scholarly journals Adeno-associated Virus (AAV) Serotypes Have Distinctive Interactions with Domains of the Cellular AAV Receptor

2017 ◽  
Vol 91 (18) ◽  
Author(s):  
Sirika Pillay ◽  
Wei Zou ◽  
Fang Cheng ◽  
Andreas S. Puschnik ◽  
Nancy L. Meyer ◽  
...  
Keyword(s):  
Protein Separation ◽  
Transduction Efficiency ◽  
Cellular Receptor ◽  
Adeno Associated Virus ◽  
Cellular Factors ◽  
Repeat Domain ◽  
Strict Requirement ◽  
Rate Limiting ◽  
Entry Receptor ◽  
Overlay Assay

ABSTRACT Adeno-associated virus (AAV) entry is determined by its interactions with specific surface glycans and a proteinaceous receptor(s). Adeno-associated virus receptor (AAVR) (also named KIAA0319L) is an essential cellular receptor required for the transduction of vectors derived from multiple AAV serotypes, including the evolutionarily distant serotypes AAV2 and AAV5. Here, we further biochemically characterize the AAV-AAVR interaction and define the domains within the ectodomain of AAVR that facilitate this interaction. By using a virus overlay assay, it was previously shown that the major AAV2 binding protein in membrane preparations of human cells corresponds to a glycoprotein with a molecular mass of 150 kDa. By establishing a purification procedure, performing further protein separation by two-dimensional electrophoresis, and utilizing mass spectrometry, we now show that this glycoprotein is identical to AAVR. While we find that AAVR is an N-linked glycosylated protein, this glycosylation is not a strict requirement for AAV2 binding or functional transduction. Using a combination of genetic complementation with deletion constructs and virus overlay assays with individual domains, we find that AAV2 functionally interacts predominantly with the second Ig-like polycystic kidney disease (PKD) repeat domain (PKD2) present in the ectodomain of AAVR. In contrast, AAV5 interacts primarily through the first, most membrane-distal, PKD domain (PKD1) of AAVR to promote transduction. Furthermore, other AAV serotypes, including AAV1 and -8, require a combination of PKD1 and PKD2 for optimal transduction. These results suggest that despite their shared dependence on AAVR as a critical entry receptor, different AAV serotypes have evolved distinctive interactions with the same receptor. IMPORTANCE Over the past decade, AAV vectors have emerged as leading gene delivery tools for therapeutic applications and biomedical research. However, fundamental aspects of the AAV life cycle, including how AAV interacts with host cellular factors to facilitate infection, are only partly understood. In particular, AAV receptors contribute significantly to AAV vector transduction efficiency and tropism. The recently identified AAV receptor (AAVR) is a key host receptor for multiple serotypes, including the most studied serotype, AAV2. AAVR binds directly to AAV2 particles and is rate limiting for viral transduction. Defining the AAV-AAVR interface in more detail is important to understand how AAV engages with its cellular receptor and how the receptor facilitates the entry process. Here, we further define AAV-AAVR interactions, genetically and biochemically, and show that different AAV serotypes have discrete interactions with the Ig-like PKD domains of AAVR. These findings reveal an unexpected divergence of AAVR engagement within these parvoviruses.

scholarly journals Adeno-associated virus Rep proteins antagonize phosphatase PP1 to counteract KAP1 repression of the latent viral genome

2018 ◽  
Vol 115 (15) ◽  
pp. E3529-E3538 ◽  
Author(s):  
Sarah Smith-Moore ◽  
Stuart J. D. Neil ◽  
Cornel Fraefel ◽  
R. Michael Linden ◽  
Mathieu Bollen ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Helper Virus ◽  
Protein Phosphatase 1 ◽  
Wild Type ◽  
Fha Domain ◽  
Adeno Associated Virus ◽  
Histone 3 ◽  
Rep Proteins ◽  
Cellular Factors

