Defective and nondefective adenovirus vectors for expressing foreign genes in vitro and in vivo

Gene ◽  
1991 ◽  
Vol 101 (2) ◽  
pp. 195-202 ◽  
Author(s):  
Massimo Levrero ◽  
Véronique Barban ◽  
Sylvie Manteca ◽  
Annick Ballay ◽  
Clara Balsamo ◽  
...  
Keyword(s):  
Adenovirus Vectors ◽  
Foreign Genes

scholarly journals In Vitro and In Vivo Biology of Recombinant Adenovirus Vectors with E1, E1/E2A, or E1/E4 Deleted

1998 ◽  
Vol 72 (3) ◽  
pp. 2022-2032 ◽  
Author(s):  
M. Lusky ◽  
M. Christ ◽  
K. Rittner ◽  
A. Dieterle ◽  
D. Dreyer ◽  
...  
Keyword(s):  
Recombinant Adenovirus ◽  
Virus Genome ◽  
Minor Role ◽  
Immunodeficient Mice ◽  
Viral Genomes ◽  
Viral Genes ◽  
Adenovirus Vectors ◽  
A Minor

ABSTRACT Isogenic, E3-deleted adenovirus vectors defective in E1, E1 and E2A, or E1 and E4 were generated in complementation cell lines expressing E1, E1 and E2A, or E1 and E4 and characterized in vitro and in vivo. In the absence of complementation, deletion of both E1 and E2A completely abolished expression of early and late viral genes, while deletion of E1 and E4 impaired expression of viral genes, although at a lower level than the E1/E2A deletion. The in vivo persistence of these three types of vectors was monitored in selected strains of mice with viral genomes devoid of transgenes to exclude any interference by immunogenic transgene-encoded products. Our studies showed no significant differences among the vectors in the short-term maintenance and long-term (4-month) persistence of viral DNA in liver and lung cells of immunocompetent and immunodeficient mice. Furthermore, all vectors induced similar antibody responses and comparable levels of adenovirus-specific cytotoxic T lymphocytes. These results suggest that in the absence of transgenes, the progressive deletion of the adenovirus genome does not extend the in vivo persistence of the transduced cells and does not reduce the antivirus immune response. In addition, our data confirm that, in the absence of transgene expression, mouse cellular immunity to viral antigens plays a minor role in the progressive elimination of the virus genome.

Increased in vitro and in vivo gene transfer by adenovirus vectors containing chimeric fiber proteins.

1997 ◽  
Vol 71 (11) ◽  
pp. 8221-8229 ◽  
Author(s):  
T J Wickham ◽  
E Tzeng ◽  
L L Shears ◽  
P W Roelvink ◽  
Y Li ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Adenovirus Vectors ◽  
Fiber Proteins ◽  
In Vivo Gene Transfer

scholarly journals Key Role of the Scavenger Receptor MARCO in Mediating Adenovirus Infection and Subsequent Innate Responses of Macrophages

mBio ◽  
2017 ◽  
Vol 8 (4) ◽  
Author(s):  
Mareike D. Maler ◽  
Peter J. Nielsen ◽  
Nicole Stichling ◽  
Idan Cohen ◽  
Zsolt Ruzsics ◽  
...  
Keyword(s):  
Alveolar Macrophages ◽  
Scavenger Receptor ◽  
Adenoviral Vectors ◽  
Adenovirus Infection ◽  
Respiratory Pathogens ◽  
Surface Receptors ◽  
Adenovirus Vectors ◽  
Macrophage Subtypes

