generation of a novel replication-incompetent adenoviral vector derived from human adenovirus type 49: manufacture on PER.C6 cells, tropism and immunogenicity.

Recombinant adenoviral vectors based on type 5 (rAd5) show great promise as a vaccine carrier. However, neutralizing activity against Ad5 is prevalent and high-titred among human populations, and significantly dampens Ad5-based vaccine modalities. The generation of alternative adenoviral vectors with low seroprevalence thus receives much research attention. Here, it is shown that a member from human adenovirus subgroup D, i.e. Ad49, does not cross-react with Ad5 neutralizing activity, making it a candidate serotype for vector development. Therefore, a plasmid system that allows formation of replication-incompetent adenovirus serotype 49 vaccine vectors (rAd49) was constructed and it was demonstrated that rAd49 can be successfully propagated to high titres on existing Ad5.E1-complementing cell lines such as PER.C6. Using an rAd49 vector carrying the luciferase marker gene, detailed seroprevalence studies were performed, demonstrating that rAd49 has low seroprevalence and neutralizing antibody titres worldwide. Also, we have initiated rAd49 vector receptor usage suggesting that rAd49 utilizes hCD46 as a cellular receptor. Finally, the immunogenicity of the rAd49 vector was assessed and it was shown that an rAd49.SIVGag vaccine induces strong anti-SIVGag CD8+ T-lymphocytes in naïve mice, albeit less than an rAd5.SIVGag vaccine. However, in mice with high anti-Ad5 immunity the rAd5.SIVGag vaccine was severely blunted, whereas the anti-SIVGag response was not significantly suppressed using the rAd49.SIVGag vaccine. These data demonstrate the potential of a replication deficient human group D adenoviral vector for vaccination purposes.

Adenoviruses, Human↗
PubMed2000

Cationic liposome conjugation to recombinant adenoviral vector reduces viral antigenicity.

Adenoviral (Ad) vectors are commonly used in gene therapy trials because of their efficiency in gene transfer. However, their use is limited by immune responses that reduce transgene expression and decrease the efficacy of repeated vector administration. In this study, we demonstrated that conjugation of Ad vector with our novel cationic liposomes could reduce viral antigenicity in vivo. Mice subcutaneously injected with liposome-conjugated Ad vector showed a 6.5-fold reduction of anti-Ad antibodies with neutralizing activity, compared to those with unconjugated Ad vector. Interestingly, we also found that the conjugated vector is less susceptible to inactivation by neutralizing antibodies in vitro and in vivo. Our results suggest that liposome conjugation reduces viral antigenicity, shields vectors from neutralizing antibody, and may allow repeated Ad vector administration.

Adenoviridae↗
PubMed2005

Electrogene transfer of an Epstein-Barr virus-based plasmid replicon vector containing the diphtheria toxin A gene suppresses mammary carcinoma growth in SCID mice.

Experimental mammary cancer therapy in mice was conducted using electrogene transfer of a non-viral EBV-based plasmid vector (reduced size of the oriP gene), containing the DT-A gene. Because the EBV-based plasmid vector exhibits high transfer efficiency and strong persistent transgene expression due to autonomous replication in human cells, it is particularly suitable as a tool for cancer gene therapy. In vitro, 79% of MDA-MB231 human mammary carcinoma cells died as a result of the EBV-based vector containing DT-A (pEB-DTA) by 48 h after transfection. DNA synthesis was also significantly decreased as compared to levels with a control vector. In vivo, mammary tumors induced by inoculation of SCID mice with MDA-MB231 cells were subsequently treated by direct injection of pEB-DTA vector or pEB-GFP vector as a control once a week for 5 weeks. After each injection, the tumors were subjected to in vivo electrogene transfer. Significantly reduced tumor volumes were observed for the pEB-DTA group in experimental week 1 and thereafter throughout the study. At necropsy, strong and extent expression of GFP was still observed in tumors receiving pEB-GFP 6 days after the last electrogene transfer. The ratio of histological necrotic area to viable area was significantly increased in the pEB-DTA-treated tumors, where levels of apoptosis were significantly higher than those observed in the pEB-GFP group. DNA synthesis showed a tendency to decrease in the pEB-DTA group but this was not significant. The incidence and multiplicity of lung metastasis were similar between the groups. There was also no difference in the density of microvessels between groups. We therefore conclude that the EBV-based plasmid vector system combined with in vivo electrogene transfer can result in efficient gene transfection and that the non-viral replicon vector containing DT-A suppresses tumor growth due to apoptotic cell death in this model.

