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At least 955 records · Page 53Linked to original sources
PubMed2005

Vector control in transportation osteogenesis.

PURPOSE: To evaluate the ability to control vectors in the technique of transportation osteogenesis using 4 principles. PATIENTS AND METHODS: Sixteen defects measuring approximately 30-180 mm were retrospectively evaluated. Vector control was attempted using a single or combination of the following principles: multiple linear vectors, exaggerated linear distraction ("sausage effect"), braced guided distraction, and reorientation osteotomies. Observations were made regarding the ability to create the desired vectors. Angular deviation from desired vectors was determined and recorded numerically by evaluating either submental vertex or occlusal radiographs, stereolithographic models, or digital radiographs. Radiographic evaluation of symmetry alone was not used to determine the desired vector because a more laterally displaced segment was desirable to maintain favorable facial balance when soft tissue deficiency was pronounced. A subjective evaluation scale was developed to supplement the numerical values. RESULTS: Vector control primarily required the use of 2 or more principles. The most common combination was that of exaggerated linear distraction and reorientation osteotomy. CONCLUSION: The use of multiple linear vectors, exaggerated linear distraction ("sausage effect"), reorientation osteotomies, and braced guided distraction greatly assisted the operator in achieving the goal of symmetrical reconstruction. Transportation osteogenesis may be considered an effective tool in the box to reconstruct patients. In cases in which vector control is achieved, the advantages of diminishing the volume of bone graft required or achieving final bony reconstruction can outweigh the disadvantages of the technique.

Adult↗
PubMed2002

Oncoretroviral and lentiviral vector-mediated gene therapy.

Oncoretroviral vectors and lentiviral vectors offer the potential for long-term gene expression by virtue of their stable chromosomal integration and lack of viral gene expression. Consequently, their integration allows passage of the transgene to all progeny cells, which makes them particularly suitable for stem cell transduction. However, a disadvantage of oncoretroviral vectors based on Moloney murine leukemia virus (MoMLV) is that cell division is required for transduction and integration, thereby limiting oncoretroviral-mediated gene therapy to actively dividing target cells. In contrast, lentiviral vectors can transduce both dividing and nondividing cells. Lentiviral vectors have been derived from either human or primate lentiviruses, with the human immunodeficiency virus (HIV) as prototype, or from nonprimate lentiviruses, such as the equine infectious anemia virus (EIAV). The ability to pseudotype oncoretroviral and lentiviral vectors with the vesicular stomatitis virus G glycoprotein (VSV-G) allowed for the production of high-titer vectors (10(9)-10(10) transducing units/ml). These high-titer vector preparations were shown to effectively cure genetic diseases in experimental animal models and constitute an essential step toward direct in vivo gene therapy applications. This chapter focuses on different methods that permit large-scale production of high-titer VSV-G pseudotyped oncoretroviral and primate or nonprimate lentiviral vectors and highlights their importance for achieving therapeutic effects in preclinical animal models.

Animals↗
PubMed2001

Power vector analysis of the optical outcome of refractive surgery.

PURPOSE: To demonstrate the power vector method of representing and analyzing spherocylindrical refractive errors. SETTING: School of Optometry, Indiana University, Bloomington, Indiana, USA. METHODS: Manifest and keratometric refractive errors were expressed as power vectors suitable for plotting as points in a 3-dimensional dioptric space. The 3 Cartesian coordinates (x, y, z) of each power vector correspond to the powers of 3 lenses that, in combination, fulfill a refractive prescription: a spherical lens of power M, a Jackson crossed cylinder of power J0 with axes at 90 degrees and 180 degrees, and a Jackson crossed cylinder of power J45 with axes at 45 degrees and 135 degrees. The Pythagorean length of the power vector, B, is a measure of overall blurring strength of a spherocylindrical lens or refractive error. Changes in refractive error due to surgery were computed by the ordinary rules of vector subtraction. RESULTS: Frequency distributions of blur strength (B) clearly demonstrate the effectiveness of refractive surgery in reducing the overall blurring effect of uncorrected refractive error. CONCLUSIONS: Power vector analysis also revealed a reduction in the astigmatic component of these refractive errors. Paired comparisons revealed that the change in manifest astigmatism due to surgery was well correlated with the change in keratometric astigmatism. Power vectors aid the visualization of complex changes in refractive error by tracing a trajectory in a uniform dioptric space. The Cartesian components of a power vector are mutually independent, which simplifies mathematical and statistical analysis of refractive errors. Power vectors also provide a natural link to a more comprehensive optical description of ocular refractive imperfections in terms of wavefront aberration functions and their description by Zernike polynomials.