ABSTRACT The scavenger receptor MARCO is expressed in several subsets of naive tissue-resident macrophages and has been shown to participate in the recognition of various bacterial pathogens. However, the role of MARCO in antiviral defense is largely unexplored. Here, we investigated whether MARCO might be involved in the innate sensing of infection with adenovirus and recombinant adenoviral vectors by macrophages, which elicit vigorous immune responses in vivo. Using cells derived from mice, we show that adenovirus infection is significantly more efficient in MARCO-positive alveolar macrophages (AMs) and in AM-like primary macrophage lines (Max Planck Institute cells) than in MARCO-negative bone marrow-derived macrophages. Using antibodies blocking ligand binding to MARCO, as well as gene-deficient and MARCO-transfected cells, we show that MARCO mediates the rapid adenovirus transduction of macrophages. By enhancing adenovirus infection, MARCO contributes to efficient innate virus recognition through the cytoplasmic DNA sensor cGAS. This leads to strong proinflammatory responses, including the production of interleukin-6 (IL-6), alpha/beta interferon, and mature IL-1α. These findings contribute to the understanding of viral pathogenesis in macrophages and may open new possibilities for the development of tools to influence the outcome of infection with adenovirus or adenovirus vectors. IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo. The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors. IMPORTANCE Macrophages play crucial roles in inflammation and defense against infection. Several macrophage subtypes have been identified with differing abilities to respond to infection with both natural adenoviruses and recombinant adenoviral vectors. Adenoviruses are important respiratory pathogens that elicit vigorous innate responses in vitro and in vivo. The cell surface receptors mediating macrophage type-specific adenovirus sensing are largely unknown. The scavenger receptor MARCO is expressed on some subsets of naive tissue-resident macrophages, including lung alveolar macrophages. Its role in antiviral macrophage responses is largely unexplored. Here, we studied whether the differential expression of MARCO might contribute to the various susceptibilities of macrophage subtypes to adenovirus. We demonstrate that MARCO significantly enhances adenovirus infection and innate responses in macrophages. These results help to understand adenoviral pathogenesis and may open new possibilities to influence the outcome of infection with adenoviruses or adenovirus vectors.

scholarly journals Long-Term Transgene Expression in Proliferating Cells Mediated by Episomally Maintained High-Capacity Adenovirus Vectors

2004 ◽  
Vol 78 (1) ◽  
pp. 9-22 ◽  
Author(s):  
Florian Kreppel ◽  
Stefan Kochanek
Keyword(s):  
Transgene Expression ◽  
High Capacity ◽  
Target Cells ◽  
Resting Cells ◽  
Proliferating Cells ◽  
Daughter Cells ◽  
Adenovirus Vectors

ABSTRACT High-capacity “gutless” adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

scholarly journals Effect of Preexisting Immunity on an Adenovirus Vaccine Vector: In Vitro Neutralization Assays Fail To Predict Inhibition by Antiviral Antibody In Vivo

2009 ◽  
Vol 83 (11) ◽  
pp. 5567-5573 ◽  
Author(s):  
Susan L. Pichla-Gollon ◽  
Shih-Wen Lin ◽  
Scott E. Hensley ◽  
Marcio O. Lasaro ◽  
Larissa Herkenhoff-Haut ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Neutralizing Antibody ◽  
Human Adenovirus ◽  
T Cell Response ◽  
Fundamental Properties ◽  
High Prevalence ◽  
Antiviral Antibody ◽  
Adenovirus Vectors

ABSTRACT A major obstacle to the use of adenovirus vectors derived from common human serotypes, such as human adenovirus 5 (AdHu5), is the high prevalence of virus-neutralizing antibodies in the human population. We previously constructed a variant of chimpanzee adenovirus 68 (AdC68) that maintained the fundamental properties of the carrier but was serologically distinct from AdC68 and resisted neutralization by AdC68 antibodies. In the present study, we tested whether this modified vector, termed AdCDQ, could induce transgene product-specific CD8+ T cells in mice with preexisting neutralizing antibody to wild-type AdC68. Contrary to our expectation, the data show conclusively that antibodies that fail to neutralize the AdCDQ mutant vector in vitro nevertheless impair the vector's capacity to transduce cells and to stimulate a transgene product-specific CD8+ T-cell response in vivo. The results thus suggest that in vitro neutralization assays may not reliably predict the effects of virus-specific antibodies on adenovirus vectors in vivo.

