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At least 343 records · Page 19Linked to original sources
PubMed2004

The significance of controlled conditions in lentiviral vector titration and in the use of multiplicity of infection (MOI) for predicting gene transfer events.

BACKGROUND: Although lentiviral vectors have been widely used for in vitro and in vivo gene therapy researches, there have been few studies systematically examining various conditions that may affect the determination of the number of viable vector particles in a vector preparation and the use of Multiplicity of Infection (MOI) as a parameter for the prediction of gene transfer events. METHODS: Lentiviral vectors encoding a marker gene were packaged and supernatants concentrated. The number of viable vector particles was determined by in vitro transduction and fluorescent microscopy and FACs analyses. Various factors that may affect the transduction process, such as vector inoculum volume, target cell number and type, vector decay, variable vector - target cell contact and adsorption periods were studied. MOI between 0-32 was assessed on commonly used cell lines as well as a new cell line. RESULTS: We demonstrated that the resulting values of lentiviral vector titre varied with changes of conditions in the transduction process, including inoculum volume of the vector, the type and number of target cells, vector stability and the length of period of the vector adsorption to target cells. Vector inoculum and the number of target cells determine the frequencies of gene transfer event, although not proportionally. Vector exposure time to target cells also influenced transduction results. Varying these parameters resulted in a greater than 50-fold differences in the vector titre from the same vector stock. Commonly used cell lines in vector titration were less sensitive to lentiviral vector-mediated gene transfer than a new cell line, FRL 19. Within 0-32 of MOI used transducing four different cell lines, the higher the MOI applied, the higher the efficiency of gene transfer obtained. CONCLUSION: Several variables in the transduction process affected in in vitro vector titration and resulted in vastly different values from the same vector stock, thus complicating the use of MOI for predicting gene transfer events. Commonly used target cell lines underestimated vector titre. However, within a certain range of MOI, it is possible that, if strictly controlled conditions are observed in the vector titration process, including the use of a sensitive cell line, such as FRL 19 for vector titration, lentivector-mediated gene transfer events could be predicted.

Journal Article↗
PubMed2026

Evaluation of transduction properties and vaccine efficacy of a simian adenovirus type 25-based vector.

Although human adenovirus serotype 5 (Ad5) is widely used as a vaccine vector for infectious diseases due to its high transduction efficiency, pre-existing immunity to Ad5 in many people reduces vaccine efficacy. To address this limitation, simian Ad vectors, such as ChAdOx1 and ChAdOx2, have been explored as alternative vaccine platforms. ChAdOx2 is based on simian Ad25 (SAd25), but the fundamental characteristics of gene transduction by SAd25-based vectors have not been fully elucidated. This study aimed to characterize the gene transduction efficiency, tissue distribution, and immunogenicity of an SAd25-based vector in comparison with those of the Ad5 vector following various routes of administration. Compared with intravenous administration of the Ad5 vector, intravenous administration of the SAd25 vector showed distinct biodistribution patterns, including reduced liver accumulation and predominant expression in the lung. Transduction by the SAd25 vector was not inhibited by human serum, whereas transduction by the Ad5 vector was inhibited, indicating that the SAd25 vector, but not the Ad5 vector, can evade pre-existing Ad immunity. Although intramuscular administration of the SAd25 vector induced lower transgene product-specific antibody production than intramuscular administration of the Ad5 vector, gene expression and Ad genome distribution mediated by the SAd25 vector, but not the Ad5 vector, were localized only to the muscle at the administration site. Intranasal administration of the SAd25 vector induced an antigen-specific antibody response in serum more rapidly than intranasal administration of the Ad5 vector. The SAd25 vector induced antigen-specific antibody production in bronchoalveolar lavage fluid (BALF) that was comparable to that induced by the Ad5 vector. These findings provide essential insights into the biological characteristics of the SAd25 vector, supporting its potential as a safe and effective vaccine vector.

