scholarly journals Widespread and targeted gene expression by systemic AAV vectors: Production, purification, and administration

2018 ◽  
Author(s):  
Rosemary C Challis ◽  
Sripriya Ravindra Kumar ◽  
Ken Y Chan ◽  
Collin Challis ◽  
Min J Jang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Culture ◽  
Nervous System ◽  
Experimental Design ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Biomedical Applications ◽  
Viral Dose ◽  
Targeted Gene Expression ◽  
Time Required

ABSTRACTWe recently developed novel AAV capsids for efficient and noninvasive gene transfer across the central and peripheral nervous systems. In this protocol, we describe how to produce and systemically administer AAV-PHP viruses to label and/or genetically manipulate cells in the mouse nervous system and organs including the heart. The procedure comprises three separate stages: AAV production, intravenous delivery, and evaluation of transgene expression. The protocol spans eight days, excluding the time required to assess gene expression, and can be readily adopted by laboratories with standard molecular and cell culture capabilities. We provide guidelines for experimental design and choosing the capsid, cargo, and viral dose appropriate for the experimental aims. The procedures outlined here are adaptable to diverse biomedical applications, from anatomical and functional mapping to gene expression, silencing, and editing.

Abstract 249: Delayed Gene Transfer Increases Transgene Expression in Vein Grafts

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Liang Du ◽  
Jingwan Zhang ◽  
Alexander Clowes ◽  
David Dichek
Keyword(s):  
Gene Expression ◽  
Blood Flow ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Ex Vivo ◽  
Arterial Blood Flow ◽  
Vein Grafts ◽  
Arterial Blood ◽  
En Face

Background Autogenous vein grafts are effective therapies for obstructive arterial disease. However, their long-term utility is limited by stenosis and occlusion. Genetic engineering of veins that prevents intimal hyperplasia and atherosclerosis could significantly improve the clinical utility of vein grafts. We recently reported that a helper-dependent adenoviral vector (HDAd) reduces atherosclerosis 4 wks after gene transfer in fat-fed rabbits and can express a therapeutic transgene (apo AI) in normal rabbit carotids for at least 48 wks. Use of HDAd for vein graft gene therapy will depend on achievement of similarly high and persistent transgene expression in grafted veins. Hypothesis We tested the hypothesis that Ad-mediated transgene expression in grafted veins (at an early time point) can be increased by varying the timing of gene transfer. Methods Rabbit external jugular veins were transduced by exposure to a beta galactosidase (b-gal)-expressing Ad: in situ either without (a) or with (b) immediate arterial grafting; c) ex vivo with grafting after overnight incubation with Ad; d) in vivo immediately after grafting and e) in vivo 4 wks after grafting (n = 6 - 19 veins/group). Transgene expression was measured in veins removed 3 d after Ad exposure by PCR quantitation of b-gal mRNA and by en-face planimetry of blue-stained area. Results B-gal transgene expression was higher in ungrafted veins than in veins grafted immediately after gene transfer (84 ± 17 vs 9.4 ± 2.0 arbitrary units (AU); P < 0.0001). Overnight incubation of veins with Ad increased gene expression ex vivo by 10-fold but neither this nor performing vector infusion immediately after grafting improved gene expression (11 ± 4.7 and 9.1 ± 1.8 AU; P > 0.9 for both vs immediately grafted veins). Delaying gene transfer until 4 wks after grafting significantly increased gene expression, to a level equivalent to transgene expression in ungrafted veins (61 ± 11 AU; P = 0.3 vs ungrafted veins). En face planimetry yielded similar results. Conclusions Exposure of a transduced vein to arterial blood flow is associated with significant loss of transgene expression. Transgene expression in grafted veins is significantly higher when gene transfer is performed 4 wks after exposure of the vein to arterial blood flow.

Stable Gene Transfer and Expression in Human Primary T-Cells by the Sleeping Beauty Transposon System.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5539-5539
Author(s):  
Xianzheng Zhou ◽  
Xin Huang ◽  
Andrew C. Wilber ◽  
Lei Bao ◽  
Dong Tuong ◽  
...  
Keyword(s):  
Gene Expression ◽  
T Cells ◽  
T Cell ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Sleeping Beauty ◽  
Stable Gene ◽  
Cell Clones ◽  
Stable Gene Expression

