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PubMed2004

Genome size and structure determine efficiency of postinternalization steps and gene transfer of capsid-modified adenovirus vectors in a cell-type-specific manner.

Adenovirus serotype 5 (Ad5) vectors containing Ad B-group fibers have become increasingly popular as gene transfer vectors because they efficiently transduce human cell types that are relatively refractory to Ad5 infection. So far, most B-group fiber-containing vectors have been first-generation vectors, deleted of E1 and/or E3 genes. Transduction with these vectors, however, results in viral gene expression and is associated with cytotoxicity and immune responses against transduced cells. To circumvent these problems, we developed fiber-chimeric Ad vectors devoid of all viral genes that were produced either by the homologous recombination of first-generation vectors or by using the Cre/lox-based helper virus system. In this study we compared early steps of infection between first-generation (35-kb genome) and Ad vectors devoid of all viral genes with genome sizes of 28 kb and 12.6 kb. All vectors possessed an Ad35-derived fiber knob domain, which uses CD46 as a primary attachment receptor. Using immortalized human hematopoietic cell lines and primary human CD34-positive hematopoietic cells, we found that the Ad genome size did not affect the efficiency of virus attachment to and internalization into cells. Furthermore, independently of the genome length and structure, all vectors migrated to the nucleus through late endosomal and lysosomal cellular compartments. However, the vector containing the short 12.6-kb genome was unable to efficiently escape from endosomes and deliver its DNA into the nucleus. Moreover, compared to other vectors, these Ad particles were less stable and had an abnormal capsid protein composition, including a lack of capsid-stabilizing protein IX. Our data indicate that the size and structure of the packaged viral genomes can affect the integrity of Ad particles, which in turn results in lower infectivity of Ad vectors.

Active Transport, Cell Nucleus↗
PubMed2005

Development and assessment of human adenovirus type 11 as a gene transfer vector.

Adenovirus vectors based on human serotype 5 (Ad5) have successfully been used as gene transfer vectors in many gene therapy-based approaches to treat disease. Despite their widespread application, many potential therapeutic applications are limited by the widespread prevalence of vector-neutralizing antibodies within the human population and the inability of Ad5-based vectors to transduce important therapeutic target cell types. In an attempt to circumvent these problems, we have developed Ad vectors based on human Ad serotype 11 (Ad11), since the prevalence of neutralizing antibodies to Ad11 in humans is low. E1-deleted Ad11 vector genomes were generated by homologous recombination in 293 cells expressing the Ad11-E1B55K protein or by recombination in Escherichia coli. E1-deleted Ad11 genomes did not display transforming activity in rodent cells. Transduction of primary human CD34+ hematopoietic progenitor cells and immature dendritic cells was more efficient with Ad11 vectors than with Ad5 vectors. Thirty minutes after intravenous injection into mice that express one of the Ad11 receptors (CD46), we found, in a pattern and at a level comparable to what is found in humans, Ad11 vector genomes in all analyzed organs, with the highest amounts in liver, lung, kidney, and spleen. Neither Ad11 genomes nor Ad11 vector-mediated transgene expression were, however, detected at 72 h postinfusion. A large number of Ad11 particles were also found to be associated with circulating blood cells. We also discovered differences in in vitro transduction efficiencies and in vivo biodistributions between Ad11 vectors and chimeric Ad5 vectors possessing Ad11 fibers, indicating that Ad11 capsid proteins other than fibers influence viral infectivity and tropism. Overall, our study provides a basis for the application of Ad11 vectors for in vitro and in vivo gene transfer and for gaining an understanding of the factors that determine Ad tropism.

Adenoviruses, Human↗
PubMed2005

Identification of unique reciprocal and non reciprocal cross packaging relationships between HIV-1, HIV-2 and SIV reveals an efficient SIV/HIV-2 lentiviral vector system with highly favourable features for in vivo testing and clinical usage.

