Intracellular trafficking of adeno-associated virus vectors: routing to the late endosomal compartment and proteasome degradation (original) (raw)
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2014
following MLV/VSV-G administration (27.7% erythroid, 18.6% myeloid); whereas 12.9% were positive after gene transfer with the lentiviral vectors (10.9% erythroid, 15.1% myeloid). Data from hematopoietic progenitor cell assays performed on samples from adults administered 1-2x10 8 producer cells/animal demonstrated that 10-30% of myeloid and erythroid progenitor cell colonies were transduced. Additionally, we demonstrated up to 1% of circulating peripheral blood mononuclear cells and granulocytes contain the transgene following producer cell administration. Markedly fewer non-hematopoietic tissues were PCR positive for vector sequences in the newborn and adult animals following direct intramarrow administration. The results of these studies indicate: (1) successful in vivo gene transfer in fetal, newborn, and adult rhesus monkey hematopoietic progenitor cells;
Infectious Entry Pathway of Adeno-Associated Virus and Adeno-Associated Virus Vectors
Journal of Virology, 2000
We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A1 prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that ...
Production of adeno-associated virus vectors for in vitro and in vivo applications
Scientific Reports
Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Of note, we achieved titers of 10 10-10 11 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. Our study provides an improved protocol for a more economical and efficient purified AAV preparation. Delivering a gene of interest to cells or animals has become an essential technique in biomedical research. In recent years, adeno-associated viruses (AAVs) have been used for many in vitro and in vivo applications due to their high transduction efficiency, safety, and extended stable gene expression. Furthermore, several recent clinical trials demonstrated the full potential of AAVs for human gene therapy 1-4. AAVs belong to the Parvoviridae family and are small non-enveloped viruses containing a linear singlestranded (ss) DNA 5. Wild-type AAVs, as part of their lysogenic cycle, can integrate into the AAVS1 site of human chromosome 19 or rarely at random locations 6. AAVs engineered for research or gene therapy, however, do not incorporate into the genome and instead form episomal concatemers in the host cell nucleus 7. These head-to-tail circular concatemers remain intact in non-dividing cells but are lost during mitosis. Thus post-mitotic tissues, such as neurons and cardiomyocytes, may express transgenes over several months 8. AAVs are ideal for gene therapy due to their low immunogenicity, restricted generation of neutralizing antibodies, and replication defectiveness. AAV production requires cytopathogenic effects, which only occur after co-infection with a helper adenovirus or herpesvirus 8,9. Helper viruses are difficult to remove and may induce undesired effects such as inflammation in the host. Current AAV expression systems avoid using helper viruses and include the plasmid pHelper instead 8 , containing essential genes such as E2A and E4. Human embryonic kidney cells (HEK) 293T cells, which express SV40 large T antigen, supply additional necessary proteins 9. This AAV production system is referred to as 'helper-free' and consists of three different plasmids encoding essential
A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells
Journal of Virology, 2013
Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families. FIG 3 Acidic endosomal pH is not required for efficient penetration of HAdV-C2. (A) Baf A1 (50 nM), niclosamide (5 M), and NH 4 Cl (25 mM) efficiently neutralize acidic compartments. HeLa-Ohio cells were treated with the indicated concentrations of the drugs for 60 min, incubated with DND-99 LysoTracker (red) to stain acidic compartments, fixed and stained with Hoechst dye (blue), and analyzed by confocal microscopy. Control cells for Baf A1 and niclosamide were treated with DMSO, and the control for NH 4 Cl was pH 8.2 culture medium. Images shown represent maximum projections of individual stacks (DND-99) or a single confocal section (Hoechst). Scale bar ϭ 10 m. (B) Penetration efficiency of HAdV-C2 in Baf A1-treated cells. Cells were permeabilized with SLO 20 min after virus internalization. The plot shows percentage of virus particles positive for the anti-Alexa-488 antibody (one dot represents one cell). Error bars represent the means Ϯ SEMs. Numbers of cells and viruses analyzed are indicated. (C) Penetration efficiency in niclosamide-treated cells. Cells were permeabilized with SLO at 60 min after virus internalization. Differences between control-and niclosamide-treated samples in the wt infection were statistically highly significant (two-tailed Mann-Whitney test). (D) Penetration efficiency in NH 4 Cl-treated cells. Cells were permeabilized with SLO at 30 min after virus internalization. Although differences between control and NH 4 Cl-treated cells in the wt infection were statistically highly significant, only in a minority of NH 4 Cl-treated cells was the wt virus penetration efficiency similar to that of TS1. (E) Two representative transmission electron micrographs of control cells 30 min p.i., showing three cytoplasmic HAdV-C2 particles (top, arrowheads) and an endosomal virus (bottom, arrow). Scale bars ϭ 200 nm. (F) Quantification of HAdV-C2 particles in the cytosol of DMSO (control)-, Baf A1, or niclosamide-treated cells 30 min p.i., as a percentage of total particles associated with the cells based on transmission EM analyses. One dot represents one cell.