Animals↗
PubMed1996

Vector-borne diseases and the basic reproduction number: a case study of African horse sickness.

The basic reproduction number, R0, can be used to determine factors important in the ability of a disease to invade or persist. We show how this number can be derived or estimated for vector-borne diseases with different complicating factors. African horse sickness is a viral disease transmitted mainly by the midge Culicoides imicola. We use this as an example of such a vector-transmitted disease where latent periods, seasonality in vector populations, and multiple host types may be important. The effect of vector population dynamics which are dependent on either host or vector density are also addressed. If density-dependent constraints on vector population density are less severe, R0 is more sensitive to vector mortality and the virus development rate. Host-dependent vector dynamics change the relationship between R0 and host population size. Seasonality can either increase or decrease the estimate of R0, depending on the lag between the peak of the midge population and the infective host population. The relative abundance of two host types is a factor in the ability of a disease to invade, but the strength of this factor depends on the differences between the hosts in recovery from infection, mortality and transmission. Removal of a reservoir host may increase R0.

African Horse Sickness↗
PubMed2006

Gateway-compatible vectors for plant functional genomics and proteomics.

Gateway cloning technology facilitates high-throughput cloning of target sequences by making use of the bacteriophage lambda site-specific recombination system. Target sequences are first captured in a commercially available "entry vector" and are then recombined into various "destination vectors" for expression in different experimental organisms. Gateway technology has been embraced by a number of plant laboratories that have engineered destination vectors for promoter specificity analyses, protein localization studies, protein/protein interaction studies, constitutive or inducible protein expression studies, gene knockdown by RNA interference, or affinity purification experiments. We review the various types of Gateway destination vectors that are currently available to the plant research community and provide links and references to enable additional information to be obtained concerning these vectors. We also describe a set of "pEarleyGate" plasmid vectors for Agrobacterium-mediated plant transformation that translationally fuse FLAG, HA, cMyc, AcV5 or tandem affinity purification epitope tags onto target proteins, with or without an adjacent fluorescent protein. The oligopeptide epitope tags allow the affinity purification, immunolocalization or immunoprecipitation of recombinant proteins expressed in vivo. We demonstrate the utility of pEarleyGate destination vectors for the expression of epitope-tagged proteins that can be affinity captured or localized by immunofluorescence microscopy. Antibodies detecting the FLAG, HA, cMyc and AcV5 tags show relatively little cross-reaction with endogenous proteins in a variety of monocotyledonous and dicotyledonous plants, suggesting broad utility for the tags and vectors.

Amino Acid Sequence↗
PubMed2002

Single-vector three-frame expression systems for affinity-tagged proteins.

An effort is presented to create expression vectors which would allow expression of an inserted gene fragment in three reading frames in a single vector from a single promoter but with three separate ribosome binding sites (RBS). Each expression frame would generate an in-frame fusion with an affinity tag to allow efficient recovery of the produced fusion proteins. In the first generation vector, three identical polyhistidyl tags (His(6)) were used as affinity tags for the three expression frames. In the second generation vector, three different tags, an albumin binding domain derived from streptococcal protein G, an IgG binding Staphylococcus aureus protein A-derived domain (Z) and a His(6) tag, were employed to allow frame-specific affinity recovery. To evaluate the systems, model genes have been inserted in three different frames in both vectors. The first vector was demonstrated to produce fusion proteins in all three frames, whereas for the second, with a much wider spacing between the RBSs and affinity tags, expression could only be demonstrated from the first two translational start sites. For both systems, the first translation start was found to be significantly favored over the others. Nevertheless, we believe that the presented results represent the first successful attempt to create single-vector three-frame expression systems, a concept that could become valuable in future combined cloning-expression vectors.

Bacterial Proteins↗
PubMed2004

Development of packaging cell lines for generation of adeno-associated virus vectors by lentiviral gene transfer of trans-complementary components.