Animals↗
PubMed2003

Comparison of wild-type and class I integrase mutant-FIV vectors in retina demonstrates sustained expression of integrated transgenes in retinal pigment epithelium.

BACKGROUND: In neonatal and adult rodent retina, substantial lentiviral vector expression has been detected primarily in retinal pigment epithelium (RPE), except in very young animals (2-5 days post-natal). In non-retinal tissues, studies of lentiviral vectors have utilized various controls. Among the most stringent are class I integrase mutants, which selectively block the integration reaction while leaving all other gag/pol-encoded functions intact. For HIV-1 vectors injected into brain, these have been used to simultaneously control for pseudotransduction and verify that long-term expression requires integration. Such experiments compare particles that differ only in a single amino acid within a single enzyme that forms a very small molar fraction of the virion. Class I integrase mutants have not been described for feline immunodeficiency virus (FIV) integrase, or tested in the eye for any lentiviral vector. METHODS: We compared subretinally and intravitreally injected FIV vectors and followed animals for up to 7 months, a duration that exceeds prior studies. We also compared the wild-type (WT) vector with one incorporating a single class I amino acid mutation in FIV integrase (D66V). A mock vector (packaging construct absent) was an alternative control. All vectors were vesicular stomatitis virus glycoprotein G (VSV-G)-pseudotyped and were injected on day 7 of life. One group of animals received either subretinal or intravitreal injections of WT vector in the right eyes. Control left eyes were injected with mock vector. These animals were sacrificed at 2 or 7 days post-injection. A second group received subretinal injections of either WT vector or equivalent D66V vector (reverse transcriptase-normalized to WT), and were analyzed after 2, 3 and 7 months. All eyes were scored for marker gene (beta-galactosidase) expression by an observer blinded to vector assignments. RESULTS: Subretinal FIV vector injections were much more effective than intravitreal injections. The RPE was the principal retinal layer transduced by the WT vector, and at least 50% of the area of the retina expressed the marker gene at 3 and 7 months. Occasional cells in inner retinal layers also expressed beta-galactosidase at these time points. The sustained retinal expression produced by subretinally injected vector was blocked by the D66V mutation. CONCLUSIONS: These results show that class I integrase mutant FIV vectors are useful control vectors, and that VSV-G-pseudotyped FIV vectors produce extensive retinal expression for at least 215 days, the longest duration yet reported for lentiviral vectors in retina. Transgene expression is mostly restricted to RPE after post-natal day 7 in rats, suggesting that FIV vectors could be used to target RPE for gene therapy.

Age Factors↗
PubMed2004

Factors influencing the titer and infectivity of lentiviral vectors.

Lentiviral vectors have undergone several generations of design improvement to enhance their biosafety and expression characteristics, and have been approved for use in human clinical studies. Most preclinical studies with these vectors have employed easily assayed marker genes for the purpose of determining vector titers and transduction efficiencies. Naturally, the adaptation of these vector systems to clinical use will increasingly involve the transfer of genes whose products may not be easily measured, meaning that the determination of vector titer will be more complicated. One method for determining vector titer that can be universally employed on all human immunodeficiency virus type 1-based lentiviral vector supernatants involves the measurement of Gag (p24) protein concentration in vector supernatants by immunoassay. We have studied the effects that manipulation of several variables involved in vector design and production by transient transfection have on vector titer and infectivity. We have determined that manipulation of the amount of transfer vector, packaging, and envelope plasmids used to transfect the packaging cells does not alter vector infectivity, but does influence vector titer. We also found that modifications to the transfer vector construct, such as replacing the internal promoter or transgene, do not generally alter vector infectivity, whereas inclusion of the central polypurine tract in the transfer vector increases vector infectivity on HEK293 cells and human umbilical cord blood CD34+ hematopoietic progenitor cells (HPCs). The infectivities of vector supernatants can also be increased by harvesting at early time points after the initiation of vector production, collection in serum-free medium, and concentration by ultracentrifugation. For the transduction of CD34+ HPCs, we found that the simplest method of increasing vector infectivity is to pseudotype vector particles with the RD114 envelope instead of vesicular stomatitis virus G glycoprotein (VSV-G).