Astigmatism↗
PubMed1998

Similarity of strain- and route-dependent murine responses to an adenovirus vector using the homologous thrombopoietin cDNA as the reporter genes.

Replication-deficient adenovirus (Ad) vectors are effective in transferring genes in vivo, but their use is associated with significant variation in the extent and/or duration of expression observed among different strains of experimental animals and different routes of administration of the vector. We have minimized the variables of the heterologous transgene and animal-to-animal variation by constructing an Ad vector encoding murine thrombopoietin (mTPO, AdmTPO), a homologous protein that induces a physiologic response (elevation of blood platelet levels) that can be followed sequentially over time in the same animal. Using the C57BL/6 and BALB/c stains, liver administration was accomplished by intravenous administration and skeletal muscle administration by direct injection. Despite the use of a homologous cDNA as a transgene, the Ad genome was rapidly lost from the liver after intravenous administration over the first 1 to 2 weeks, with no difference in pattern of decline between the C57BL/6 and BALB/c strains. Both strains exhibited a cytotoxic T lymphocyte (CTL) response directed against the AdmTPO vector. Consistent with the decline in vector genome over time, the initial high levels of mTPO mRNA in the liver declined to an undetectable level within 2 weeks. Platelet counts peaked at 8- to 10-fold above baseline within the first 2 weeks, and then gradually declined, returning to normal level by 50 to 60 days. Intravenous administration of the AdmTPO vector to beta2-microglobulin-deficient mice resulted in a longer persistence of elevated platelets levels, although the eventual return of platelet levels to normal in these mice suggests the elimination of the Ad vector cannot be explained solely by CTL response. Although the intramuscular administration of the AdmTPO vector resulted in platelet levels with a lower peak and minor differences over time compared with the intravenous route, the C57BL/6 and BALB/c strains demonstrated the same rapid loss of Ad genome and mTPO mRNA levels in the muscle as in the liver. Together, these observations suggest that simplifying the experimental design by eliminating the variable of host response to a heterologous transgene, and by following the consequences of gene transfer in the same animals over time, there can be remarkable similarity in strain- and route-dependent responses to an Ad vector.

Adenoviridae↗
PubMed2004

Population vector analysis of primate prefrontal activity during spatial working memory.

Population vectors were used to examine information represented by a population of prefrontal activity and its temporal change during spatial working memory processes while monkeys performed ODR and R-ODR tasks. In the ODR task, monkeys made a saccade to the cue location after the delay, whereas in the R-ODR task, they made a saccade 90 degrees clockwise from the cue location. We first constructed population vectors using cue- and response-period activity. The directions of population vectors were similar to the cue directions and the saccade target directions, respectively, indicating that population vectors correctly represented information regarding directions of visual cues and saccade targets. We then calculated population vectors during a 250 ms time-window from the cue presentation to the end of the response period. In the ODR task, all population vectors were directed toward the cue direction. However, in the R-ODR task, the population vector gradually rotated during the delay period from the cue direction to the saccade direction. These results indicate that spatial information represented by a population of prefrontal activity can be shown as the direction of the population vector and that its temporal change during spatial working memory tasks can be depicted as the temporal change of the vector's direction.

Animals↗
PubMed1993

Ischaemia and reperfusion induced transient QRS vector changes: relationship to size of the ischaemic territory.

