scholarly journals Activation of p38 and ERK Signaling during Adenovirus Vector Cell Entry Lead to Expression of the C-X-C Chemokine IP-10

2002 ◽  
Vol 76 (4) ◽  
pp. 1559-1568 ◽  
Author(s):  
Lee Anne Tibbles ◽  
Jason C. L. Spurrell ◽  
Gloria P. Bowen ◽  
Qiang Liu ◽  
Mindy Lam ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Mrna Expression ◽  
Inflammatory Responses ◽  
Human Gene ◽  
Adenovirus Vector ◽  
Endosomal Escape ◽  
Human Gene Therapy ◽  
Serotype 5 ◽  
Adenovirus Vectors

ABSTRACT The use of adenovirus vectors for human gene therapy is limited by potent inflammatory responses that result in significant morbidity. In kidney-derived epithelial cells (REC), activation of extracellular signal-regulated kinase 1/2 (ERK) and p38 kinase (p38) pathways occurred within 20 min of transduction with the serotype 5 adenovirus vector AdCMVβgal. Inhibition of ERK and p38 with U0126 and SB203580, respectively, reduced the expression of IP-10 mRNA following transduction with AdCMVβgal. To determine the role of the coxsackievirus-adenovirus receptor (CAR) or αv integrins in the activation of ERK and p38 and the expression of IP-10, REC cells were transduced with the fiber-modified and RGD-deleted adenovirus vectors AdL.F(RAEK-HA) and AdL.PB(HA), respectively. Compared with the wild-type capsid vector Ad5Luc, transduction with AdL.F(RAEK-HA) and AdL.PB(HA) resulted in reduced ERK-p38 activation and less IP-10 mRNA expression. The decreased IP-10 expression induced by the tropism-modified vectors was due to diminished transduction, since increasing multiplicity of infection resulted in increased IP-10 expression. Inhibition of adenovirus penetration with bafilomycin A1 or ammonium chloride attenuated the activation of ERK-p38 and IP-10 mRNA expression following infection, suggesting that endosomal escape was required to trigger these pathways. In vivo, direct inhibition of ERK and p38 signaling pathways inhibited adenovirus vector-induced IP-10 expression in mouse liver 1 h following transduction. These results demonstrate the importance of signaling via ERK and p38 in the early host response to adenovirus vectors and will permit the development of novel strategies to improve the safety and efficacy of these agents in human gene therapy.

scholarly journals Elimination of both E1 and E2 from adenovirus vectors further improves prospects for in vivo human gene therapy.

1996 ◽  
Vol 70 (6) ◽  
pp. 4173-4178 ◽  
Author(s):  
M I Gorziglia ◽  
M J Kadan ◽  
S Yei ◽  
J Lim ◽  
G M Lee ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Human Gene ◽  
Human Gene Therapy ◽  
Adenovirus Vectors

scholarly journals Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

1999 ◽  
Vol 73 (7) ◽  
pp. 6048-6055 ◽  
Author(s):  
Mario I. Gorziglia ◽  
Claudia Lapcevich ◽  
Soumitra Roy ◽  
Qiang Kang ◽  
Mike Kadan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Liver Toxicity ◽  
Adenovirus Vector ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Adenovirus Vectors ◽  
Virus Promoter

ABSTRACT Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a β-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of β-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the β-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.

scholarly journals In Vitro and In Vivo Gene Therapy Vector Evolution via Multispecies Interbreeding and Retargeting of Adeno-Associated Viruses

2008 ◽  
Vol 82 (12) ◽  
pp. 5887-5911 ◽  
Author(s):  
Dirk Grimm ◽  
Joyce S. Lee ◽  
Lora Wang ◽  
Tushar Desai ◽  
Bassel Akache ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Human Gene ◽  
Single Type ◽  
Alveolar Cells ◽  
Human Gene Therapy ◽  
Viral Peptide ◽  
Peptide Display ◽  
Family Shuffling

ABSTRACT Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.

scholarly journals Adeno-Associated Virus Type 2-Mediated Gene Transfer: Correlation of Tyrosine Phosphorylation of the Cellular Single-Stranded D Sequence-Binding Protein with Transgene Expression in Human Cells In Vitro and Murine Tissues In Vivo