scholarly journals Inflammation and Immune Response of Intra-Articular Serotype 2 Adeno-Associated Virus or Adenovirus Vectors in a Large Animal Model

Arthritis ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Akikazu Ishihara ◽  
Jeffrey S. Bartlett ◽  
Alicia L. Bertone
Keyword(s):  
Joint Inflammation ◽  
Joint Fluid ◽  
Transduction Efficiency ◽  
Large Animal ◽  
Synovial Cells ◽  
Gene Transduction ◽  
Adeno Associated Virus ◽  
Adenovirus Vectors

Intra-articular gene therapy has potential for the treatment of osteoarthritis and rheumatoid arthritis. To quantify in vitro relative gene transduction, equine chondrocytes and synovial cells were treated with adenovirus vectors (Ad), serotype 2 adeno-associated virus vectors (rAAV2), or self-complementary (sc) AAV2 vectors carrying green fluorescent protein (GFP). Using 6 horses, bilateral metacarpophalangeal joints were injected with Ad, rAAV2, or scAAV2 vectors carrying GFP genes to assess the in vivo joint inflammation and neutralizing antibody (NAb) titer in serum and joint fluid. In vitro, the greater transduction efficiency and sustained gene expression were achieved by scAAV2 compared to rAAV2 in equine chondrocytes and synovial cells. In vivo, AAV2 demonstrated less joint inflammation than Ad, but similar NAb titer. The scAAV2 vectors can induce superior gene transduction than rAAV2 in articular cells, and both rAAV2 and scAAV2 vectors were showed to be safer for intra-articular use than Ad vectors.

scholarly journals Transcription Initiation Activity of Adenovirus Left-End Sequence in Adenovirus Vectors with E1 Deleted

2003 ◽  
Vol 77 (2) ◽  
pp. 1633-1637 ◽  
Author(s):  
Masato Yamamoto ◽  
Julia Davydova ◽  
Koichi Takayama ◽  
Ramon Alemany ◽  
David T. Curiel
Keyword(s):  
Nucleotide Sequence ◽  
Transcription Initiation ◽  
Adenovirus Vector ◽  
Nucleotide Sequences ◽  
Alveolar Cells ◽  
Strong Activity ◽  
Adenovirus Vectors

ABSTRACT We analyzed the transcription initiation activity of the left-end sequence (first 342 bp) of the adenovirus genome in the context of an adenovirus vector with E1 deleted in in vitro and in vivo gene transfer models. While nucleotide sequences 1 to 190 and 1 to 342 showed strong activity in three out of three lung cancer cell lines, nucleotide sequence 1 to 103 showed limited activity in H358, cells which show characteristics of type 2 alveolar cells. In vivo, the transcription initiation activities of nucleotide sequence 1 to 103 in the liver and the lung were minimal, while nucleotide sequences 1 to 190 and 1 to 342 showed strong activity comparable to that of the cytomegalovirus promoter. Further understanding of the transcription initiation activity of the left-end sequence of the adenovirus genome should lead to optimization of adenovirus vectors.

Targeted Anti-Tumor Research of Mesenchymal Stem Cells Delivered Enzyme-Prodrug Gene CYP450.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3582-3582
Author(s):  
Jishi Wang ◽  
Yuan Yang ◽  
Wei Zhang ◽  
Pengxiang Guo
Keyword(s):  
Nude Mice ◽  
Recombinant Adenovirus ◽  
Annexin V ◽  
Fluorescence Microscope ◽  
Mice Model ◽  
Rt Pcr ◽  
Nude Mice Model ◽  
Adenovirus Vectors