Abstract The Sleeping Beauty (SB) transposon system is a non-viral DNA delivery system in which a transposase directs integration of an SB transposon into TA-dinucleotide sites in the genome. To determine whether the SB transposon system can mediate integration and long-term transgene expression in human primary T-cells, freshly isolated peripheral blood lymphocytes (PBLs) without prior activation were nucleofected with SB vectors carrying a DsRed reporter gene. Plasmids containing the SB transposase on the same (cis) (n=10) or separate molecule (trans) (n=8) as the SB transposon mediated long-term and stable reporter gene expression in human primary T-cells. We observed that delivery of SB transposase-encoding plasmid in trans effectively mediated stable gene expression in primary T-cells, exhibiting about a 3-fold increase (11% vs. 3% with 10 microgram plasmid on day 21) in potency in comparison with the cis vector (p&lt;0.0001). In addition, a transposase mutant construct was incapable of mediating stable gene expression in human PBLs (n=6, p&lt;0.0001), confirming that catalytic DDE domain is necessary for transposition in human primary T-cells. Immunophenotyping analysis in transposed T-cells showed that both CD4 and CD8 T-cells were transgene positive. SB-mediated high level of transgene expression in human T-cells was maintained in culture for at least 4 months without losing observable expression. Southern hybridization analysis showed a variety of transposon integrants among the 6 DsRed positive T-cell clones and no transposon sequences identifiable in the 2 DsRed negative clones. Sequencing of transposon:chromosome junctions in 5 out of 6 transposed T-cell clones confirmed that stable gene expression was due to SB-mediated transposition. In other studies, PBLs were successfully transfected using the SB transposon system and shown to stably and functionally express a fusion protein consisting of a surface receptor useful for positive T-cell selection and a “suicide” gene useful for elimination of transfected T-cells after chemotherapy. This study is the first report demonstrating that the SB transposon system can mediate stable gene transfer in human primary PBLs, which may be more advantageous for T-cell based gene therapies over widely used virus-based or conventional mammalian DNA vectors in terms of simplicity, stability, efficiency and safety.

scholarly journals Reduced Inflammatory Reactions to the Inoculation of Helper-Dependent Adenoviral Vectors in Traumatically Injured Rat Brain

2002 ◽  
Vol 22 (8) ◽  
pp. 959-970 ◽  
Author(s):  
Linglong Zou ◽  
Patricia Yotnda ◽  
Tiejun Zhao ◽  
Xiaoqing Yuan ◽  
Yan Long ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Rat Brain ◽  
Neutralizing Antibodies ◽  
Transgene Expression ◽  
First Generation ◽  
High Efficiency ◽  
Adenoviral Vectors ◽  
Tissue Loss ◽  
Inflammatory Reactions

Traumatic brain injury (TBI) causes delayed neuronal deficits that in principle could be prevented by timely intervention with therapeutic genes. However, appropriate vectors for gene transfer to the brain with TBI remain to be developed. First-generation adenoviruses (fgAd) are usually associated with inflammatory and toxic effects when inoculated into brains, despite their high efficiency of gene transfer to these tissues. In this study the authors attempted to determine whether a less immunogenic gene-transfer protocol can be established in the traumatically injured rat brain using helper-dependent adenoviruses (hdAd), a novel adenoviral construct with full deletion of viral coding sequences. Their results show that transgene expression from intrahippocampally inoculated hdAd is maintained for at least 2 months after TBI, in contrast to the much shorter duration of fgAd-mediated gene expression. There was only minimal secretion of proinflammatory IL-1β and TNF-α after inoculation of hdAd. Furthermore, the hdAd-mediated gene expression was associated with less microglial proliferation, astrocytic activation, and macrophage infiltration than observed in fgAd-inoculated brains. There was no additional tissue loss after hdAd inoculation compared with PBS injection. Although both anti-adenoviral and neutralizing antibodies were found in serum after brain inoculation of hdAd, they did not appear to affect transgene expression. The results suggest that hdAd are less immunogenic vectors than conventional adenoviral vectors, and offer improved vehicles for long-term therapeutic transgene transfer to traumatically injured brains.

scholarly journals Sources of off-target expression from recombinase-dependent AAV vectors and mitigation with cross-over insensitive ATG-out vectors

2019 ◽  
Vol 116 (52) ◽  
pp. 27001-27010 ◽  
Author(s):  
Kyle B. Fischer ◽  
Hannah K. Collins ◽  
Edward M. Callaway
Keyword(s):  
Gene Expression ◽  
Transgenic Mouse ◽  
Transgene Expression ◽  
Cell Types ◽  
Cell Type ◽  
Recognition Sites ◽  
Highly Sensitive ◽  
Plasmid Production ◽  
Targeted Gene Expression ◽  
Mouse Lines

In combination with transgenic mouse lines expressing Cre or Flp recombinases in defined cell types, recombinase-dependent adeno-associated viruses (AAVs) have become the tool of choice for localized cell-type-targeted gene expression. Unfortunately, applications of this technique when expressing highly sensitive transgenes are impeded by off-target, or “leak” expression, from recombinase-dependent AAVs. We investigated this phenomenon and find that leak expression is mediated by both infrequent transcription from the inverted transgene in recombinant-dependent AAV designs and recombination events during bacterial AAV plasmid production. Recombination in bacteria is mediated by homology across the antiparallel recombinase-specific recognition sites present in recombinase-dependent designs. To address both of these issues we designed an AAV vector that uses mutant “cross-over insensitive” recognition sites combined with an “ATG-out” design. We show that these CIAO (cross-over insensitive ATG-out) vectors virtually eliminate leak expression. CIAO vectors provide reliable and targeted transgene expression and are extremely useful for recombinase-dependent expression of highly sensitive transgenes.