Adeno-associated virus (AAV) vector system has several useful advantages with regard to in vitro and in vivo gene transfer. However, their usages have been limited by cumbersome and labor-intensive vector production in the traditional method. To overcome limitations in AAV production, in this report, we explored the possibility of generating AAV packaging cell line, 293T R/C.VA.E2A.E4. cells, by using lentivirus-mediated transduction of Rep/Cap gene of AAV-2, VA RNA, E2A, and E4 genes of Ad5 into 293T cells. In packaging cell lines, it is important that supply of the AAV vector can be stably performed for long time. We showed that the 293T R/C.VA.E2A.E4. cells have stably maintained the transduced components after more than 10 passages and yielded high-titer AAV vectors, and the titer of AAV vectors did not decline even if culture of the packaging cells was continued for long time. The Rep/Cap and E4 gene products caused no remarkable cytotoxicity. The 293T R/C.VA.E2A.E4. cells might be able to tolerate the Rep/Cap and E4 gene products, or have less copy numbers of the Rep/Cap and E4 genes than the traditional method. Moreover, we showed that the AAV vectors derived from 293T R/C.VA.E2A.E4. cells infected the primary human CD34+ haematopoietic progenitor cells with high efficiency (50-70%). In the 293T R/C.VA.E2A.E4. cells, the AAV vectors can be generated by the transfection of one AAV vector plasmid, and large-scale AAV production can be easily achieved. It is important that cumbersome, variable, and costly transfection is avoided.

Base Sequence↗
PubMed1993

Analysis of Streptococcus pyogenes promoters by using novel Tn916-based shuttle vectors for the construction of transcriptional fusions to chloramphenicol acetyltransferase.

We have developed a series of shuttle vectors based on the conjugative transposon Tn916 that have been designed for the analysis of transcriptional regulation in Streptococcus pyogenes and other gram-positive bacteria. Designated the pVIT vectors (vectors for integration into Tn916), the vectors are small, stable plasmids in Escherichia coli to facilitate the fusion of promoters from cloned S. pyogenes genes to a promoterless gene which encodes chloramphenicol acetyltransferase. The vectors each contain one or more small regions of Tn916 to direct the integration of the transcriptional fusion into the transposon via homologous recombination following transformation of S. pyogenes or other suitable gram-positive hosts. Integration can be monitored by the inactivation or replacement of an antibiotic resistance determinant in modified derivatives of Tn916. Promoter activity can then be quantitated by the determination of chloramphenicol acetyltransferase-specific activity. In addition, since integration is into loci that do not disrupt the conjugative transpositional functions of Tn916, the vectors are useful for analysis of regulation in strains that are difficult or impossible to transform and can be introduced into these strains by conjugation following transformation of an intermediate host. The promoters for the genes which encode both the M protein and protein F of S. pyogenes were active in pVIT vectors, as was the region which controls transcription of mry, a trans-acting positive regulator of M protein expression. However, neither of the two characterized promoters for mry demonstrated activity when independently analyzed in pVIT-generated partial diploid strains, suggesting that regulation of mry is more complex than predicted by current models. The broad host range of Tn916 should make the pVIT vectors useful for analysis of regulation in numerous other bacterial species.

Antigens, Bacterial↗
PubMed2006

A helper-dependent capsid-modified adenovirus vector expressing adeno-associated virus rep78 mediates site-specific integration of a 27-kilobase transgene cassette.

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.

Adenoviridae↗
PubMed2006

Optimization of feline immunodeficiency virus vectors for RNA interference.

RNA interference (RNAi) occurs naturally in plant and animal cells as a means for modulating gene expression. This process has been experimentally manipulated to achieve targeted gene silencing in cells, tissues, and animals, using a variety of vector systems. Here, we tested the hypothesis that vectors based on feline immunodeficiency virus (FIV) could be used for coexpression of reporter constructs and RNAi expression cassettes. We found, unexpectedly, in our initial constructs that placement of RNAi expression cassettes downstream from a polymerase II (pol II)-expressed reporter gene inhibited reporter expression but not vector titer. Through a series of intermediate vector constructs, we found that placement of the RNAi expression cassette relative to the Rev response element and the pol II expression cassette was critical for efficient RNAi and reporter gene expression. These results suggested that steric factors, including RNA structure and recruitment of competing transcriptional machinery, may affect gene expression from FIV vectors. In a second series of studies, we show that target sequence silencing can be achieved in cells transduced by FIV vectors coexpressing reporter genes and 3' untranslated region resident microRNAs. The optimized FIV-based RNAi expression vectors will find broad use given the extensive tropism of pseudotyped FIV vectors for many cell types in vitro and in vivo.

Cell Line, Tumor↗