BACKGROUND: Lentiviral vectors have shown immense promise as vehicles for gene delivery to non-dividing cells particularly to cells of the central nervous system (CNS). Improvements in the biosafety of viral vectors are paramount as lentiviral vectors move into human clinical trials. This study investigates the packaging relationship between gene transfer (vector) and Gag-Pol expression constructs of HIV-1, HIV-2 and SIV. Cross-packaged vectors expressing GFP were assessed for RNA packaging, viral vector titre and their ability to transduce rat primary glial cell cultures and human neural stem cells. RESULTS: HIV-1 Gag-Pol demonstrated the ability to cross package both HIV-2 and SIV gene transfer vectors. However both HIV-2 and SIV Gag-Pol showed a reduced ability to package HIV-1 vector RNA with no significant gene transfer to target cells. An unexpected packaging relationship was found to exist between HIV-2 and SIV with SIV Gag-Pol able to package HIV-2 vector RNA and transduce dividing SV2T cells and CNS cell cultures with an efficiency equivalent to the homologous HIV-1 vector however HIV-2 was unable to deliver SIV based vectors. CONCLUSION: This new non-reciprocal cross packaging relationship between SIV and HIV-2 provides a novel way of significantly increasing bio-safety with a reduced sequence homology between the HIV-2 gene transfer vector and the SIV Gag-Pol construct thus ensuring that vector RNA packaging is unidirectional.

Animals↗
PubMed2004

Strategies for retargeted gene delivery using vectors derived from lentiviruses.

With the development of the first viral vector systems 20 years ago [Mann et al., 1983; Watanabe and Temin, 1983] gene therapy strategies have come to the forefront of novel therapeutics [Cavazzana-Calvo et al., 2000]. A deeper understanding of vector biology and the molecular mechanisms of disease alongside tremendous advances in vector technology have significantly advanced the field of human gene therapy. Over the last few years several challenges needed to be overcome in order to bring gene therapy strategies closer to the clinic. These hurdles include the preparation of large amounts of stable, high titre vectors, minimising vector-related immunology and last but not least targeting infection and transgene expression to tissue or cells, which in many cases are not or only slowly dividing. Viral vectors are useful vehicles for the delivery of foreign genes into target cells, and retroviral vectors have been popular because of their ability to integrate into the host cell genome and maintain persistent gene expression. Moreover, lentiviruses, members of the retroviral family, have the ability to infect cells at both mitotic and post-mitotic stages of the cell cycle thus opening up the possibility to target non-dividing target cells and tissues. Human immunodeficiency virus (HIV) based vectors have been used in vitro and in vivo in a number of situations, however, safety concerns still exist, and therefore the development of vector systems based on primate as well as non-primate lentiviruses is ongoing. Concomitantly with lentiviral vector design, much has been learned about the incorporation of heterologous env proteins on lentiviral cores in order to combine specific targeting properties of envelope glycoproteins with the biological properties of lentiviral vectors. In this review article we will give an overview over advantages lentiviral vector systems offer. We will then discuss the current state of our understanding of the structure and function of viral envelope glycoproteins and emerging targeting strategies based on retroviral and lentiviral vector systems.

Animals↗
PubMed2002

Retroviral gene therapy vectors for prevention of excimer laser-induced corneal haze.

PURPOSE: To determine the in vivo efficacy and safety of a retroviral vector bearing an antiproliferative dominant negative mutant cyclin G1 (dnG1) construct, when used for the prevention of corneal haze after phototherapeutic keratectomy (PTK). METHODS: For in vivo efficacy studies, a 6-mm-diameter, 150-microm-deep transepithelial PTK, performed with a clinical 193-nm ArF excimer laser (VISX Star2, Santa Clara, CA) was performed on the left eyes of 20 adult New Zealand White rabbits. The surgically altered eyes were subsequently treated with eye drops containing: a retroviral vector bearing a dnG1 construct (dnG1; n = 7), a control retroviral vector (null vector) bearing only the neomycin resistance, neo(r), gene (n = 7), or a retroviral vector bearing an antisense cyclin G1 (aG1) construct (n = 6). The time of closure of the corneal epithelial defect was monitored daily with fluorescein staining. Corneal haze was evaluated before surgery and at 2, 3, and 4 weeks after surgery, with a digital imaging system. Biodistribution studies for detection of potential vector dissemination to nontarget organs were conducted by PCR-based assay. RESULTS: The re-epithelialization rate was similar among treatment groups, with complete closure of the corneal epithelial defect at 72 hours (P > 0.05). Significant corneal haze developed in the null and aG1 vector-treated groups (P <or= 0.05) at 3 to 4 weeks after PTK. In contrast, development of corneal haze was inhibited in the dnG1 vector-treated group when compared with the null vector-treated group (P < 0.05). In parallel, a dramatic reduction to complete abrogation of abnormal extracellular matrix production was noted in the dnG1 vector-treated corneas when compared with the null and aG1 vector-treated groups. Biodistribution studies showed no evidence of vector dissemination in neighboring and distant organs. CONCLUSIONS: At therapeutic doses, eye drop application of the dnG1 retroviral vector is safe and effective in inhibiting development of corneal haze after PTK in rabbits.