Antigens, CD34↗
PubMed1999

Dynamic changes in the characteristics of cationic lipidic vectors after exposure to mouse serum: implications for intravenous lipofection.

Intravenous gene delivery via cationic lipidic vectors gives systemic gene expression particularly in the lung. In order to understand the mechanism of intravenous lipofection, a systematic study was performed to investigate the interactions of lipidic vectors with mouse serum emphasizing how serum affects the biophysical and biological properties of vectors of different lipid compositions. Results from this study showed that lipidic vectors underwent dynamic changes in their characteristics after exposure to serum. Addition of lipidic vectors into serum resulted in an immediate aggregation of vectors. Prolonged incubation of lipidic vectors with serum led to vector disintegration as shown in turbidity study, sucrose-gradient centrifugation analysis and fluorescence resonance energy transfer (FRET) study. Vector disintegration was associated with DNA release and degradation as shown in EtBr intercalation assay and DNA digestion study. Serum-induced disintegration of vectors is a general phenomenon for all cationic lipidic vectors tested in this study. Yet, vectors of different lipid compositions vary greatly in the rate of disintegration. There is an inverse correlation between the disintegration rate of lipidic vectors and their in vivo transfection efficiency. Vectors with a rapid rate of disintegration such as those containing dioleoyl-phosphatidylethanolamine (DOPE) poorly stayed in the lung and were barely active in transfecting cells. In contrast, cholesterol-containing vectors that had a rapid aggregation and a slow disintegration were highly efficient in transfecting cells in vivo. The results of this study explain why cationic lipidic vectors of different lipid compositions have a dramatic difference in their in vivo transfection efficiency. These results also suggest that the study of the interactions of lipidic vectors with serum may serve as a predictive model for the in vivo efficiency of a lipidic vector. Further study of the numerous interactions of lipidic vectors with serum might lead to the development of a vector which can deliver a gene to target cells in a tissue-specific manner.

Animals↗
PubMed2004

Reducing mobilization of simian immunodeficiency virus based vectors by primer complementation.

BACKGROUND: Human gene therapy vectors based on primate lentiviruses harbour in contrast to oncoretroviruses the risk of vector mobilization by human immunodeficiency viruses (HIV). Infection of cells transduced with a lentiviral vector by HIV could lead to packaging of the lentiviral vector RNA into HIV particles and transfer of the vector. METHODS: A new approach based on primer complementation was developed to reduce the risk of vector mobilization. The primer binding site (PBS) of an SIV-based vector was mutated abolishing tRNA primer binding and thus blocking reverse transcription. This block was efficiently by-passed during vector production by providing an artificial tRNA matching the mutated PBS with titers reaching 10(6) infectious units/ml. RESULTS: Primer-complemented SIV vectors were mobilized from transduced cells by HIV-1 >150-fold less efficiently than vectors with wild-type PBS. Mobilization of the primer-complemented SIV vector by SIV was inhibited to a lesser extent indicating reduced efficacy of the primer complementation approach for preventing mobilization of lentiviral vectors by homologous virus. The analysis of the PBS of the vector DNA in target cells transduced with vectors containing mutated PBS in the absence of a matched tRNA suggests that formation of heterozygous particles followed by priming on the helper RNA and strand switch during reverse transcription can lead to mobilization of the primer-complemented vector by SIV, but not HIV-1. Although self-inactivating vectors were more efficient in preventing vector mobilization by HIV-1 than primer-complemented vectors, mobilization remained undectable only if both approaches were combined. CONCLUSIONS: The primer complementation approach should further reduce the risk of mobilization of self-inactivating SIV-based vectors by HIV-1 and thus increase their safety.

Animals↗