Fibrinolysis, 1996
The plasminogen activation (PA) system is involved in vascular remodelling. Modulating its activity in vascular cells might be a way to interfere in processes such as angiogenesis and restenosis. Adenoviral vectors have become a favourable tool for direct gene transfer into vascular cells. In the interest of using adenoviral vectors to modulate plasminogen activator activity and endothelial and smooth muscle cell migration, we studied the effects of endothelial and smooth muscle cell transduction in vitro and in the umbilical vein ex vivo with a replication-defective adenoviral vector containing the 13-galactosidase gene (AdCMVLacZ). Segments of the umbilical vein were infected with AdCMVLacZ (109-10~°pfu/ml). After 48 h strong 13-galactosidase expression could be observed in the vessel wall, which was restricted to the endothelial layer. Although some heterogeneity in the transduction throughout the vessel could be seen, 13-galactosidase expression was detectable for 21 days in explant. Infection of human vascular endothelial cells (HUVEC) with recombinant adenoviral vectors is a dose and time dependent process. To achieve 100% infection of cultured HUVEC after a 30 min infection period, adenovirus concentrations ranging from 10 ~° to 5.10 '° pfu/ml are required. To achieve a similar infection of HUVSMC a concentration of 2.109 to 10 '° is sufficient. Expression of I~-galactosidase can be detected for at least 14 days. Effects of transduction of HUVEC with AdCMVLacZ on proliferation, morphology and monolayer integrity were also studied. At high virus concentrations (> 10 '9 pfu/ml) an inhibitory effect on cell proliferation was detected and cell morphology displayed many giant cells, often polynuclear, in these cultures. Infection of confluent HUVEC monolayers had, apart from a rapid transient effect during the infection procedure, little effect on the permeability of these monolayers. Only when the monolayers were infected using concentrations of 10 '° pfu/ml or more, an increase in permeability of these monolayers could be observed.
Journal of Virological Methods, 2006
We have developed a simple protocol to transfect mammalian cells using linear polyethylenimine (PEI). Our linear PEI protocol is as effective as commercial reagents in the transfection of HeLa cells and XDC293 cells, a derivative of HEK293 cells, but at a fraction of the cost. Greater than 90% of XDC293 cells and 98% of HeLa cells transfected using our method were positive for EGFP expression as determined by flow cytometery. Our protocol should be useful for many different applications such as large-scale production of recombinant protein and viruses, which requires transient transfection of mammalian cells in large batches. We have used this protocol to produce recombinant adeno-associated virus (AAV) in XDC293 cells and in HeLa cells. This requires transient expression of three adenovirus gene-products (E2A, E4orf6, and VA RNAs) as well as the AAV replication (Rep78, Rep68, Rep52, and Rep40) and capsid (VP1, VP2, and VP3) proteins. Production of a recombinant AAV that expresses green fluorescent protein was assessed by quantitative PCR and by transduction of HeLa cells. Linear PEI is a better transfection reagent than calcium phosphate for the production of recombinant AAV in both HEK293 and HeLa cells. In addition, when both HeLa and XDC293 cells were by our method, HeLa cells in the absence of E1A generated threefold more recombinant AAV than XDC293 cells, which constitutively express E1A.
Journal of Virology, 2004
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 CD34positive 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. FIG. 4. Intracellular trafficking of Cy3-labeled HD5/35 and ⌬5/35 vectors in MO7e cells. Cells were infected with indicated Ad vectors (red) for 15 min and 2 h. Then, cells were fixed and stained to detect colocalization of virus with cathepsin B-positive endosomal compartments (green) as described in the legend of Fig. 3A. Confocal images of cells were taken in both green and red channels. Colocalization of virus with cathepsin B-positive endosomes appears as yellow. Representative fields are shown.