3T3 Cells↗
PubMed2002

Gene transfer in ovarian cancer cells: a comparison between retroviral and lentiviral vectors.

Local gene therapy could be a therapeutic option for ovarian carcinoma, a life-threatening malignancy, because of disease containment within the peritoneal cavity in most patients. Lentiviral vectors, which are potentially capable of stable transgene expression, may be useful to vehicle therapeutic molecules requiring long-term production in these tumors. To investigate this concept, we used lentiviral vectors to deliver the enhanced green fluorescent protein (EGFP) gene to ovarian cancer cells. Their efficiency of gene transfer was compared with that of a retroviral vector carrying the same envelope. In vitro, both vectors infected ovarian cancer cells with comparable efficiency under standard culture conditions; however, the lentiviral vector was much more efficient in transducing growth-arrested cells when compared with the retroviral vector. Gene transfer was fully neutralized by an anti-VSV-G antibody, and in vitro stability was similar. In vivo, the lentiviral vector delivered the transgene 10-fold more efficiently to ovarian cancer cells growing i.p. in SCID mice, as evaluated by real-time PCR analysis of the tumors. Confocal microscopy analysis of tumor sections showed a dramatic difference at the level of transgene expression, because abundant EGFP(+) cells were detected only in mice receiving the lentiviral vector. Quantitative analysis by flow cytometry confirmed this and indicated 0.05 and 5.6% EGFP(+) tumor cells after administration of the retroviral and lentiviral vector, respectively. Injection of ex vivo transduced tumor cells, sorted for EGFP expression, indicated that the lentiviral vector was considerably more resistant to in vivo silencing in comparison with the retroviral vector. Finally, multiple administrations of a murine IFN-alpha(1)-lentiviral vector to ovarian carcinoma-bearing mice significantly prolonged the animals' survival, indicating the therapeutic efficacy of this approach. These findings indicate that lentiviral vectors deserve attention in the design of future gene therapy approaches to ovarian cancer aimed at achieving long-term expression of therapeutic genes.

Adenoviridae↗
PubMed2003

[Construction of the plant expression vectors containing the multiepitope gene of Toxoplasma gondii].

OBJECTIVE: To construct the plant expression vectors containing the multiepitope gene of Toxoplasma gondii (TGMG). METHODS: 1. TGMG was subcloned into pBAC55 vector to construct the intermediate plasmid pB35MG. The E35S/TGMG/NOS3' fragment was cleaved from pB35MG and ligated into the plant binary vector pCAMBIA2300 to construct the plant expression vector pC35MG. 2. Tomato fruit-specific E81. 1 promoter was introduced to pB35MG to construct pB35E1MG vector. The E35SE81. 1/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pC35E1MG. 3. Tomato fruit-specific E82.2 promoter was inserted to pB35MG to construct pBE2MG vector. The E82.2/TGMG/NOS3' fragment was subcloned into pCAMBIA2300 to construct the plant expression vector pCE2MG. The insert gene TGMG in the vectors pB35MG, pC35E1MG and pCE2MG were confirmed by sequencing. 4. pC35MG, pC35E1MG and pCE2MG were introduced into Agrobacterium tumefaciens strain LBA4404 competent cell. RESULTS: Digestion with restriction enzymes proved that all recombinant vectors had the inserts with expected length of the target fragments. And the sequencing results were confirmed correct. CONCLUSION: The TGMG intermediate vectors pB35MG, pB35E1MG and pBE2MG and the plant expression vectors pC35MG, pC35E1MG and pCE2MG were constructed successfully, and the three plant expression vectors were introduced into Agrobacterium tumefaciens.

Agrobacterium tumefaciens↗
PubMed1994

Expression of human glucocerebrosidase in murine macrophages: identification of efficient retroviral vectors.

