scholarly journals Single intra-articular injection of adeno-associated virus results in stable and controllable in vivo transgene expression in normal rat knees

2011 ◽  
Vol 19 (8) ◽  
pp. 1058-1065 ◽  
Author(s):  
K.A. Payne ◽  
H.H. Lee ◽  
A.M. Haleem ◽  
C. Martins ◽  
Z. Yuan ◽  
...  
Keyword(s):  
Transgene Expression ◽  
Adeno Associated Virus ◽  
Normal Rat

scholarly journals Lack of Repeat Transduction by Recombinant Adeno-Associated Virus Type 5/5 Vectors in the Mouse Airway

2007 ◽  
Vol 81 (22) ◽  
pp. 12360-12367 ◽  
Author(s):  
Stephanie G. Sumner-Jones ◽  
Deborah R. Gill ◽  
Stephen C. Hyde
Keyword(s):  
Transgene Expression ◽  
Lung Diseases ◽  
Repeated Administration ◽  
Transduction Efficiency ◽  
Viral Gene ◽  
Rapid Rise ◽  
Adeno Associated Virus ◽  
Subsequent Failure

ABSTRACT While recombinant adeno-associated virus (rAAV) vectors promote long-term transgene expression in the lungs and other organs, the goal of correcting chronic inherited lung diseases such as cystic fibrosis with this type of viral gene transfer vector is limited by the requirement of achieving stable potent transgene expression, potentially requiring vector readministration. Here we evaluated the abilities of rAAV type 5/5 (rAAV5/5) vectors based on the genome and capsid of AAV5 to efficiently transduce the lungs and nasal epithelium of mice after repeated administration. Transduction efficiency as judged by reporter gene expression was markedly reduced on a second rAAV5/5 administration and effectively abolished on a third. Varying the period between administrations from 8 to 36 weeks did not allow efficient repeated administration. A rapid rise in anti-AAV5 antibodies was noted after rAAV5/5 vector administration that was sustained for the entire period of investigation (in some cases exceeding 9 months). Furthermore, this antibody response and subsequent failure to repeatedly administer the vector were not rescued by the in vivo expression of CTLA4Ig from an rAAV5/5 vector. These results suggest that without the development of an effective and clinically acceptable immunosuppression strategy, treatments for chronic diseases that require repeated administration of rAAV5/5 vectors will be unsuccessful.

scholarly journals Conjugate-Based Targeting of Recombinant Adeno-Associated Virus Type 2 Vectors by Using Avidin-Linked Ligands

2002 ◽  
Vol 76 (24) ◽  
pp. 12900-12907 ◽  
Author(s):  
Selvarangan Ponnazhagan ◽  
Gandham Mahendra ◽  
Sanjay Kumar ◽  
John A. Thompson ◽  
Mark Castillas,
Keyword(s):  
Gene Therapy ◽  
Growth Factor ◽  
Virus Type ◽  
Transgene Expression ◽  
Future Application ◽  
Adeno Associated Virus ◽  
Tissue Specific ◽  
Prokaryotic Expression System

ABSTRACT The development of targeted vectors, capable of tissue-specific transduction, remains one of the important aspects of vector modification for gene therapy applications. Recombinant adeno-associated virus type 2 (rAAV-2)-based vectors are nonpathogenic, have relatively low immunogenicity, and are capable of long-term transgene expression. AAV-2 vectors bind primarily to heparan sulfate proteoglycan (HSPG), a receptor that is present in many tissues and cell types. Because of the widespread expression of HSPG on many tissues, targeted transduction in vivo appears to be limited with AAV-2 vectors. Thus, development of strategies to achieve transductional targeting will have a profound benefit in the future application of these vectors. We report here a novel conjugate-based targeting method to enhance tissue-specific transduction of AAV-2-based vectors. The present report utilized a high-affinity biotin-avidin interaction as a molecular bridge to cross-link purified targeting ligands, produced genetically as fusion proteins to core-streptavidin, in a prokaryotic expression system. Conjugation of the bispecific targeting protein to the vector was achieved by biotinylating purified rAAV-2 without abolishing the capsid structure, internalization, and subsequent transgene expression. The tropism-modified vectors, targeted via epidermal growth factor receptor (EGFR) or fibroblast growth factor 1α receptor (FGFR1α), resulted in a significant increase in transduction efficiency of EGFR-positive SKOV3.ip1 cells and FGFR1α-positive M07e cells, respectively. Further optimization of this method of targeting should enhance the potential of AAV-2 vectors in ex vivo and in vivo gene therapy and may form the basis for developing targeting methods for other AAV serotype capsids.

scholarly journals A Limited Number of Transducible Hepatocytes Restricts a Wide-Range Linear Vector Dose Response in Recombinant Adeno-Associated Virus-Mediated Liver Transduction

