Disease-Inducible Transgene Expression from a Recombinant Adeno-Associated Virus Vector in a Rat Arthritis Model

ABSTRACT Rheumatoid arthritis (RA) is a systemic autoimmune disease affecting 1% of the world’s population, with significant morbidity and mortality. In this study, we investigated a recombinant adeno-associated virus (rAAV) vector for its potential application in RA gene therapy. rAAV encoding Escherichia coliβ-galactosidase was injected into rat joints which had already been induced into acute arthritis after local lipopolysaccharide (LPS) administration, and the efficiency of in vivo transduction was evaluated. We observed a striking correlation between vector transgene expression and disease severity in arthritic joints. The inflammatory reaction peaked at 3 to 7 days after LPS treatment, and, at the same time, 95% of the synoviocytes had high-level transgene expression. Gene expression diminished to the basal level (5%) when the inflammation subsided at 30 days after LPS treatment. More importantly, the diminished transgene expression could be efficiently reactivated by a repeated insult. The transgene expression in normal joints transduced with rAAV remained low for a long period of time (30 days) but could still be induced to high levels (95%) at 3 to 7 days after LPS treatment. This is the first demonstration of disease state-regulated transgene expression. These findings strongly support the feasibility of therapeutic as well as preventative gene transfer approaches for RA with rAAV vectors containing therapeutic genes, which are expected to respond primarily to the disease state of the target tissue.

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

Journal of Virology ◽

10.1128/jvi.00526-06 ◽

2006 ◽

Vol 80 (12) ◽

pp. 6192-6194 ◽

Cited By ~ 61

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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The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

Blood ◽

10.1182/blood.v85.2.319.319 ◽

1995 ◽

Vol 85 (2) ◽

pp. 319-329 ◽

Cited By ~ 66

Author(s):

S Dziennis ◽

RA Van Etten ◽

HL Pahl ◽

DL Morris ◽

TL Rothstein ◽

...

Keyword(s):

Gene Expression ◽

Transgenic Mice ◽

Transgene Expression ◽

Heterologous Gene Expression ◽

Myeloid Cells ◽

Reporter Genes ◽

Regulatory Elements ◽

Specific Expression ◽

High Level

Abstract CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.

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Plasmid DNA-based Alphavirus Vaccines

Vaccines ◽

10.3390/vaccines7010029 ◽

2019 ◽

Vol 7 (1) ◽

pp. 29 ◽

Cited By ~ 8

Author(s):

Kenneth Lundstrom

Keyword(s):

Tumor Cells ◽

Plasmid Dna ◽

Transgene Expression ◽

Tumor Regression ◽

Infectious Agents ◽

Application Range ◽

Mammalian Cell Lines ◽

High Level ◽

Rna Replicon

Alphaviruses have been engineered as vectors for high-level transgene expression. Originally, alphavirus-based vectors were applied as recombinant replication-deficient particles, subjected to expression studies in mammalian and non-mammalian cell lines, primary cell cultures, and in vivo. However, vector engineering has expanded the application range to plasmid DNA-based delivery and expression. Immunization studies with DNA-based alphavirus vectors have demonstrated tumor regression and protection against challenges with infectious agents and tumor cells in animal tumor models. The presence of the RNA replicon genes responsible for extensive RNA replication in the RNA/DNA layered alphavirus vectors provides superior transgene expression in comparison to conventional plasmid DNA-based expression. Immunization with alphavirus DNA vectors revealed that 1000-fold less DNA was required to elicit similar immune responses compared to conventional plasmid DNA. In addition to DNA-based delivery, immunization with recombinant alphavirus particles and RNA replicons has demonstrated efficacy in providing protection against lethal challenges by infectious agents and tumor cells.

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469. Sustainable High Level of Transgene Expression In Vivo by Transgene Expression Cassette Dissociated with Bacterial DNA Sequence

Molecular Therapy ◽

10.1016/s1525-0016(16)43299-5 ◽

2002 ◽

Vol 5 (5) ◽

pp. S153-S154

Keyword(s):

Dna Sequence ◽

Transgene Expression ◽

Expression Cassette ◽

Bacterial Dna ◽

High Level

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

Journal of Virology ◽

10.1128/jvi.01010-07 ◽

2007 ◽

Vol 81 (22) ◽

pp. 12360-12367 ◽

Cited By ~ 21

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.

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304. The Inhibitory Effect of Various Transfection Agents on rAAV Vector-Mediated Transgene Expression Both In Vitro and In Vivo

Molecular Therapy ◽

10.1016/s1525-0016(16)35317-5 ◽

2014 ◽

Vol 22 ◽

pp. S117

Keyword(s):

Transgene Expression ◽

Inhibitory Effect ◽

Raav Vector

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Regulated hAAT Expression from a Novel rAAV Vector and Its Application in the Prevention of Type 1 Diabetes

Journal of Clinical Medicine ◽

10.3390/jcm8091321 ◽

2019 ◽

Vol 8 (9) ◽

pp. 1321 ◽

Cited By ~ 2

Author(s):

Hongxia Ma ◽

Yuanqing Lu ◽

Keith Lowe ◽

Lonneke van der Meijden-Erkelens ◽

Clive Wasserfall ◽

...

Keyword(s):

Type 1 Diabetes ◽

Transgene Expression ◽

Viral Vectors ◽

Fine Tuning ◽

Vector System ◽

Regulation System ◽

Raav Vector

We, and others, have previously achieved high and sustained levels of transgene expression from viral vectors, such as recombinant adeno-associated virus (rAAV). However, regulatable transgene expression may be preferred in gene therapy for diseases, such as type 1 diabetes (T1D) and rheumatoid arthritis (RA), in which the timing and dosing of the therapeutic gene product play critical roles. In the present study, we generated a positive feedback regulation system for human alpha 1 antitrypsin (hAAT) expression in the rAAV vector. We performed quantitative kinetics studies in vitro and in vivo demonstrating that this vector system can mediate high levels of inducible transgene expression. Transgene induction could be tailored to occur rapidly or gradually, depending on the dose of the inducing drug, doxycycline (Dox). Conversely, after withdrawal of Dox, the silencing of transgene expression occurred slowly over the course of several weeks. Importantly, rAAV delivery of inducible hAAT significantly prevented T1D development in non-obese diabetic (NOD) mice. These results indicate that this Dox-inducible vector system may facilitate the fine-tuning of transgene expression, particularly for hAAT treatment of human autoimmune diseases, including T1D.

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