OBJECTIVE: The aim was to investigate QRS vector changes during the first 30 min of coronary occlusion or the early phase of reperfusion with special reference to location and size of myocardium at risk. METHODS: 24 h experiments were performed in closed chest anaesthetised pigs. QRS vectors were studied by computerised vectorcardiography via Frank leads. Occlusion of the left anterior descending coronary artery followed by reperfusion was induced in 23 pigs and a sustained occlusion in 20 pigs: left anterior descending coronary artery in seven, right coronary artery in eight, and left circumflex coronary artery in five. Myocardium at risk was measured in postmortem autoradiograms. Eight animals were excluded. RESULTS: Four minutes after occlusion, QRS(mean) deviated towards the ischaemic region in 34/35 animals and returned thereafter at varying speeds. In half of the reperfused animals, deviation of QRS vectors towards the ischaemic territory was also observed during the first minutes of reperfusion. A paradoxical increase in QRS vector changes, "reperfusion peak", was recorded during the initial minutes of reperfusion in 12/19 animals. Maximum spatial QRS vector magnitude increased in all right coronary or left circumflex coronary occlusion animals compared to 6/25 in left anterior descending coronary occlusion animals. QRS vector difference, change in spatial QRS vector angle, and maximum change in QRS azimuth 4 min after occlusion correlated significantly with extent of myocardium at risk. CONCLUSIONS: Marked directional and quantitative QRS vector changes, with significant relation to size and location of myocardium at risk, were recorded during the initial minutes of ischaemia. The transient increase in QRS vector changes during the first minutes of reperfusion deserves further exploration as a new indicator of reperfusion.

Animals↗
PubMed1996

Ray vector fields, prismatic effect, and thick astigmatic optical systems.

The application of the concept of ray vector fields to optical systems is reexamined. Paraxial or linear optics defines a four-dimensional ray vector field for any optical system: the vector field maps the incident ray vector into the emergent ray vector. In the case of thin systems, including thin astigmatic lenses, one can define a vector field of reduced dimensionality: the vector field is two-dimensional and maps the ray's incident position into the change in reduced direction. When the index of refraction is the same before and after a thin system, the change in reduced direction is the reduced deflection through the system or the reduced prismatic effect. Contrary to what has recently been claimed, this type of two-dimensional vector field does not apply in general to thick systems. However, a number of different types of two-dimensional vector fields can be defined for various particular classes of optical systems. Thick systems differ qualitatively from thin systems. They do not have equivalent thin lenses and cannot generally be replaced by thin lenses. Equations are derived for the change in reduced direction and deflection for a ray through optical systems in general and through separated two- and three-lens systems in particular.

Astigmatism↗
PubMed1998

Identification of operational clues to dry weight prescription in hemodialysis using bioimpedance vector analysis. The Italian Hemodialysis-Bioelectrical Impedance Analysis (HD-BIA) Study Group.

In patients undergoing hemodialysis (HD) cyclic body fluid changes are estimated by body weight variations, which may be misleading. Conventional bioelectrical impedance analysis (BIA) produces biased estimates of fluids in HD due to the assumption of constant tissue hydration. We used an assumption-free assessment of hydration based on direct measurements of the impedance vector. The impedance vector (standard BIA at 50 kHz frequency) was measured in 1367 HD patients, ages 16 to 89 years with BMI 17 to 31 kg/m2, 1116 asymptomatic (680 M and 436 F), and 251 with recurrent HD hypotension (118 M and 133 F) before and after two HD sessions (thrice weekly bicarbonate dialysis, 210 to 240 min) removing 2.7 kg fluid. The vector distribution of HD patients was compared to 726 healthy subjects with the same age and BMI range. Individual vector measurements (resistance and reactance components) were plotted on the gender specific 50th, 75th and 95th percentiles of the vector distribution in the healthy population (reference tolerance ellipses) as a resistance-reactance graph (RXc graph). The wet-dry weight cycling of HD patients was represented on the resistance-reactance plane with a definite, cyclical, backward-forward displacement of the impedance vector. The vectors of patients with HD hypotension were less steep and more often shifted to the right, out of the reference 75% tolerance ellipse, than asymptomatic patients. A wet-dry weight prescription, based on BIA indications, would bring the vectors of patients back into the 75% reference ellipse, where tissue electrical conductivity is restored. Whether HD patients with vector cycling within the normal third quartile ellipse have better outcome awaits confirmation by longitudinal evaluation.