Adeno-associated virus (AAV) is a small human Dependovirus whose low immunogenicity and capacity for long-term persistence have led to its widespread use as vector for gene therapy. Despite great recent successes in AAV-based gene therapy, further improvements in vector technology may be hindered by an inadequate understanding of various aspects of basic AAV biology. AAV is unique in that its replication is largely dependent on a helper virus and cellular factors. In the absence of helper virus coinfection, wild-type AAV establishes latency through mechanisms that are not yet fully understood. Challenging the currently held model for AAV latency, we show here that the corepressor Krüppel-associated box domain-associated protein 1 (KAP1) binds the latent AAV2 genome at the rep ORF, leading to trimethylation of AAV2-associated histone 3 lysine 9 and that the inactivation of KAP1 repression is necessary for AAV2 reactivation and replication. We identify a viral mechanism for the counteraction of KAP1 in which interference with the KAP1 phosphatase protein phosphatase 1 (PP1) by the AAV2 Rep proteins mediates enhanced phosphorylation of KAP1-S824 and thus relief from KAP1 repression. Furthermore, we show that this phenomenon involves recruitment of the NIPP1 (nuclear inhibitor of PP1)–PP1α holoenzyme to KAP1 in a manner dependent upon the NIPP1 FHA domain, identifying NIPP1 as an interaction partner for KAP1 and shedding light on the mechanism through which PP1 regulates cellular KAP1 activity.

scholarly journals Cloning of an Avian Adeno-Associated Virus (AAAV) and Generation of Recombinant AAAV Particles

2003 ◽  
Vol 77 (12) ◽  
pp. 6799-6810 ◽  
Author(s):  
Ioannis Bossis ◽  
John A. Chiorini
Keyword(s):  
Nucleotide Substitution ◽  
Amino Acid Level ◽  
The Other ◽  
Transduction Efficiency ◽  
Entire Genome ◽  
Single Nucleotide ◽  
Adeno Associated Virus ◽  
Reading Frame ◽  
Palindromic Structure ◽  
The Relationship

ABSTRACT Recent studies have proposed that adeno-associated viruses (AAVs) are not evolutionarily linked to other mammalian autonomous parvoviruses but are more closely linked to the autonomous parvoviruses of birds. To better understand the relationship between primate and avian AAVs (AAAVs), we cloned and sequenced the genome of an AAAV (ATCC VR-865) and generated recombinant AAAV particles. The genome of AAAV is 4,694 nucleotides in length and has organization similar to that of other AAVs. The entire genome of AAAV displays 56 to 65% identity at the nucleotide level with the other known AAVs. The AAAV genome has inverted terminal repeats of 142 nucleotides, with the first 122 forming the characteristic T-shaped palindromic structure. The putative Rep-binding element consists of a tandem (GAGY)4 repeat, and the putative terminal resolution site (trs), CCGGT/CG, contains a single nucleotide substitution relative to the AAV2 trs. The Rep open reading frame of AAAV displays 50 to 54% identity at the amino acid level with the other AAVs, with most of the diversity clustered at the carboxyl and amino termini. Comparison of the capsid proteins of AAAV and the primate dependoviruses indicate that divergent regions are localized to surface-exposed loops. Despite these sequence differences, we were able to produce recombinant AAAV particles carrying a lacZ reporter gene by cotransfection in 293T cells and were able to examine transduction efficiency in both chicken primary cells and several cell lines. Our findings indicate that AAAV is the most divergent AAV described to date but maintains all the characteristics unique to the genera of dependovirus.

scholarly journals Comparison of the transduction efficiency of tyrosine-mutant adeno-associated virus serotype vectors in kidney

2012 ◽  
Vol 40 (1) ◽  
pp. 53-55 ◽  
Author(s):  
Yan F. Qi ◽  
Qiu H. Li ◽  
Vinayak Shenoy ◽  
Michael Zingler ◽  
Joo Y. Jun ◽  
...  
Keyword(s):  
Transduction Efficiency ◽  
Adeno Associated Virus ◽  
Virus Serotype

scholarly journals Hepatocellular Carcinoma Is a Natural Target for Adeno-Associated Virus (AAV) 2 Vectors

Cancers ◽  
2022 ◽  
Vol 14 (2) ◽  
pp. 427
Author(s):  
Nadja Meumann ◽  
Christian Schmithals ◽  
Leroy Elenschneider ◽  
Tanja Hansen ◽  
Asha Balakrishnan ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Tumor Tissue ◽  
Transduction Efficiency ◽  
Mouse Tumor ◽  
Mechanistic Studies ◽  
Human Patient ◽  
Adeno Associated Virus ◽  
Efficient Delivery ◽  
Stage Disease ◽  
Natural Target