1998 ◽  
Vol 72 (2) ◽  
pp. 1593-1599 ◽  
Author(s):  
Keyun Qing ◽  
Benjawan Khuntirat ◽  
Cathryn Mah ◽  
Dagmar M. Kube ◽  
Xu-Shan Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Dna Synthesis ◽  
Progenitor Cells ◽  
Transgene Expression ◽  
Human Gene ◽  
Adeno Associated Virus ◽  
Human Gene Therapy

ABSTRACT Although the adeno-associated virus type 2 (AAV)-based vector system has gained attention as a potentially useful alternative to the more commonly used retroviral and adenoviral vectors for human gene therapy, the single-stranded nature of the viral genome, and consequently the rate-limiting second-strand viral DNA synthesis, significantly affect its transduction efficiency. We have identified a cellular tyrosine phosphoprotein, designated the single-stranded D sequence-binding protein (ssD-BP), which interacts specifically with the D sequence at the 3′ end of the AAV genome and may prevent viral second-strand DNA synthesis in HeLa cells (K. Y. Qing et al., Proc. Natl. Acad. Sci. USA 94:10879–10884, 1997). In the present studies, we examined whether the phosphorylation state of the ssD-BP correlates with the ability of AAV to transduce various established and primary cells in vitro and murine tissues in vivo. The efficiencies of transduction of established human cells by a recombinant AAV vector containing the β-galactosidase reporter gene were 293 > KB > HeLa, which did not correlate with the levels of AAV infectivity. However, the amounts of dephosphorylated ssD-BP which interacted with the minus-strand D probe were also as follows: 293 > KB > HeLa. Predominantly the phosphorylated form of the ssD-BP was detected in cells of the K562 line, a human erythroleukemia cell line, and in CD34+ primary human hematopoietic progenitor cells; consequently, the efficiencies of AAV-mediated transgene expression were significantly lower in these cells. Murine Sca-1+ lin − primary hematopoietic stem/progenitor cells contained predominantly the dephosphorylated form of the ssD-BP, and these cells could be efficiently transduced by AAV vectors. Dephosphorylation of the ssD-BP also correlated with expression of the adenovirus E4orf6 protein, known to induce AAV gene expression. A deletion mutation in the E4orf6 gene resulted in a failure to catalyze dephosphorylation of the ssD-BP. Extracts prepared from mouse brain, heart, liver, lung, and skeletal-muscle tissues, all of which are known to be highly permissive for AAV-mediated transgene expression, contained predominantly the dephosphorylated form of the ssD-BP. Thus, the efficiency of transduction by AAV vectors correlates well with the extent of the dephosphorylation state of the ssD-BP in vitro as well as in vivo. These data suggest that further studies on the cellular gene that encodes the ssD-BP may promote the successful use of AAV vectors in human gene therapy.

scholarly journals Genome Editing Technologies: From Bench Side to Bedside

Acta Medica ◽  
2018 ◽  
Vol 49 (3) ◽  
pp. 30
Author(s):  
Fulya Yaylacıoğlu Tuncay ◽  
Pervin Rukiye Dinçer
Keyword(s):  
Gene Therapy ◽  
Animal Models ◽  
Genome Editing ◽  
Clinical Studies ◽  
Human Gene ◽  
Human Gene Therapy ◽  
Novel Technologies ◽  
Preclinical And Clinical Studies

The development of genome editing technologies has given the chance to researchers to manipulate any genomic sequences precisely. This ability is very useful for creating animal models to study human diseases in vivo; for easy creation of isogenic cell lines to study in vitro and most importantly for overcoming many disadvantages that the researchers faced during the human gene therapy trials. Here we review the basic mechanisms of genome editing technology and the four genome-editing platforms. We also discuss the applications of these novel technologies in preclinical and clinical studies in four groups according to the mechanism used, and lastly, summarize the problems in these technologies.

Advanced Methods of Adenovirus Vector Production for Human Gene Therapy: Roller Bottles, Microcarriers, and Hollow Fibers

2003 ◽  
Vol 2 (5) ◽  
pp. 75-81 ◽  
Author(s):  
Alexander Kotov ◽  
Tatyana Isayeva ◽  
Olga Kotova ◽  
Victor Krasnykh
Keyword(s):  
Gene Therapy ◽  
Human Gene ◽  
Adenovirus Vector ◽  
Hollow Fibers ◽  
Human Gene Therapy ◽  
Roller Bottles
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