Adolescent↗
PubMed2004

Effects of cyclooxygenase-2 antisense vector on proliferation of human cholangiocarcinoma cells.

OBJECTIVE: To transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing cholangiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis. METHODS: QBC939 cells were transfected with antisense vector of human COX-2 gene using LipoVec transfecting technique. Transfected cells were selected with G418; COX-2 mRNA was examined using reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein expression was detected by immunocytochemistry using isozyme selective antibodies. The proliferative status of transfected cells was measured by using methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry. RESULTS: RT-PCR showed a lower COX-2 mRNA level in antisense vector transfected cells and immunocytochemistry showed a weaker COX-2 protein expression in antisense vector transfected cells. The antisense vector transfected cells proliferative index decreased significantly (P < 0.01), the percentage of S phase decreased remarkably (P < 0.05) in antisense vector transfected cells (9.27% +/- 1.91%) compared with that in QBC939 cells without transfection(16.35% +/- 2.87%), and the percentage of G0/G1 phase increased remarkably (P < 0.05) in antisense vector transfected cells (75.16% +/- 4.13%) compared with that in QBC939 cells without transfection (57.31% +/- 10.16%). Transfection with antisense vector of human COX-2 gene had no significant influence on the apoptosis in QBC939 cells (P > 0.05). CONCLUSION: Transfection with antisense vector of human COX-2 gene could inhibit the proliferation of human cholangiocarcinoma QBC939 cells.

Apoptosis↗
PubMed2006

Exploiting the potential of vector control for disease prevention.

Although vector control has proven highly effective in preventing disease transmission, it is not being used to its full potential, thereby depriving disadvantaged populations of the benefits of well tried and tested methods. Following the discovery of synthetic residual insecticides in the 1940s, large-scale programmes succeeded in bringing many of the important vector-borne diseases under control. By the late 1960s, most vector-borne diseases--with the exception of malaria in Africa--were no longer considered to be of primary public health importance. The result was that control programmes lapsed, resources dwindled, and specialists in vector control disappeared from public health units. Within two decades, many important vector-borne diseases had re-emerged or spread to new areas. The time has come to restore vector control to its key role in the prevention of disease transmission, albeit with an increased emphasis on multiple measures, whether pesticide-based or involving environmental modification, and with a strengthened managerial and operational capacity. Integrated vector management provides a sound conceptual framework for deployment of cost-effective and sustainable methods of vector control. This approach allows for full consideration of the complex determinants of disease transmission, including local disease ecology, the role of human activity in increasing risks of disease transmission, and the socioeconomic conditions of affected communities.

Animals↗
PubMedDate not supplied

Herpes simplex virus vector-mediated dystrophin gene transfer and expression in MDX mouse skeletal muscle.

BACKGROUND: Duchenne muscular dystrophy (DMD) results from mutations that prevent the expression of functional dystrophin in muscle fibers. Herpes simplex virus type-1 (HSV-1) represents a potentially useful vector for treatment of DMD because it has the capacity to accommodate the 14-kb full-length dystrophin cDNA and can efficiently transduce muscle cells. We have tested the ability of first- and second-generation replication-defective HSV vectors to deliver full-length dystrophin to dystrophin-deficient mdx muscle cells in vitro and in vivo. METHODS: First-generation replication-defective HSV vectors harboring full-length or truncated (Becker) dystrophin expression cassettes and lacking a single viral immediate-early (IE) gene were constructed and tested by immunofluorescence and immunoblotting for their ability to direct dystrophin expression in infected mdx cells in culture. To reduce vector cytotoxicity and safety concerns, a second-generation dystrophin vector missing additional IE genes was constructed and tested in vitro and in vivo. RESULTS: Dystrophin expression was observed in infected mdx myotubes in vitro in all cases. Confocal microscopy showed exclusive localization of full-length dystrophin to the cell membrane whereas the Becker variant was also found abundantly throughout the cytoplasm. Dystrophin expression in mdx mice was restored in muscle cells near the site of vector injection. CONCLUSION: Highly defective HSV-1 vectors which lack the ability to spread systemically and are greatly reduced in toxicity for infected cells, thus removing an impediment to prolonged transgene expression, can direct the delivery and proper expression of full-length dystrophin whose considerable size is compatible with few other modes of delivery. These vectors may offer a legitimate opportunity toward the development of effective gene therapy treatments for DMD.