Gaucher's disease is an autosomal recessive disorder characterized by a functional deficiency in beta-glucocerebrosidase enzymatic activity and the resultant accumulation of the glycolipid glucocerebroside in macrophages. Due to the nature of the affected cells, Gaucher's disease is an excellent candidate for gene therapy of hematopoietic stem cells and autologous bone marrow transplantation of transduced cells using retroviral vectors containing the glucocerebrosidase (GC) gene. In order to identify a retroviral vector capable of high levels of expression of the GC gene in macrophages, we have used the murine myeloid leukemia cell line, M1, a cell line that can be differentiated with interleukin-6 (IL-6) from blasts to macrophages. Two vectors use the Moloney murine leukemia virus (MoMLV) enhancer/promoter (LG vector) or the myeloproliferative sarcoma virus (MPSV) enhancer/MoMLV promoter (MG vector), both located in the viral long-terminal repeat (LTR); the third vector uses the phosphoglycerate kinase (PGK) promoter located internally in the vector (PG vector). The amphotropic PA317 and GP+am12 packaging cell lines were used as virus producer cells, and the GP+am12 cell line demonstrated higher titers, higher levels of GC protein expression, and specific GC enzymatic activity as well as higher transduction efficiencies for all three vectors. The LG retroviral vector was the most efficient in transducing the M1 cells. On average, higher levels of RNA and protein expression were seen in the M1 clones transduced with the LG vector, and these levels increased after differentiation. Thus, the LG retroviral vector in which the expression of the GC gene is driven by the MoMLV LTR enhancer/promoter is the best vector of the three studied for future studies for gene therapy of Gaucher's disease and other hematopoietic disorders that involve macrophages.

Animals↗
PubMed1996

Increased levels of spliced RNA account for augmented expression from the MFG retroviral vector in hematopoietic cells.

A persistent obstacle in the use of vector systems for gene therapy has been the inability to attain high-level expression of the target gene in primary cells in vivo. The MFG retroviral vector was designed to yield improved expression over the widely used N2 or LN vectors; however, the molecular basis for this effect has not been examined. Using the human glucocerebrosidase (GC) enzyme as a reporter, we compared expression from the MFG and N2 vector backbones in transduced murine hematopoietic cells after syngeneic bone marrow transplantation. Reporter enzyme activities in primary spleen colonies of transplanted mice were seven-fold higher per vector copy in cells transduced with the (MFG-based) MGC vector than in cells bearing the (N2-based) G2 vector. In spleen colonies harboring the MGC vector, the ratio of spliced to unspliced vector RNA was increased four-fold relative to the G2 vector transcripts in Northern blot analyses. Further analyses indicated that MGC-transduced cells contained five-fold higher levels of spliced RNA per vector copy. Since translation of spliced RNA species (in which the complex secondary structure of the packaging signal has been excised) is likely to proceed with enhanced efficiency, the augmented levels of spliced RNA produced by MFG may represent the key element of increased protein expression from this vector. These findings suggest that the MFG retroviral vector may provide higher level expression of target genes used in human gene therapy.

Animals↗
PubMed1996

Neonatal cotton rats do not exhibit destructive immune responses to adenoviral vectors.

Recombinant adenoviruses are being considered for treatment of cystic fibrosis lung disease. The utility of this vector system in adult recipients is limited by the formation of destructive humoral and cellular immune responses to the vector and vector-infected cells. We hypothesized that the host immune responses could be avoided by administering the vector early in development when the immune system is naive. This was evaluated in the context of newborn and adult cotton rats administered E1-deleted recombinant adenoviral vectors into the trachea. The results with adult cotton rats were consistent with previous studies in that gene expression was extremely high in the conducting airway when evaluated 3 days after administration of the vector. However, expression diminished to undetectable levels within 2 weeks and was associated with substantial inflammation and formation of neutralizing antibodies. The level of expression achieved in newborn cotton rats was equally high with reporter gene expression detected in both the conducting airway and alveolar cells. The immune response in the newborn cotton rats was substantially blunted with diminished CD4+ T cell activation, based on in vitro studies, as well as essentially no formation of neutralizing antibody. This translated to a substantially improved outcome with transgene expression persisting for over 6 months. The window of optimal immune tolerance was within 3 days of birth. Vector was readministered into animals 8-10 weeks after they had received the neonatal challenge of the initial vector. This second delivery of vector was efficient, consistent with the low neutralizing antibody in these animals. However, the animals mounted a full T cell response which correlated with extinction of the transgene from the vector administered at birth, as well as that of the second vector. Neutralizing antibodies did eventually develop following the second vector administration. These studies indicate that immune responses can be avoided if gene therapy is administered early in development. However, this so-called "tolerance' is broken when the animal receives a second vector after the immune system matures.

