Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle

ABSTRACT Recombinant adeno-associated virus (rAAV) vectors are promising human gene transfer vectors, because they mediate long-term gene expression in vivo. The vector DNA form responsible for sustained gene expression has not been clearly defined, but it has been presumed that the vector integrates to some degree and persists in this manner. Using two independent methods, we were unable to identify rAAV integrants in mouse muscle. In the first approach, we were unable to recover host cell-vector DNA junctions from a lambda phage library generated using transduced mouse muscle DNA that contained a high vector copy number. Following this result, we devised a PCR assay based on the principle that integrated rAAV vector sequences could be amplified using primers specific for mouse interspersed repetitive sequences (B1 elements). Using this assay, we analyzed transduced mouse muscle DNA isolated from 6 to 57 weeks after injection and did not detect amplification above background levels. Based on the demonstrated sensitivity of the assay, these results suggested that >99.5% of vector DNA was not integrated. Additional analyses using a novel DNA exonuclease showed that the majority of the rAAV vector DNA in muscle persisted over time as transcriptionally active monomeric and concatameric episomes.

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CD40 Ligand-Dependent Activation of Cytotoxic T Lymphocytes by Adeno-Associated Virus Vectors In Vivo: Role of Immature Dendritic Cells

Journal of Virology ◽

10.1128/jvi.74.17.8003-8010.2000 ◽

2000 ◽

Vol 74 (17) ◽

pp. 8003-8010 ◽

Cited By ~ 81

Author(s):

Yi Zhang ◽

Narendra Chirmule ◽

Guang-ping Gao ◽

James Wilson

Keyword(s):

Gene Expression ◽

Immune Response ◽

Dendritic Cells ◽

Cd40 Ligand ◽

Broad Host Range ◽

Mouse Bone Marrow ◽

Adeno Associated Virus ◽

Ctl Response ◽

Cell Immunity

ABSTRACT Recombinant adeno-associated virus type 2 (rAAV) is being explored as a vector for gene therapy because of its broad host range, good safety profile, and persistent transgene expression in vivo. However, accumulating evidence indicates that administration of AAV vector may initiate a detectable cellular and humoral immune response to its transduced neo-antigen in vivo. To elucidate the cellular basis of the AAV-mediated immune response, C57BL/6 mouse bone marrow-derived immature and mature dendritic cells (DCs) were infected with AAV encoding β-galactosidase (AAV-lacZ) and adoptively transferred into mice that had received an intramuscular injection of AAV-lacZ 10 days earlier. Unexpectedly, C57BL/6 mice but not CD40 ligand-deficient (CD40L−/−) mice adoptively transferred with AAV-lacZ-infected immature DCs developed a β-galactosidase-specific cytotoxic T-lymphocyte (CTL) response that markedly diminished AAV-lacZ-transduced gene expression in muscle fibers. In contrast, adoptive transfer of AAV-lacZ-infected mature DCs failed to elicit a similar CTL response in vivo. Our findings indicate, for the first time, that immature DCs may be able to elicit a CD40L-dependent T-cell immunity to markedly diminish AAV-lacZ transduced gene expression in vivo when a sufficient number of DCs capturing rAAV vector and/or its transduced gene products is recruited.

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Identification of an Adeno-Associated Virus Rep Protein Binding Site in the Adenovirus E2a Promoter

Journal of Virology ◽

10.1128/jvi.79.1.28-38.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 28-38 ◽

Cited By ~ 10

Author(s):

John M. Casper ◽

Jennifer M. Timpe ◽

John David Dignam ◽

James P. Trempe

Keyword(s):