Animals↗
PubMedDate not supplied

Stabilization of transgenes delivered by recombinant adenovirus vectors through extrachromosomal replication.

BACKGROUND: A major limitation of adenovirus-mediated gene therapy for metabolic and inherited diseases is the instability of transgene expression in vivo. This instability results, at least in part, from the inability of the vector genome to maintain the transgene through replication or integration. In this study we evaluated the possibility of stabilization of an adenovirus-delivered transgene by non-adenovirus replicative elements. METHODS: We have developed a novel system for the maintenance of transgenes delivered by adenovirus vectors through extrachromosomal replication. In its initial configuration, this system combines the Epstein-Barr virus (EBV) replicative elements, a tetracycline (Tc)-inducible expression system, and the Cre-lox recombination system in the context of a single E1/E3/E4-deleted adenovirus vector. Induction of Cre expression initiates a Cre-mediated recombination, resulting in the excision of a fragment of the vector genome and its circularization into an EBV-based episome. RESULTS: In vitro studies have demonstrated that excision of the circular episome can occur in a cell-free system as well as in cultured cells transfected with plasmid DNA or transduced by a virus vector carrying the episome-excising cassette. PCR studies have shown that in proliferating, non-permissive, cultured primate cells the episome generated from the adenovirus vector is maintained much more stably than the genome of the parent vector. This episome was also able to replicate in mammalian cells. CONCLUSION: Together these studies demonstrate the feasibility of this approach for the stabilization of transgenes delivered to dividing cells by adenovirus vectors.

Adenoviridae↗
PubMedDate not supplied

Concentration of viral vectors by co-precipitation with calcium phosphate.

BACKGROUND: The envelope glycoproteins, surface unit (SU) and transmembrane (TM) of the murine leukemia virus (MLV) are not covalently linked and tend to dissociate upon high-speed centrifugation, leading to loss of vector infectivity. This study describes a gentle and simple method to concentrate MLV vectors or HIV vectors pseudotyped with MLV envelopes. Having a fast and inexpensive method to concentrate large volumes of vector supernatant will facilitate in vivo experiments and clinical trials that require high titer vector stocks. METHODS: The methods employed in the study were co-precipitation of viral supernatant with calcium phosphate, low-speed centrifugation, dialysis, and infection assays with Lac-Z transducing vectors. RESULTS: Murine leukemia virus vectors and HIV vectors pseudotyped with vesicular stomatitis virus glycoprotein (VSV.G) or MLV envelopes were concentrated successfully using the calcium phosphate co-precipitation method. Parameters that influence virus yield and the reproducibility of the method were investigated. The optimized protocol involves virus harvest in serum-free media, co-precipitation using 60mM calcium chloride, pelleting at 2,000 g, resuspending the pellet in a small volume of 0.1M EDTA-saline, and dialysis against saline to remove EDTA. Volumes were decreased from 300 ml to 10 ml, with 50-100% recovery, and titers can be concentrated up to 1,000-fold. CONCLUSIONS: The calcium phosphate co-precipitation method to concentrate virus is applicable to retrovirus and lentivirus preparations. It uses simple techniques and does not require expensive equipment. Multiple rounds of co-precipitation can be carried out if required.

Animals↗
PubMedDate not supplied

Comparison of bicistronic retroviral vectors containing internal ribosome entry sites (IRES) using expression of human interleukin-12 (IL-12) as a readout.