Adenoviridae↗
PubMed2000

Location of the vector of jaw muscle force in mammals.

Previous work has suggested that the third molar lies just in front of the point where the resultant vector of jaw muscle force, estimated from dissections, intersects the tooth row. This point meets the jaw such that the vector is 30% of jaw length from the jaw joint. Thus, the vector divides the jaw in the ratio of 3:7 when measurements are taken perpendicular to the vector. In practice, however, distances along mammalian jaws are typically measured on an easily determined line such as a line from one end of the tooth row to the other. The position of the jaw joint is then projected onto this line. As a rule, such a line is not perpendicular to the vector and so the distance from the projection of the joint, out to the rear of the third molar (and the vector's intersection), is different in different mammals. Rarely is this distance 30% of total jaw length. However, when the location of the vector's intersection is measured along the tooth row, this position varies directly with the inclination of the vector; a vector inclined posteriorly intersects the tooth row far from the projection of the joint and an anterior vector's intersection is relatively close. Only a vector perpendicular to the line from one end of the tooth row to the other intersects at 30%. This obvious point suggests a way to test the above hypotheses when the inclination of the vector is not known exactly. The predicted relationship between the distance to the molar, as a percentage of the total jaw length, and the approximate inclination of the vector derived from muscle weights (posterior or anterior depending on whether the temporalis or the masseter/pterygoid, respectively, is dominant) was observed in a sample of 46 different mammals.

Animals↗
PubMed2004

The fundamental thumb-tip force vectors produced by the muscles of the thumb.

A rigorous description of the magnitude and direction of the 3D force vector each thumb muscle produces at the thumb-tip is necessary to understand the biomechanical consequences to pinch of a variety of paralyses and surgical procedures (such as tendon transfers). In this study, we characterized the 3D force vector each muscle produces at the thumb-tip, and investigated if these thumb-tip force vectors scaled linearly with tendon tension. In 13 cadaver specimens, we measured the output 3D thumb-tip force vector produced by each tendon acting on the thumb, plus two common tendon transfers, as a function of input tendon tension. After fixing the hand to a rigid frame, we mounted the thumb by configuring it in standardized key or opposition pinch posture and coupling the thumb-tip to a rigidly held 6 degree-of-freedom force/torque sensor. Linear actuators applied tension to the distal tendons of the four extrinsic thumb muscles, and to six Nylon cords reproducing the lines of action of (i) the four intrinsic thumb muscles and (ii) two alternative tendon transfers commonly used to restore thumb opposition following low median nerve palsy. Each computer-controlled linear actuator ramped tendon tension from zero to 1/3 of predicted maximal muscle force expected at each tendon, and back to zero, while we measured the 3D force vector at the thumb-tip. In test/re-test trials, we saw thumb-tip force vectors were quite sensitive to mounting procedure, but also sensitive to variations in the seating of joint surfaces. We found that: (i) some thumb-tip force vectors act in unexpected directions (e.g., the opponens force vector is parallel to the distal phalanx), (ii) the two tendon transfers produced patently different force vectors, and (iii) for most muscles, thumb-tip force vectors do not scale linearly with tendon tension--likely due to load-dependent viscoelastic tendon paths, joint seating and/or bone motion. Our 3D force vector data provide the first quantitative reference descriptions of the thumb-tip force vectors produced by all thumb muscles and two tendon transfers. We conclude that it may not be realistic to assume in biomechanical models that thumb-tip force vectors scale linearly with tendon tensions, and that our data suggest the thumb may act as a "floating digit" affected by load-dependent trapezium motion.

Aged↗
PubMed2002

Gene therapy of HIV-1 infection using lentiviral vectors expressing anti-HIV-1 genes.