2002 ◽  
Vol 76 (22) ◽  
pp. 11343-11349 ◽  
Author(s):  
Hiroyuki Nakai ◽  
Clare E. Thomas ◽  
Theresa A. Storm ◽  
Sally Fuess ◽  
Sharon Powell ◽  
...  
Keyword(s):  
Dose Response ◽  
Transgene Expression ◽  
Linear Increase ◽  
Factor Ix ◽  
Adeno Associated Virus ◽  
Linear Dose ◽  
Human Factor Ix ◽  
Wide Range ◽  
High Doses

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors are promising vehicles for achieving stable liver transduction in vivo. However, the mechanisms of liver transduction are not fully understood, and furthermore, the relationships between rAAV dose and levels of transgene expression, total number of hepatocytes transduced, and proportion of integrated vector genomes have not been well established. To begin to elucidate the liver transduction dose response with rAAV vectors, we injected mice with two different human factor IX or Escherichia coli lacZ-expressing AAV serotype 2-based vectors at doses ranging between 4.0 × 108 and 1.1 × 1013 vector genomes (vg)/mouse, in three- to sixfold increments. A 2-log-range linear dose-response curve of transgene expression was obtained from 3.7 × 109 to 3.0 × 1011 vg/mouse. Vector doses above 3.0 × 1011 vg/mouse resulted in disproportionately smaller increases in both the number of transduced hepatocytes and levels of transgene expression, followed by saturation at doses above 1.8 × 1012 vg/mouse. In contrast, a linear increase in the number of vector genomes per hepatocyte was observed up to 1.8 × 1012 vg/mouse concomitantly with enhanced vector genome concatemerization, while the proportion of integrated vector genomes was independent of the vector dose. Thus, the mechanisms that restrict a wide-range linear dose response at high doses likely involve decreased functionality of vector genomes and restriction of transduction to fewer than 10% of total hepatocytes. Such information may be useful to determine appropriate vector doses for in vivo administration and provides further insights into the mechanisms of rAAV transduction in the liver.

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 Macrophage Depletion via Clodronate Pretreatment Reduces Transgene Expression from AAV Vectors In Vivo

Viruses ◽  
2021 ◽  
Vol 13 (10) ◽  
pp. 2002 ◽  
Author(s):  
Darrick L. Yu ◽  
Natalie S. M. Chow ◽  
Byram W. Bridle ◽  
Sarah K. Wootton
Keyword(s):  
Gene Delivery ◽  
Transgene Expression ◽  
Adenoviral Vectors ◽  
White Pulp ◽  
Systemic Delivery ◽  
Delivery Vehicle ◽  
Adeno Associated Virus ◽  
Clodronate Liposome ◽  
Therapy Studies

Adeno-associated virus is a popular gene delivery vehicle for gene therapy studies. A potential roadblock to widespread clinical adoption is the high vector doses required for efficient transduction in vivo, and the potential for subsequent immune responses that may limit prolonged transgene expression. We hypothesized that the depletion of macrophages via systemic delivery of liposome-encapsulated clodronate would improve transgene expression if given prior to systemic AAV vector administration, as has been shown to be the case with adenoviral vectors. Contrary to our expectations, clodronate liposome pretreatment resulted in significantly reduced transgene expression in the liver and heart, but permitted moderate transduction of the white pulp of the spleen. There was a remarkable localization of transgene expression from the red pulp to the center of the white pulp in clodronate-treated mice compared to untreated mice. Similarly, a greater proportion of transgene expression could be observed in the medulla located in the center of the lymph node in mice treated with clodronate-containing liposomes as compared to untreated mice where transgene expression was localized primarily to the cortex. These results underscore the highly significant role that the immune system plays in influencing the distribution and relative numbers of transduced cells in the context of AAV-mediated gene delivery.

scholarly journals Adeno-Associated Virus Type 2-Mediated Gene Transfer: Role of Cellular T-Cell Protein Tyrosine Phosphatase in Transgene Expression in Established Cell Lines In Vitro and Transgenic Mice In Vivo

2003 ◽  
Vol 77 (4) ◽  
pp. 2741-2746 ◽  
Author(s):  
Keyun Qing ◽  
Weiming Li ◽  
Li Zhong ◽  
Mengqun Tan ◽  
Jonathan Hansen ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Dna Synthesis ◽  
Transgene Expression ◽  
Tyrosine Phosphatase ◽  
Cell Protein ◽  
Transduction Efficiency ◽  
Adeno Associated Virus