Abstract Abstract 3582 Poster Board III-519 Objective Cytochrome P450(CYP450-CYP1A2 /CYP2B6/CYP2C9) was transfected into human bone marrow-derived mesenchymal stem cells (hBMSCs), and the targed anti-tumor effect of BMSC-CYP450 cooperated with enzyme-prodrug(Dacarbazine (DTIC)/Cyclophosphamide (CPA)) was measured to provide laboratory data base for gene directed enzyme prodrug targeted anti-tumor therapy (GDEPT) which used BMSC as vehicles. Methods We respectively cloned CYP1A2/CYP2B6/CYP2C9 cDNA from human liver and constructed recombinant adenovirus vectors(pAd5CMV-NpA-CYP1A2/ pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9) which titer was 1×1012 pfu/mL. These hBMSCs were separated, cultured, purified, and detected by morphology, flow cytometry, osteogenic, adipogenic and chondrogenic induction, and RT-PCR(A surface marker for the identification of MSCs-the neural ganglioside GD2 gene). The tropism of BMSCs for cancer cells was detected by Transwell inserts technique. These recombinant vectors were transferred into BMSCs and A375/K562 cells, and the expression of EGFP and CYP1A2/CYP2B6/CYP2C9 was detected by fluorescence microscope, RT-PCR and Western blot respectively. Inverted microscope, MTT and Annexin V-FITC/PI detected the anti-tumor effect of CYP450 recombinant adenovirus vectors combined with chemotherapeutic prodrug DTIC/CPA in vitro. A human melanoma(A375) BALB/c nude mice model and a human myelocytic leukemia(K562) BALB/c nude mice model was constructed and detected by immuno-histochemistry analysis. The CYP1A2 gene tranfected BMSCs were injected into the A375 BALB/c nude mice model in combination with DTIC through caudal vein, while CYP2B6/CYP2C9 gene tranfected BMSCs were injected into K562 BALB/c nude mice model in combination with CPA in the same way. The measurement of tumors size, fluorescence microscope and TUNEL were used to detect anti-tumor effect of BMSCs-CYP1A2 cooperating with DTIC and BMSCs-CYP2B6/CYP2C9 with CPA in vivo. Results We constructed the recombinant adenovirus vectors pAd5CMV-NpA-CYP1A2/pAd5CMV-NpA-CYP2B6/pAd5CMV-NpA-CYP2C9 and pAd5CMV-NpA-EGFP successfully. BMSCs was separated successfully, and it respectively showed that BMSCs can migrate through the polycarbonate filter toward K562 and A375 cells in the lower chamber in vitro. Fluorescence microscope detected the expression of EGFP, while both RT-PCR and Western blot detected high expression of CYP1A2/CYP2B6/CYP2C9 in gene-transfected group cells. Inverted microscope, MTT and Annexin V-FITC/PI confirmed that BMSCs transferred with CYP1A2/CYP2B6/CYP2C9 recombinant adenovirus vectors could activate DTIC/CPA and increase its anti-tumor effect(In the DTIC/CPA concentration(0.05 mmol/L/2.5 mmol/L) which BMSCs was relatively safe, the cell apoptosis was (38.38±2.27)% (P<0.01), (42.69±2.03)% (P<0.01) and (39. 51±1.94)% (P<0.01) in BMSCs-CYP1A2+A375 group, BMSCs-CYP2B6+K562 group and BMSCs-CYP2C9+K562 group respectively. ). A375 and K562 BALB/c nude mice model was constructed successfully. The sizes of the tumor in the nude mice treated with transfected BMSCs and DTIC/CPA were significantly smaller than control case and changed along with concentration(P< 0.01, P< 0.05).BMSCs was congregated to tumor site in fluorescence microscope. Apoptosis of tumor cells was conspicuously more in BMSCs-CYP1A2+A375/BMSCs-CYP2B6+K562/BMSCs-CYP2C9+K562 treatment group than in control group by TUNEL. Conclusion BMSCs had the tropism for cancer cells in vitro and vivo. DTIC can be catalyzed by CYP2E1/CYP1A2, while CPA by CYP2B6/CYP2C9 in vitro and vivo. BMSC-based enzyme prodrug system of CYP2E1/CYP1A2 and DTIC can induce A375 cells apoptosis, while BMSC-based enzyme prodrug system of CYP2B6/CYP2C9 and CPA can induce K562 cells apoptosis in vitro and targetedly in vivo. Disclosures: No relevant conflicts of interest to declare.

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