The use of vectors based on primate lentiviruses for gene therapy of human immunodeficiency virus type 1 (HIV-1) infection has many potential advantages over the previous murine retroviral vectors used for delivery of genes that inhibit replication of HIV-1. First, lentiviral vectors have the ability to transduce dividing and nondividing cells that constitute the targets of HIV-1 infection such as resting T cells, dendritic cells, and macrophages. Lentiviral vectors can also transfer genes to hematopoietic stem cells with a superior gene transfer efficiency and without affecting the repopulating capacity of these cells. Second, these vectors could be potentially mobilized in vivo by the wild-type virus to secondary target cells, thus expanding the protection to previously untransduced cells. And finally, lentiviral vector backbones have the ability to block HIV-1 replication by several mechanisms that include sequestration of the regulatory proteins Tat and Rev, competition for packaging into virions, and by inhibition of reverse transcription in heterodimeric virions with possible generation of nonfunctional recombinants between the vector and viral genomes. The inhibitory ability of lentiviral vectors can be further increased by expression of anti-HIV-1 genes. In this case, the lentiviral vector packaging system has to be modified to become resistant to the anti-HIV-1 genes expressed by the vector in order to avoid self-inhibition of the vector packaging system during vector production. This review focuses on the use of lentiviral vectors as the main agents to mediate inhibition of HIV-1 replication and discusses the different genetic intervention strategies for gene therapy of HIV-1 infection.

Genes, Viral↗
PubMed2005

Patterns of bioelectrical impedance vector distribution by body mass index and age: implications for body-composition analysis.

BACKGROUND: Bioelectrical impedance analysis (BIA) gives resistance (R) and reactance (Xc). R and Xc normalized for body height (H) can be plotted as a bivariate vector (H(2)/Xc versus H(2)/R). Vector BIA is useful for studying the determinants of BIA results. OBJECTIVE: We investigated the effect of age on BIA results and its relevance to body-composition analysis in a large database of impedance vector distributions stratified by age, sex, and body mass index (BMI). DESIGN: Mean bivariate vector distribution patterns (95% confidence ellipses) were examined in a German population of 15605 children and adolescents and 213294 adults. Children and adolescents were divided into 3 age groups with up to 5 BMI categories. In adults, 5 BMI categories were stratified into 7 age groups. RESULTS: Mean impedance vectors were shorter in children than in adults. The vector distribution pattern was influenced by sex, BMI, and age, with shorter vectors in females than in males and longer vectors with increasing BMI. Consistent with a decrease in body cell mass with increasing age, there was a downward slope in the mean vector with age as a result of a decrease in the H(2)/Xc vector component. By contrast, there was no age-dependent increase in the H(2)/R vector component. In women of the same BMI at different ages, H(2)/R and percentage fat mass tended to decrease with age. CONCLUSIONS: The lack of an age-dependent increase in the H(2)/R vector component renders conventional BIA unsuitable for an examination of the age-related increase in body fat mass. By contrast, the increase in the H(2)/Xc vector component with advancing age suggests the potential of BIA to depict the age-related decrease in body cell mass.

Adolescent↗
PubMed2002

Effect of genetic background and culture conditions on the production of herpesvirus-based gene therapy vectors.

Herpes simplex virus type-1 (HSV-1) represents a unique vehicle for the introduction of foreign DNA to cells of a variety of tissues. The nature of the vector, the cell line used for propagation of the vector, and the culture conditions will significantly impact vector yield. An ideal vector should be deficient in genes essential for replication as well as those that contribute to its cytotoxicity. Advances in the engineering of replication-defective HSV-1 vectors to reduce vector-associated cytotoxicity and attain sustained expression of target genes make HSV-1 an attractive gene-delivery vehicle. However, the yield of the less-cytotoxic vectors produced in standard tissue-culture systems is at least three order of magnitudes lower than that achieved with wild-type virus. The low overall yield and the complexity involved in the preparation of HSV vectors at high concentrations represent major obstacles in use of replication-defective HSV-derived vectors in gene therapy applications. In this work, the dependence of the vector yield on the genetic background of the virus is examined. In addition, we investigated the production of the least toxic, lowest-yield vector in a CellCube bioreactor system. After initial optimization of the operational parameters of the cellcube system, we were able to attain virus yields similar to or better than those values attained using the tissue culture flask system for vector production with significant savings with respect to time, labor, and cost.

Bioreactors↗
PubMed1999

Dose-dependent transduction of vesicular stomatitis virus G protein-pseudotyped retrovirus vector into human solid tumor cell lines and murine fibroblasts.