ABSTRACT The use of adeno-associated virus type 2 (AAV) vectors has gained attention as a potentially useful alternative to the more commonly used retrovirus and adenovirus vectors for human gene therapy. However, the transduction efficiency of AAV vectors varies greatly in different cells and tissues in vitro and in vivo. We have documented that a cellular protein that binds the immunosuppressant drug FK506, termed the FK506-binding protein (FKBP52), interacts with the single-stranded D sequence within the AAV inverted terminal repeats, inhibits viral second-strand DNA synthesis, and consequently limits high-efficiency transgene expression (K. Qing, J. Hansen, K. A. Weigel-Kelley, M. Tan, S. Zhou, and A. Srivastava, J. Virol., 75: 8968-8976, 2001). FKBP52 can be phosphorylated at both tyrosine and serine/threonine residues, but only the phosphorylated forms of FKBP52 interact with the D sequence. Furthermore, the tyrosine-phosphorylated FKBP52 inhibits AAV second-strand DNA synthesis by greater than 90%, and the serine/threonine-phosphorylated FKBP52 causes ∼40% inhibition, whereas the dephosphorylated FKBP52 has no effect on AAV second-strand DNA synthesis. In the present study, we have identified that the tyrosine-phosphorylated form of FKBP52 is a substrate for the cellular T-cell protein tyrosine phosphatase (TC-PTP). Deliberate overexpression of the murine wild-type (wt) TC-PTP gene, but not that of a cysteine-to-serine (C-S) mutant, caused tyrosine dephosphorylation of FKBP52, leading to efficient viral second-strand DNA synthesis and resulting in a significant increase in AAV-mediated transduction efficiency in HeLa cells in vitro. Both wt and C-S mutant TC-PTP expression cassettes were also used to generate transgenic mice. Primitive hematopoietic stem/progenitor cells from wt TC-PTP-transgenic mice, but not from C-S mutant TC-PTP-transgenic mice, could be successfully transduced by recombinant AAV vectors. These studies corroborate the fact that tyrosine phosphorylation of the cellular FKBP52 protein strongly influences AAV transduction efficiency, which may have important implications in the optimal use of AAV vectors in human gene therapy.

scholarly journals Utility of microminipigs for evaluating liver-mediated gene expression in the presence of neutralizing antibody against vector capsid

Gene Therapy ◽  
2020 ◽  
Vol 27 (9) ◽  
pp. 427-434
Author(s):  
Ryota Watano ◽  
Tsukasa Ohmori ◽  
Shuji Hishikawa ◽  
Asuka Sakata ◽  
Hiroaki Mizukami
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Transgene Expression ◽  
Imaging System ◽  
Standard Dose ◽  
Neutralizing Antibody ◽  
Aav Vector ◽  
Adeno Associated Virus ◽  
In Vivo Imaging System

Abstract Adeno-associated virus (AAV) vectors can transduce hepatocytes efficiently in vivo in various animal species, including humans. Few reports, however, have examined the utility of pigs in gene therapy. Pigs are potentially useful in preclinical studies because of their anatomical and physiological similarity to humans. Here, we evaluated the utility of microminipigs for liver-targeted gene therapy. These pigs were intravenously inoculated with an AAV8 vector encoding the luciferase gene, and gene expression was assessed by an in vivo imaging system. Robust transgene expression was observed almost exclusively in the liver, even though the pig showed a low-titer of neutralizing antibody (NAb) against the AAV8 capsid. We assessed the action of NAbs against AAV, which interfere with AAV vector-mediated gene transfer by intravascular delivery. When a standard dose of vector was administered intravenously, transgene expression was observed in both NAb-negative and low-titer (14×)-positive subjects, whereas gene expression was not observed in animals with higher titers (56×). These results are compatible with our previous observations using nonhuman primates, indicating that pigs are useful in gene therapy experiments, and that the role of low-titer NAb in intravenous administration of the AAV vector shows similarities across species.

scholarly journals High-Level Transgene Expression in Nonhuman Primate Liver with Novel Adeno-Associated Virus Serotypes Containing Self-Complementary Genomes

2006 ◽  
Vol 80 (12) ◽  
pp. 6192-6194 ◽  
Author(s):  
Guang-Ping Gao ◽  
You Lu ◽  
Xun Sun ◽  
Julie Johnston ◽  
Roberto Calcedo ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Nonhuman Primate ◽  
Transgene Expression ◽  
Second Generation ◽  
The Novel ◽  
Adeno Associated Virus ◽  
Cynomolgus Macaques ◽  
High Level

ABSTRACT Adeno-associated virus (AAV) vectors are being considered for in vivo applications of gene therapy in the treatment of a variety of disorders. This study evaluates the biology of second-generation vectors based on the novel serotypes AAV7 and AAV8 and containing self-complementary genomes in the nonhuman primate liver. Stable levels of transgene expression were achieved in cynomolgus macaques and suggest efficiencies at least 2 log higher than what could be achieved with AAV2 vectors using traditional single-stranded genomes. Analysis of DNAs from tissues revealed high levels of vector in the liver that appeared proportional to the relative amounts of transgene expression.

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