BACKGROUND: Many gene therapy applications require the co-ordinated delivery of more than one reading frame. We wished to systematically compare IRES in the context of a retroviral vector to determine which was the most effective for protein production and viral titre. To do this we monitored expression of IL-12, as co-ordinated expression of both p35 and p40 subunits is required for production of the active heterodimer. METHODS: Retroviral vectors were constructed to express human IL-12 in which an IRES initiates translation of the p40 subunit, with the IRES optimally aligned to the initiation codon of p40. Vectors containing an IRES from either polio virus (PV), encephalomyocarditis virus (EMCV), foot and mouth disease virus (FMDV) or murine leukaemia virus (MLV) were compared with a vector expressing IL-12 as a single protein (Flexi-12; in which the two IL-12 subunits are linked by a peptide). RESULTS: All vectors produced high titre virus and directed synthesis of IL-12 in target cells. The bicistronic vectors containing the IRES from EMCV and PV were the most effective in infected 3T3 cells, producing up to 40 ng IL-12/10(6) cells/48 h, similar to the 50 ng IL-12/10(6) cells/48 h obtained with Flexi-12. The IRES from PV was the most efficient in human melanoma cells. CONCLUSIONS: Bicistronic retroviral vectors have been constructed that effectively transduce target cells and produce high levels of protein. Target cell specificity of IRES function was observed. The combination of Flexi-12 and the IRES from PV will be useful in the generation of vectors expressing IL-12 with a second protein such as IL-2 for transduction of melanoma cells.

Aphthovirus↗
PubMedDate not supplied

Retroviral transduction of human CD34+ cells on fibronectin fragment CH-296 is inhibited by high concentrations of vector containing medium.

BACKGROUND: The objective of the present study was to optimize conditions for retroviral transduction of human cord blood (CB) CD34+ cells and to reveal mechanisms which interfere with efficient gene transfer. METHODS: An MSCV based retroviral vector with the gene for enhanced green fluorescent protein (MGIN) produced by GP+envAM12 (amphotropic envelope), PG13 (gibbon ape leukemia virus envelope) or 293GPG (vesicular stomatitis virus envelope) cell lines was used to transduce cord blood CD34+ cells on Retronectin (fibronectin fragment CH-296) in three different ways: either in vector containing medium (VCM), in fresh medium on Retronectin pre-loaded with vector or in VCM on Retronectin pre-loaded with vector. RESULTS: Paradoxically, the transduction efficiency obtained with pre-load of vector onto Retronectin alone was higher than pre-load plus VCM for PG13-MGIN (67.9 +/- 6.0% vs 24.9 +/- 8.0%) and AM12-MGIN (47.5 +/- 5.8% vs 38.7 +/- 2.2%). Further experiments showed that transduction on Retronectin pre-loaded with PG13-MGIN or AM12-MGIN was inhibited by the presence of the same VCM at high concentrations, but not by the presence of a VCM with a different receptor specificity. If no pre-load of vector was performed, the highest transduction efficiencies were seen when VCMs were diluted 1:10 (MOIs of 3). The inhibitory effect of high titer PG13-MGIN VCM was confirmed in more primitive CD34+CD38low cells and in NOD/SCID repopulating cells, and was also seen in experiments with bone marrow CD34+ cells. CONCLUSIONS: Retroviral transduction of CB CD34+ cells on Retronectin is inhibited by high titer PG13 and GP+envAM12 vector containing medium. Efficient gene transfer to human hematopoietic cells can be obtained by preload alone of the vector onto Retronectin. These findings are of importance for the design of transduction protocols for repopulating hematopoietic cells.

Antigens, CD34↗
PubMed2002

The role of selectins and integrins in adenovirus vector-induced neutrophil recruitment to the liver.