Although therapeutic options are gradually improving, the overall prognosis for patients with hepatocellular carcinoma (HCC) is still poor. Gene therapy-based strategies are developed to complement the therapeutic armamentarium, both in early and late-stage disease. For efficient delivery of transgenes with antitumor activity, vectors demonstrating preferred tumor tropism are required. Here, we report on the natural tropism of adeno-associated virus (AAV) serotype 2 vectors for HCC. When applied intravenously in transgenic HCC mouse models, similar amounts of vectors were detected in the liver and liver tumor tissue. In contrast, transduction efficiency, as indicated by the level of transgene product, was moderate in the liver but was elevated up to 19-fold in mouse tumor tissue. Preferred transduction of HCC compared to hepatocytes was confirmed in precision-cut liver slices from human patient samples. Our mechanistic studies revealed that this preference is due to the improved intracellular processing of AAV2 vectors in HCC, resulting, for example, in nearly 4-fold more AAV vector episomes that serve as templates for gene transcription. Given this background, AAV2 vectors ought to be considered to strengthen current—or develop novel—strategies for treating HCC.

scholarly journals CRISPR-Targeted CAR Gene Insertion Using Cas9/RNP and AAV6 Enhances Anti-AML Activity of Primary NK Cells

2021 ◽  
Author(s):  
Meisam Naeimi Kararoudi ◽  
Shibi Likhite ◽  
Ezgi Elmas ◽  
Kenta Yamamoto ◽  
Maura Schwartz ◽  
...  
Keyword(s):  
Nk Cells ◽  
Efficient Method ◽  
Myeloid Leukemia ◽  
Human Peripheral Blood ◽  
Transduction Efficiency ◽  
Proof Of Concept ◽  
Activation Markers ◽  
Adeno Associated Virus ◽  
Gene Insertion ◽  
Signaling Domains

Human peripheral blood natural killer (NK) cells have intense antitumor activity and have been used successfully in several clinical trials. Modifying NK cells with a chimeric antigen receptor (CAR) can improve their targeting and increase specificity. Recently, we described an efficient method for gene targeting in NK cells using Cas9/ribonucleoprotein (RNP) complexes. Here we combined this approach with single stranded (ss) or self-complementary (sc) Adeno-associated virus (AAV)-mediated gene delivery for gene insertion into a safe-harbor locus using a wide variety of homology arms for homology repair (HR) and non-homologous directed CRISPR-assisted insertion tagging (CRISPaint) approaches. For proof-of-concept, we generated mCherry-expressing primary NK cells and determined that sc vectors with 300bp homology 30 arms had optimal transduction efficiency. Then, we generated CD33-targeting CAR NK cells with differing transmembrane and signaling domains (CD4/4-1BB+CD3ζ and NKG2D/2B4+CD3ζ) and expanded them on CSTX002 feeder cells. Expansion kinetics were unaltered and the expanded NK cells maintained high CAR expression (mean 68% CAR+). The CD33-CAR-NK cells showed increased activation markers and enhanced antileukemic activity with improved killing kinetics against CD33-positive acute myeloid leukemia (AML) cell lines and primary samples. Using targeted sequencing we demonstrated the accuracy of CAR gene insertion in human primary NK cells genome. Site-directed insertion using RNP and scAAV6 is an efficient method for stable genetic transfer into primary NK cells that has broad potential for fundamental discovery and therapeutic applications.

Optimization of Meniscus Cell Transduction Using Lentivirus and Adeno-Associated Virus for Gene Editing and Tissue Engineering Applications

Cartilage ◽  
2019 ◽  
pp. 194760351988032
Author(s):  
Paolo Arrigoni ◽  
Jacob C. Ruprecht ◽  
Dawn A.D. Chasse ◽  
Katherine A. Glass ◽  
Benjamin Andress ◽  
...  
Keyword(s):  
Gene Editing ◽  
Fluorescent Protein ◽  
Meniscus Repair ◽  
Transduction Efficiency ◽  
Viral Gene ◽  
Isolated Cells ◽  
Adeno Associated Virus ◽  
Meniscus Tissue ◽  
Meniscus Cells

Objectives The utilization of viral vectors to deliver genes of interest directly to meniscus cells and promote long-term modulation of gene expression may prove useful to enhance meniscus repair and regeneration. The objective of this study was to optimize and compare the potential of lentivirus (LV) and adeno-associated virus (AAV) to deliver transgenes to meniscus cells in both intact meniscus tissue and isolated primary cells in monolayer. Design Porcine meniscus tissue explants and primary meniscus cells in monolayer were transduced with LV or self-complementary AAV2 (scAAV2) encoding green fluorescent protein (GFP). Following transduction, explants were enzymatically digested to isolate meniscus cells, and monolayer cells were trypsinized. Isolated cells were analyzed by flow cytometry to determine percent transduction. Results LV and scAAV2 showed a high transduction efficiency in monolayer meniscus cells. scAAV2 was most effective at transducing cells within intact meniscus tissue but the efficiency was less than 20%. Outer zone meniscus cells were more readily transduced by both LV and scAAV2 than the inner zone cells. Higher virus titers and higher cell density resulted in improved transduction efficiency. Polybrene was necessary for the highest transduction efficiency with LV, but it reduced scAAV2 transduction. Conclusions Both LV and scAAV2 efficiently transduce primary meniscus cells but only scAAV2 can modestly transduce cells embedded in meniscus tissue. This work lays the foundation for viral gene transfer to be utilized to deliver bioactive transgenes or gene editing machinery, which can induce long-term and tunable expression of therapeutic proteins from tissue-engineered constructs for meniscus repair and regeneration.