INSULATORS AND CHROMOSOME-TYPE VECTORS FOR GENE TRANSFER

2013 ◽  
Vol 25 (1) ◽  
pp. 318
Author(s):  
Lluis Montoliu ◽  
Almudena Fernández ◽  
Davide Seruggia ◽  
Diego Muñoz ◽  
Cristina Vicente
Keyword(s):  
Gene Expression ◽  
Gene Transfer ◽  
Transgene Expression ◽  
Expression Patterns ◽  
Regulatory Elements ◽  
Host Genome ◽  
Yeast Cells ◽  
Dna Molecules ◽  
Position Effects ◽  
Artificial Chromosomes

Vertebrate genomes are functionally and structurally organised as gene expression domains. These domains contain all regulatory elements that are required for the gene (or genes) to be expressed correctly, according to a given pattern in time and space. The use of genomic-type DNA constructs in animal transgenesis ensures optimal transgene expression, once inserted into the host genome, because their large size include most if not all the regulatory elements that are needed for a given gene in order to be expressed correctly. Ideally, the entire gene expression domain is included. Large heterologous DNA molecules can be easily manipulated in bacterial or yeast cells, through the use of bacterial artificial chromosomes (BACs) or yeast artificial chromosomes (YACs), respectively. YACs are the vectors that allow the manipulation of the larger DNA molecules, in excess of 1 Mb (1000 kb). Some mammalian loci (i.e. the APP locus, ~450 kb) greatly exceed the maximum size for inserts that can be accommodated into BACs. Therefore, YACs are currently the only available robust and reliable solution to work with these large genes. The use of YACs, and/or BACs, usually ensures optimal transgene expression, because the heterologous constructs carry all that is required to resume the correct expression of the locus irrespective of the location where the transgene eventually lands in the host genome. Adjacent gene expression domains in genomes are often insulated by boundary elements, or insulators that insulate each domain and maintain the expression program of each gene according to their own set of regulatory elements, blocking any nondesirable interaction from its neighbouring locus. The function of these boundaries is, hence, to insulate gene expression domains in genomes allowing the protected locus to be expressed according to internal regulatory elements, without suffering from the adverse effects of flanking loci and without transmitting the effect of the internal regulatory elements beyond the protected domain. Insulators can act as enhancer blockers, preventing a distal enhancer from interacting with a given promoter, when placed in between, and/or as barriers, preventing the chromosomal position effects associated with random insertion of gene expression constructs in host genomes. In addition, insulators are known to contribute to the chromatin and nuclear structural organisation. A variety of molecular mechanisms has been associated with boundary function, probably reflecting the diversity of functional elements that can efficiently insulate genomic sequences. Insulator elements can be used in biotechnological applications, as spacers, as boundaries, in any gene expression construct to be used in gene transfer experiments, preventing the inappropriate expression patterns of transgenes and insulating them from neighbouring sequences surrounding the place of insertion in the host genomes.

Identification of adeno-associated viral vectors suitable for intestinal gene delivery and modulation of experimental colitis

2012 ◽  
Vol 302 (3) ◽  
pp. G296-G308 ◽  
Author(s):  
Steven Polyak ◽  
Annette Mach ◽  
Stacy Porvasnik ◽  
Lisa Dixon ◽  
Thomas Conlon ◽  
...  
Keyword(s):  
Gene Expression ◽  
Small Intestine ◽  
Gene Transfer ◽  
Real Time ◽  
Real Time Pcr ◽  
Transgene Expression ◽  
Intestinal Inflammation ◽  
Experimental Colitis ◽  
Mucosal Inflammation

Effective gene transfer with sustained gene expression is an important adjunct to the study of intestinal inflammation and future therapy in inflammatory bowel disease. Recombinant adeno-associated virus (AAV) vectors are ideal for gene transfer and long-term transgene expression. The purpose of our study was to identify optimal AAV pseudotypes for transduction of the epithelium in the small intestine and colon, which could be used for studies in experimental colitis. The tropism and transduction efficiencies of AAV pseudotypes 1–10 were examined in murine small intestine and colon 8 wk after administration by real-time PCR and immunohistochemistry. The clinical and histopathological effects of IL-10-mediated intestinal transduction delivered by AAVrh10 were examined in the murine IL-10−/− enterocolitis model. Serum IL-10 levels and IL-10 expression were followed by ELISA and real-time PCR, respectively. AAV pseudotypes 4, 7, 8, 9, and 10 demonstrated optimal intestinal transduction. Transgene expression was sustained 8 wk after administration and was frequently observed in enteroendocrine cells. Long-term IL-10 gene expression and serum IL-10 levels were observed following AAV transduction in an IL-10−/− model of enterocolitis. Animals treated with AAVrh10-IL-10 had lower disease activity index scores, higher colon weight-to-length ratios, and lower microscopic inflammation scores. This study identifies novel AAV pseudotypes with small intestine and colon tropism and sustained transgene expression capable of modulating mucosal inflammation in a murine model of enterocolitis.