Adenovirus vectors for human gene therapy induce early host inflammatory responses in transduced tissues that limit gene transfer efficiency and can result in significant morbidity. The present study aimed to elucidate the cellular mechanisms underlying the acute inflammation induced by adenovirus vectors in the liver. Leukocyte rolling and adhesion in response to an intravenously administered adenovirus vector was examined by intravital microscopy in mouse liver. Adenovirus vectors significantly increased leukocyte rolling and adhesion in the postsinusoidal venules within minutes of transduction. Unlike other inflammatory states in the liver, no leukocyte retention was seen in the sinusoids in response to adenovirus vector administration. Inhibition of P-selectin, alpha(4)-integrin, and E-selectin was necessary to completely block leukocyte rolling and subsequent adhesion. The administration of an anti-alpha(4)-integrin antibody alone significantly reduced leukocyte adhesion. In contrast, adenovirus vector-induced leukocyte adhesion was unchanged in CD18-knockout mice. Depletion of circulating neutrophils eliminated leukocyte rolling and adhesion in response to adenovirus vector transduction in the liver. In conclusion, adenovirus vectors induce rapid neutrophil-mediated inflammation in the post-sinusoidal venules by selectins and alpha(4)-integrin but surprisingly not by CD18.

Adenoviridae↗
PubMed1996

Direct gene delivery to synovium. An evaluation of potential vectors in vitro and in vivo.

OBJECTIVE: To assess the abilities of various vectors to transfer genes to the synovial lining of joints. METHODS: Vectors derived from retrovirus, adenovirus, and herpes simplex virus as well as cationic liposomes and naked plasmid DNA were evaluated. Each construct contained the lac Z marker gene; and one retroviral construct, and one plasmid also contained a gene encoding human interleukin-1 receptor antagonist. Gene expression was under the control of the human cytomegalovirus promoter in all vectors except the retrovirus, where the endogenous 5' long terminal repeat was retained as the promoter. Cultures of rabbit synovial fibroblasts were exposed to these vectors and stained with X-gal to identify lac Z+ cells. Vectors were then injected directly into rabbits' knee joints, and gene transfer and expression were assessed by X-gal staining and polymerase chain reaction (PCR). RESULTS: Adenovirus was a highly effective vector both in vitro and in vivo, with lac Z gene expression persisting for at least 28 days. However, an inflammatory response was noted in vivo. Cells infected in vitro and in vivo with herpes simplex virus also expressed the lac Z gene at high levels, but expression was limited by cytotoxicity. Retroviruses, in contrast, were effective only under in vitro conditions, permitting cell division. Liposomes gave variable in vitro results; when injected into joints in vivo, gene expression was low and was detectable for only a few days, even though a PCR signal persisted for at least 28 days. Unexpectedly, plasmid DNA was captured by the synoviocytes and expressed transiently following intraarticular injection. CONCLUSION: None of the vectors was ideal for in vivo gene delivery to synovium, although adenovirus was clearly the most effective of those tested. Retroviruses, although poor vectors for in vivo gene delivery, are well suited for ex vivo gene transfer to the synovial lining of joints.

Adenoviridae↗
PubMed2006

Effect of medium sugar source on the production of retroviral vectors for gene therapy.

The production of gene therapy retroviral vectors presents many difficulties, mainly due to vector instability and low cell productivities hampering the attainment of high titers of infectious viral vectors. The objective of this work is to increase the production titers of retroviral vectors by manipulating the sugar carbon sources used in bioreaction. Four sugars were tested (glucose, galactose, sorbitol, and fructose) on an established Moloney murine leukemia virus (MoMLV) producer cell line. Galactose and sorbitol did not support cell growth or vector production. Glucose supplemented at 25 mM supported the highest cell growth; however, the use of glucose or fructose at 83 and 140 mM have shown to improve the infectious vector titer three to fourfold. The reasons for the titer improvements were further analyzed and, although, the cell-specific productivity in viral transgene RNA and reverse transcriptase were augmented 5- and 6-fold for glucose at 140 mM and 14- and 16-fold for fructose at 140 mM, comparing with glucose at 25 mM, these increases did not seem sufficient to account for the 14- (140 mM glucose) and 32- (140 mM fructose) fold increment obtained for the infectious particles-specific productivity. Further accounting the enhancement in the titers was the improvement in the viral stability, the half-life of the vectors was enhanced by 30-60%. This resulted in a product quality with a superior ratio of infectious to total particles, thus reducing the most problematic contaminant in the production of retroviral vectors, non-infectious retroviral particles.

Carbohydrates↗