We examined the transduction efficiency of a VSV-G (vesicular stomatitis virus G protein)-pseudotyped vector encoding beta-galactosidase (lacZ) into human solid tumor cell lines and murine fibroblasts, compared with that of an amphotropic vector carrying the same RNA sequence. The ratio of cells transduced with the VSV-G-pseudotyped vector corresponded closely to 1 - e(-m.o.i.), as predicted from a Poisson distribution of transduction to the entire cellular population, while this was not the case for the amphotropic vector. Here m.o.i. (multiplicity of infection) is defined as the ratio of input infectious units (titrated on the corresponding cell line) to the number of cells used for the transduction. At high m.o.i.s (values greater than 3), the VSV-G-pseudotyped vector transduced approximately 95% of the culture population of all cell lines examined. The transduction efficiency of the amphotropic vector, however, was not dose-dependent and reached a plateau or even decreased, especially at high m.o.i.; this may be attributable at least in part to the presence of envelope protein and noninfectious particles that compete for the receptor of infectious amphotropic virus. The copy number of integrated vector proviral DNA and the expression level of lacZ increased almost linearly with the dose of the VSV-G-pseudotyed vector, which could readily achieve multiple transduction of more than 10 copies per cell and afforded about 100-fold more transgene product than could be achieved with the amphotropic vector. These features of both the VSV-G-pseudotyped vector and the amphotropic vector were essentially unaffected by purification using centrifugation. These properties of the vector should be highly advantageous for gene transfer into entire populations of human tumor cell lines at a designed dosage.

Animals↗
PubMed2001

Kinetic analyses of stability of simple and complex retroviral vectors.

Simple and complex retroviral vectors derived from Moloney murine leukemia virus (MLV) and human immunodeficiency virus type 1 (HIV-1), respectively, are useful tools for gene transfer studies. However, factors affecting the stability of these vectors have not been carefully investigated. Here we studied the stability factors on vesicular stomatitis viral envelope glycoprotein (VSV-G)-pseudotyped MLV- and HIV-1-derived vectors. Analysis of the ratio of defective particles versus infectious units using electron microscopy and a functional transduction assay revealed that both vectors consisted of high numbers of defective particles ( approximately 100-350:1), which could be reduced ( approximately 10-20:1) by centrifugation. Frequent freeze-and-thaw rapidly decreased vector titer in the first three to five cycles and stabilized thereafter. Both viral vectors were sensitive to temperatures above 37 degrees C but more stable at temperatures below 37 degrees C, exhibiting a two-phase inactivation kinetic starting with a steep inactivation phase, followed by a more leveled phase. Interestingly, HIV-1-derived vectors were significantly more stable than MLV-derived vectors at higher temperatures (>37 degrees C). Both vectors were rapidly destabilized at pH either below or above 7.0. Incubation with human or mouse serum significantly inhibited VSV-G-pseudotyped vector activities. Preheated human serum still reduced vector half-lives to approximately 50% (150 min), suggesting that certain inactivation factors are not heat-labile. Analyses of these stability factors may improve future production and applications of retroviral and lentiviral vectors.

Animals↗
PubMed2006

Helminths as vectors of pathogens in vertebrate hosts: a theoretical approach.

Pathogens frequently use vectors to facilitate transmission between hosts and, for vertebrate hosts, the vectors are typically ectoparasitic arthropods. However, other parasites that are intimately associated with their hosts may also be ideal candidate vectors; namely the parasitic helminths. Here, we present empirical evidence that helminth vectoring of pathogens occurs in a range of vertebrate systems by a variety of helminth taxa. Using a novel theoretical framework we explore the dynamics of helminth vectoring and determine which host-helminth-pathogen characteristics may favour the evolution of helminth vectoring. We use two theoretical models: the first is a population dynamic model amalgamated from standard macro- and microparasite models, which serves as a framework for investigation of within-host interactions between co-infecting pathogens and helminths. The second is an evolutionary model, which we use to predict the ecological conditions under which we would expect helminth vectoring to evolve. We show that, like arthropod vectors, helminth vectors increase pathogen fitness. However, unlike arthropod vectors, helminth vectoring increases the pathogenic impact on the host and may allow the evolution of high pathogen virulence. We show that concomitant infection of a host with a helminth and pathogen are not necessarily independent of one another, due to helminth vectoring of microparasites, with profound consequences for pathogen persistence and the impact of disease on the host population.

Animals↗