Gene Expression ◽

Random Sequence ◽

Gene Promoter ◽

Helper Virus ◽

Host Cells ◽

Mobility Shift ◽

Adeno Associated Virus ◽

Rep Protein

ABSTRACT Adeno-associated virus (AAV) and other parvoviruses inhibit proliferation of nonpermissive cells. The mechanism of this inhibition is not thoroughly understood. To learn how AAV interacts with host cells, we investigated AAV's interaction with adenovirus (Ad), AAV's most efficient helper virus. Coinfection with Ad and AAV results in an AAV-mediated inhibition of Ad5 gene expression and replication. The AAV replication proteins (Rep) activate and repress gene expression from AAV and heterologous transcription promoters. To investigate the role of Rep proteins in the suppression of Ad propagation, we performed chromatin immunoprecipitation analyses that demonstrated in vivo AAV Rep protein interaction with the Ad E2a gene promoter. In vitro binding of purified AAV Rep68 protein to the Ad E2a promoter was characterized by electrophoretic mobility shift assays (Kd = 200 ± 25 nM). A 38 bp, Rep68-protected region (5′-TAAGAGTCAGCGCGCAGTATTTACTGAAGAGAGCCT-3′) was identified by DNase I footprint analysis. The 38-bp protected region contains the weak E2a TATA box, sequence elements that resemble the Rep binding sites identified by random sequence oligonucleotide selection, and the transcription start site. These results suggest that Rep binding to the E2a promoter contributes to the inhibition of E2a gene expression from the Ad E2a promoter and may affect Ad replication.

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Genomic Stability of Self-Complementary Adeno-Associated Virus 2 during Early Stages of Transduction in Mouse Muscle In Vivo

Human Gene Therapy ◽

10.1089/hum.2005.16.ft-104 ◽

2005 ◽

Vol 0 (0) ◽

pp. 050727070950001

Author(s):

Changchun Ren ◽

Sanjay Kumar ◽

Denise R. Shaw ◽

Selvarangan Ponnazhagan

Keyword(s):

Genomic Stability ◽

Adeno Associated Virus ◽

Mouse Muscle ◽

Early Stages

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A Chimeric Protein Containing the N Terminus of the Adeno-Associated Virus Rep Protein Recognizes Its Target Site in an In Vivo Assay

Journal of Virology ◽

10.1128/jvi.74.5.2372-2382.2000 ◽

2000 ◽

Vol 74 (5) ◽

pp. 2372-2382 ◽

Cited By ~ 28

Author(s):

Toni Cathomen ◽

Delphine Collete ◽

Matthew D. Weitzman

Keyword(s):

Gene Expression ◽

Mammalian Cells ◽

Target Site ◽

Yeast Genome ◽

Chimeric Proteins ◽

Adeno Associated Virus ◽

Amino Terminal ◽

N Terminus

ABSTRACT The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and inSaccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.

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Liver Transduction with Recombinant Adeno-Associated Virus Is Primarily Restricted by Capsid Serotype Not Vector Genotype

Journal of Virology ◽

10.1128/jvi.80.1.426-439.2006 ◽

2006 ◽

Vol 80 (1) ◽

pp. 426-439 ◽

Cited By ~ 75

Author(s):

Dirk Grimm ◽

Kusum Pandey ◽

Hiroyuki Nakai ◽

Theresa A. Storm ◽

Mark A. Kay

Keyword(s):

Current Model ◽

Factor Ix ◽

In Vivo Studies ◽

Adeno Associated Virus ◽

Copy Numbers ◽

Human Factor Ix ◽

Dna Copy Numbers ◽

Vector Dna

ABSTRACT We and others have recently reported highly efficient liver gene transfer with adeno-associated virus 8 (AAV-8) pseudotypes, i.e., AAV-2 genomes packaged into AAV-8 capsids. Here we studied whether liver transduction could be further enhanced by using viral DNA packaging sequences (inverted terminal repeats [ITRs]) derived from AAV genotypes other than 2. To this end, we generated two sets of vector constructs carrying expression cassettes embedding a gfp gene or the human factor IX (hfIX) gene flanked by ITRs from AAV genotypes 1 through 6. Initial in vitro analyses of gfp vector DNA replication, encapsidation, and cell transduction revealed a surprisingly high degree of interchangeability among the six genotypes. For subsequent in vivo studies, we cross-packaged the six hfIX variants into AAV-8 and infused mice via the portal vein with doses of 5 × 1010 to 1.8 × 1012 particles. Notably, all vectors expressed comparably high plasma hFIX levels within a dose cohort over the following 6 months, concurrent with the finding of equivalent vector DNA copy numbers per cell. Partial hepatectomies resulted in ∼80% drops of hFIX levels and vector DNA copy numbers in all groups, indicating genotype-independent persistence of predominantly episomal vector DNA. Southern blot analyses of total liver DNA in fact confirmed the presence of identical and mostly nonintegrated molecular vector forms for all genotypes. We conclude that, unlike serotypes, AAV genotypes are not critical for efficient hepatocyte transduction and can be freely substituted. This corroborates our current model for AAV vector persistence in the liver and provides useful information for the future design and application of recombinant AAV.