scholarly journals Hemocyte-targeted gene expression in the female malaria mosquito using the hemolectin promoter from Drosophila

2019 ◽  
Author(s):  
Emilie Pondeville ◽  
Nicolas Puchot ◽  
Jean-Philippe Parvy ◽  
Guillaume Carrissimo ◽  
Mickael Poidevin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Anopheles Gambiae ◽  
Transgene Expression ◽  
Regulatory Region ◽  
Malaria Mosquito ◽  
Immune Markers ◽  
Good Tool ◽  
Immune Genes ◽  
Viral Nature ◽  
Targeted Gene Expression

AbstractHemocytes, the immune cells in mosquitoes, participate in immune defenses against pathogens including malaria parasites. Mosquito hemocytes can also be infected by arthropod-borne viruses but the pro- or anti-viral nature of this interaction is unknown. Although there has been progress on hemocyte characterization during pathogen infections in mosquitoes, the specific contribution of hemocytes to immune responses and the hemocyte-specific functions of immune genes and pathways remain unresolved due to the lack of genetic tools to manipulate gene expression in these cells specifically. Here, we used the Gal4-UAS system to characterize the activity of the Drosophila hemocyte-specific hemolectin promoter in the adults of Anopheles gambiae, the malaria mosquito. We established an hml-Gal4 driver line that we further crossed to a fluorescent UAS responder line, and examined the expression pattern in the adult progeny driven by the hml promoter. We show that the hml regulatory region drives hemocyte-specific transgene expression in a subset of hemocytes, and that transgene expression is triggered after a blood meal. The hml promoter drives transgene expression in differentiating prohemocytes as well as in differentiated granulocytes. Analysis of different immune markers in hemocytes in which the hml promoter drives transgene expression revealed that this regulatory region could be used to study phagocytosis as well as melanization. Finally, the hml promoter drives transgene expression in hemocytes in which o’nyong’nyong virus replicates. Altogether, the hml promoter constitutes a good tool to drive transgene expression in hemocyte only and to analyze the function of these cells and the genes they express during pathogen infection in Anopheles gambiae.

scholarly journals The Effect of Rosmarinic Acid in Neural Differentiation of Wartons Jelly-derived Mesenchymal Stem Cells in Two Dimensional and Three Dimensional Cultures using Chitosan-based Hydrogel

2021 ◽  
Author(s):  
Mohsen Salmanvandi ◽  
◽  
Seyed Mohammad Amin Haramshahi ◽  
Elahe Mansoori ◽  
Akram Alizadeh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Culture ◽  
Nervous System ◽  
Rosmarinic Acid ◽  
Three Dimensional ◽  
3D Culture ◽  
Neural Tissue ◽  
Two Dimensional ◽  
Neural Tissue Engineering

Numerous studies have shown the positive effects of rosmarinic acid on the nervous system. Rosmarinic acid as a herbal compound with anti-inflammatory effects can prevent the destructive effect of inflammation on the nervous system. Furthermore, various studies have emphasized the advantages of three dimensional (3D) culture over the two dimensional (2D) culture of cells. In this study, thermosensitive chitosan-based hydrogel as a 3D scaffold with the combination of chitosan (CH), beta-glycerol phosphate(βGP) and hydroxyl ethyl cellulose (HEC) CH-βGP-HEC loaded with rosmarinic acid was used to induce neuronal differentiation in human Wharton jelly stem cells. Also, cells were divided into eight groups in order to evaluate the effect of 3D cell culture and to compare gene expression in different induction conditions. The results of gene expression analysis showed the highest expression of neuronal markers in WJMSCs cultured in CH-βGP-HEC loaded with differentiation medium and rosmarinic acid. According to the results of gene expression, rosmarinic acid alone have positively effect on the induction of expression of neural markers. This positive effect is enhanced by cell culture in 3D conditions. This study shows that rosmarinic acid can be considered an inexpensive and available compound for use in neural tissue engineering. The results of this study indicate that rosmarinic acid can be considered a cheap and available compound for use in neural tissue engineering. The results of our study also emphasize the need to study stem cell differentiation under 3D culture conditions.

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