scholarly journals Dosage Thresholds and Influence of Transgene Cassette in Adeno-Associated Virus–Related Toxicity

2018 ◽  
Vol 29 (11) ◽  
pp. 1235-1241 ◽  
Author(s):  
Hanen Khabou ◽  
Chloé Cordeau ◽  
Laure Pacot ◽  
Sylvain Fisson ◽  
Deniz Dalkara
Keyword(s):  
Adeno Associated Virus ◽  
Transgene Cassette

scholarly journals A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette

2006 ◽  
Vol 80 (23) ◽  
pp. 11699-11709 ◽  
Author(s):  
Hongjie Wang ◽  
André Lieber
Keyword(s):  
Fluorescent Protein ◽  
Adenovirus Vector ◽  
Viral Gene ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Site Specific ◽  
Vector Integration ◽  
Site Specific Integration ◽  
Integration Sites ◽  
Transgene Cassette

ABSTRACT Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular “nicking” enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.

scholarly journals Site-Specific Integration in Mammalian Cells Mediated by a New Hybrid Baculovirus–Adeno-Associated Virus Vector

1998 ◽  
Vol 72 (6) ◽  
pp. 5025-5034 ◽  
Author(s):  
Fabio Palombo ◽  
Andrea Monciotti ◽  
Alessandra Recchia ◽  
Riccardo Cortese ◽  
Gennaro Ciliberto ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Ex Vivo ◽  
High Titer ◽  
Human Fibroblasts ◽  
Pcr Amplification ◽  
Adeno Associated Virus ◽  
Site Specific ◽  
Hybrid Vectors ◽  
Site Specific Integration ◽  
Transgene Cassette

ABSTRACT Baculovirus can transiently transduce primary human and rat hepatocytes, as well as a subset of stable cell lines. To prolong transgene expression, we have developed new hybrid vectors which associate key elements from adeno-associated virus (AAV) with the elevated transducing capacity of baculovirus. The hybrid vectors contain a transgene cassette composed of the β-galactosidase (β-Gal) reporter gene and the hygromycin resistance (Hygr) gene flanked by the AAV inverted terminal repeats (ITRs), which are necessary for AAV replication and integration in the host genome. Constructs were derived both with and without the AAVrep gene under the p5 and p19 promoters cloned in different positions with respect to the baculovirus polyheidrin promoter. A high-titer preparation of baculovirus-AAV (Bac-AAV) chimeric virus containing the ITR–Hygr–β-Gal sequence was obtained with insect cells only when the rep gene was placed in an antisense orientation to the polyheidrin promoter. Infection of 293 cells with Bac-AAV virus expressing the rep gene results in a 10- to 50-fold increase in the number of Hygr stable cell clones. Additionally, rep expression determined the localization of the transgene cassette in the aavs1 site in approximately 41% of cases as detected by both Southern blotting and fluorescent in situ hybridization analysis. Moreover, site-specific integration of the ITR-flanked DNA was also detected by PCR amplification of the ITR-aavs1 junction in transduced human fibroblasts. These data indicate that Bac-AAV hybrid vectors can allow permanent, nontoxic gene delivery of DNA constructs for ex vivo treatment of primary human cells.

scholarly journals Herpes Simplex Virus Type 1/Adeno-Associated Virus Hybrid Vectors Mediate Site-Specific Integration at the Adeno-Associated Virus Preintegration Site, AAVS1, on Human Chromosome 19

2002 ◽  
Vol 76 (14) ◽  
pp. 7163-7173 ◽  
Author(s):  
Thomas Heister ◽  
Irma Heid ◽  
Mathias Ackermann ◽  
Cornel Fraefel
Keyword(s):  
Adeno Associated Virus ◽  
Site Specific ◽  
Hybrid Vectors ◽  
Site Specific Integration ◽  
Cell Genome ◽  
Simplex Virus ◽  
Transgene Cassette ◽  
Hsv 1

ABSTRACT Herpes simplex virus type 1 (HSV-1)-based amplicon vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 vectors is their instability of transgene expression. By contrast, vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV vectors is the small transgene capacity (∼4.6 kb). In this study, we constructed HSV/AAV hybrid vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the hybrid vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the hybrid vector. All hybrid vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in hybrid vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV hybrid vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.

Immune Electron Microscopy (IEM) of a Canine Adeno-Associated Virus

1974 ◽  
Vol 32 ◽  
pp. 256-257
Author(s):  
Gunter F. Thomas ◽  
M. David Hoggan
Keyword(s):  
Electron Microscopy ◽  
Cell Cultures ◽  
Complement Fixation ◽  
Hepatitis Virus ◽  
Wide Distribution ◽  
Immune Electron Microscopy ◽  
Adeno Associated Virus ◽  
Virus Like Particle ◽  
Dog Kidney ◽  
Green Monkeys

In 1968, Sugimura and Yanagawa described a small 25 nm virus like particle in association with the Matsuda strain of infectious canine hepatitis virus (ICHV). Domoto and Yanagawa showed that this particle was dependent on ICHV for its replication in primary dog kidney cell cultures (PDK) and was resistant to heating at 70°C for 10 min, and concluded that it was a canine adeno-associated virus (CAAV). Later studies by Onuma and Yanagawa compared CAAV with the known human serotypes (AAV 1, 2, 3) and AAV-4, known to be associated with African Green Monkeys. Using the complement fixation (CF) test, they found that CAAV was serologically related to AAV-3 and had wide distribution in the dog population of Japan.

1509: Adeno-Associated Virus-Mediated Human IL-10 Gene Transfer Suppresses the Development of Experimental Autoimmune Orchitis

2005 ◽  
Vol 173 (4S) ◽  
pp. 409-409
Author(s):  
Masami Watanabe ◽  
Atsushi Nagai ◽  
Norihiro Kusumi ◽  
Yasutomo Nasu ◽  
Hiromi Kumon ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Adeno Associated Virus ◽  
Autoimmune Orchitis ◽  
Experimental Autoimmune

1011: Suppression of Renal Cell Carcinoma Lung Metastasis by Adeno-Associated Virus (AAV) Mediated Gene Transfer of a Soluble Receptor of VEGF

2004 ◽  
Vol 171 (4S) ◽  
pp. 267-267
Author(s):  
Ichiro Yoshimura ◽  
Yasunori Mizuguchi ◽  
Akira Miyajima ◽  
Tomohiko Asano ◽  
Hiroaki Mizukami ◽  
...  
Keyword(s):  
Renal Cell Carcinoma ◽  
Gene Transfer ◽  
Cell Carcinoma ◽  
Lung Metastasis ◽  
Renal Cell ◽  
Soluble Receptor ◽  
Adeno Associated Virus
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