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Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5 Element for Recombinant AAV Vector Site-Specific Integration

Journal of Virology ◽

10.1128/jvi.01956-07 ◽

2007 ◽

Vol 82 (5) ◽

pp. 2590-2593 ◽

Cited By ~ 4

Author(s):

Mickaël Guilbaud ◽

Gilliane Chadeuf ◽

Fabio Avolio ◽

Achille François ◽

Philippe Moullier ◽

...

Keyword(s):

Human Chromosome ◽

Virus Type ◽

Promoter Region ◽

Raav Vector ◽

Adeno Associated Virus ◽

Site Specific ◽

Site Specific Integration ◽

Human Chromosome 19

ABSTRACT The p5 promoter region of the adeno-associated virus type 2 (AAV-2) rep gene has been described as essential for Rep-mediated site-specific integration (RMSSI) of plasmid sequences in human chromosome 19. We report here that insertion of a full-length or minimal p5 element between the viral inverted terminal repeats does not significantly increase RMSSI of a recombinant AAV (rAAV) vector after infection of growth-arrested or proliferating human cells. This result suggests that the p5 element may not improve RMSSI of rAAV vectors in vivo.

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[50] Cross-species comparison of in vivo reporter gene expression after recombinant adeno-associated virus-mediated retinal transduction

Methods in Enzymology - Vertebrate Phototransduction and the Visual Cycle, Part B ◽

10.1016/s0076-6879(00)16762-x ◽

2000 ◽

pp. 777-789 ◽

Cited By ~ 25

Author(s):

Jean Bennett ◽

Vibha Anand ◽

Gregory M. Acland ◽

Albert M. Maguire

Keyword(s):

Gene Expression ◽

Reporter Gene ◽

Reporter Gene Expression ◽

Species Comparison ◽

Adeno Associated Virus ◽

Cross Species Comparison

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HNRNPA1-induced spliceopathy in a transgenic mouse model of myotonic dystrophy

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1907297117 ◽

2020 ◽

Vol 117 (10) ◽

pp. 5472-5477 ◽

Cited By ~ 2

Author(s):

Moyi Li ◽

Yan Zhuang ◽

Ranjan Batra ◽

James D. Thomas ◽

Mao Li ◽

...

Keyword(s):

Myotonic Dystrophy ◽

Rna Binding ◽

Rna Binding Proteins ◽

Disease Model ◽

Adeno Associated Virus ◽

Mouse Muscle ◽

Protein Levels ◽

Hereditary Disorders ◽

Splicing Patterns

Studies on myotonic dystrophy type 1 (DM1) have led to the RNA-mediated disease model for hereditary disorders caused by noncoding microsatellite expansions. This model proposes that DM1 disease manifestations are caused by a reversion to fetal RNA processing patterns in adult tissues due to the expression of toxic CUG RNA expansions (CUGexp) leading to decreased muscleblind-like, but increased CUGBP1/ETR3-like factor 1 (CELF1), alternative splicing activities. Here, we test this model in vivo, using the mouse HSALR poly(CUG) model for DM1 and recombinant adeno-associated virus (rAAV)-mediated transduction of specific splicing factors. Surprisingly, systemic overexpression of HNRNPA1, not previously linked to DM1, also shifted DM1-relevant splicing targets to fetal isoforms, resulting in more severe muscle weakness/myopathy as early as 4 to 6 wk posttransduction, whereas rAAV controls were unaffected. Overexpression of HNRNPA1 promotes fetal exon inclusion of representative DM1-relevant splicing targets in differentiated myoblasts, and HITS-CLIP of rAAV-mycHnrnpa1-injected muscle revealed direct interactions of HNRNPA1 with these targets in vivo. Similar to CELF1, HNRNPA1 protein levels decrease during postnatal development, but are elevated in both regenerating mouse muscle and DM1 skeletal muscle. Our studies suggest that CUGexp RNA triggers abnormal expression of multiple nuclear RNA binding proteins, including CELF1 and HNRNPA1, that antagonize MBNL activity to promote fetal splicing patterns.

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

Gene Therapy ◽

10.1038/s41434-020-0125-0 ◽

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.

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