scholarly journals An Exon-Specific Small Nuclear U1 RNA (ExSpeU1) Improves Hepatic OTC Expression in a Splicing-Defective spf/ash Mouse Model of Ornithine Transcarbamylase Deficiency

2020 ◽  
Vol 21 (22) ◽  
pp. 8735
Author(s):  
Dario Balestra ◽  
Mattia Ferrarese ◽  
Silvia Lombardi ◽  
Nicole Ziliotto ◽  
Alessio Branchini ◽  
...  
Keyword(s):  
Mouse Model ◽  
Protein Function ◽  
Disease Onset ◽  
Fold Increase ◽  
Ornithine Transcarbamylase ◽  
Transduction Efficiency ◽  
Ornithine Transcarbamylase Deficiency ◽  
Adeno Associated Virus ◽  
Early Proof

OTC splicing mutations are generally associated with the severest and early disease onset of ornithine transcarbamylase deficiency (OTCD), the most common urea cycle disorder. Noticeably, splicing defects can be rescued by spliceosomal U1snRNA variants, which showed their efficacy in cellular and animal models. Here, we challenged an U1snRNA variant in the OTCD mouse model (spf/ash) carrying the mutation c.386G > A (p.R129H), also reported in OTCD patients. It is known that the R129H change does not impair protein function but affects pre-mRNA splicing since it is located within the 5′ splice site. Through in vitro studies, we identified an Exon Specific U1snRNA (ExSpeU1O3) that targets an intronic region downstream of the defective exon 4 and rescues exon inclusion. The adeno-associated virus (AAV8)-mediated delivery of the ExSpeU1O3 to mouse hepatocytes, although in the presence of a modest transduction efficiency, led to increased levels of correct OTC transcripts (from 6.1 ± 1.4% to 17.2 ± 4.5%, p = 0.0033). Consistently, this resulted in increased liver expression of OTC protein, as demonstrated by Western blotting (~3 fold increase) and immunostaining. Altogether data provide the early proof-of-principle of the efficacy of ExSpeU1 in the spf/ash mouse model and encourage further studies to assess the potential of RNA therapeutics for OTCD caused by aberrant splicing.

scholarly journals Characterization of Tissue Tropism Determinants of Adeno-Associated Virus Type 1

2003 ◽  
Vol 77 (4) ◽  
pp. 2768-2774 ◽  
Author(s):  
Bernd Hauck ◽  
Weidong Xiao
Keyword(s):  
Amino Acids ◽  
Transduction Efficiency ◽  
Antigenic Determinants ◽  
Tissue Tropism ◽  
Heparin Binding ◽  
Adeno Associated Virus ◽  
Heparin Binding Domain

ABSTRACT Muscle is an attractive target for gene delivery because of its mass and because vectors can be delivered in a noninvasive fashion. Adeno-associated virus (AAV) has been shown to be effective for muscle-targeted gene transfer. Recent progress in characterization of AAV serotype 1 (AAV1) and AAV6 demonstrated that these two AAV serotypes are far more efficient in transducing muscle than is the traditionally used AAV2. Since all cis elements are identical in these vectors, the potential determinants for their differences in transducing muscle appear to be located within the AAV capsid proteins. In the present study, a series of AAV capsid mutants were generated to identify the major regions affecting AAV transduction efficiency in muscle. Replacement of amino acids 350 to 736 of AAV2 VP1 with the corresponding amino acids from VP1 of AAV1 resulted in a hybrid vector that behaved very similarly to AAV1 in vitro and in vivo in muscle. Characterization of additional mutants carrying smaller regions of the AAV1 VP1 amino acid sequence in the AAV2 capsid protein suggested that amino acids 350 to 430 of VP1 function as a major tissue tropism determinant. Further analysis showed that the heparin binding domain and the major antigenic determinants in the AAV capsid region were not necessary for the efficiency of AAV1 transduction of muscle.

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.

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