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      An acidic oligopeptide displayed on AAV2 improves axial muscle tropism after systemic delivery

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          Abstract

          Background

          The appropriate tropism of adeno-associated virus (AAV) vectors that are systemically injected is crucial for successful gene therapy when local injection is not practical. Acidic oligopeptides have been shown to enhance drug delivery to bones.

          Methods

          In this study six-L aspartic acids (D6) were inserted into the AAV2 capsid protein sequence between amino acid residues 587 and 588. 129SVE mice were injected with double-stranded wild-type- (WT-) or D6-AAV2 mCherry expression vectors (3.24 x 10 10 vg per animal) via the superficial temporal vein within 24 hours of birth.

          Results

          Fluorescence microscopy and quantitative polymerase chain reaction confirmed higher levels of mCherry expression in the paraspinal and gluteus muscles in the D6-AAV2 injected mice. The results revealed that although D6-AAV2 was less efficient in the transduction of immortalized cells stronger mCherry signals were detected over the spine and pelvis by live imaging in the D6-AAV2-injected mice than were detected in the WT-AAV2-injected mice. In addition, D6-AAV2 lost the liver tropism observed for WT-AAV2.

          Conclusions

          An acidic oligopeptide displayed on AAV2 improves axial muscle tropism and decreases liver tropism after systemic delivery. This modification should be useful in creating AAV vectors that are suitable for gene therapy for diseases involving the proximal muscles.

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          Most cited references22

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          Membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type 2 virions.

          The human parvovirus adeno-associated virus (AAV) infects a broad range of cell types, including human, nonhuman primate, canine, murine, and avian. Although little is known about the initial events of virus infection, AAV is currently being developed as a vector for human gene therapy. Using defined mutant CHO cell lines and standard biochemical assays, we demonstrate that heparan sulfate proteoglycans mediate both AAV attachment to and infection of target cells. Competition experiments using heparin, a soluble receptor analog, demonstrated dose-dependent inhibition of AAV attachment and infection. Enzymatic removal of heparan but not chondroitin sulfate moieties from the cell surface greatly reduced AAV attachment and infectivity. Finally, mutant cell lines that do not produce heparan sulfate proteoglycans were significantly impaired for both AAV binding and infection. This is the first report that proteoglycan has a role in cellular attachment of a parvovirus. Together, these results demonstrate that membrane-associated heparan sulfate proteoglycan serves as the viral receptor for AAV type 2, and provide an explanation for the broad host range of AAV. Identification of heparan sulfate proteoglycan as a viral receptor should facilitate development of new reagents for virus purification and provide critical information on the use of AAV as a gene therapy vector.
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            Production and purification of serotype 1, 2, and 5 recombinant adeno-associated viral vectors.

            Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1 x 10(12) to 1 x 10(13)vector genomes/ml. Copyright 2002 Elsevier Science (USA)
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              The genome of self-complementary adeno-associated viral vectors increases Toll-like receptor 9-dependent innate immune responses in the liver.

              Although adeno-associated viral (AAV) vectors have been successfully used in hepatic gene transfer for treatment of hemophilia and other diseases in animals, adaptive immune responses blocked long-term transgene expression in patients on administration of single-stranded AAV serotype-2 vector. More efficient vectors have been developed using alternate capsids and self-complimentary (sc) genomes. This study investigated their effects on the innate immune profile on hepatic gene transfer to mice. A mild and transient up-regulation of myeloid differentiation primary response gene (88), TLR9, TNF-α, monocyte chemotactic protein-1, IFN-γ inducible protein-10, and IFN-α/β expression in the liver was found after single-stranded AAV vector administration, regardless of the capsid sequence. In contrast, scAAV vectors induced higher increases of these transcripts, upregulated additional proinflammatory genes, and increased circulating IL-6. Neutrophil, macrophage, and natural killer cell liver infiltrates were substantially higher on injection of scAAV. Some but not all of these responses were Kupffer cell dependent. Independent of the capsid or expression cassette, scAAV vectors induced dose-dependent innate responses by signaling through TLR9. Increased innate responses to scAAV correlated with stronger adaptive immune responses against capsid (but not against the transgene product). However, these could be blunted by transient inhibition of TLR9.
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                Author and article information

                Journal
                Genet Vaccines Ther
                Genet Vaccines Ther
                Genetic Vaccines and Therapy
                BioMed Central
                1479-0556
                2012
                18 June 2012
                : 10
                : 3
                Affiliations
                [1 ]Departments of Pediatrics and Medical Genetics, National Taiwan University Hospital and National Taiwan University College of Medicine, Taipei, Taiwan
                [2 ]Department of Pediatrics, Div. Cell and Molecular Therapy and Pediatric Cardiology, University of Florida, Gainesville, FL, USA
                [3 ]Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA
                [4 ]Department of Pathology and Laboratory Medicine and Neuroscience, University of Florida, Gainesville, FL, USA
                [5 ]Departments of Orthopaedics and Rehabilitation and Molecular Genetics & Microbiology, University of Florida, Gainesville, FL, USA
                [6 ]Department of Pediatrics and Medical Genetics, National Taiwan University Hospital, 7 Chung-Shan South Road, Taipei, 100, Taiwan
                Article
                1479-0556-10-3
                10.1186/1479-0556-10-3
                3416570
                22709483
                5faa810a-389f-4f86-a2a9-b807435d1053
                Copyright ©2012 Lee et al.; licensee Biomed Central Ltd.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 29 April 2012
                : 18 June 2012
                Categories
                Research

                Genetics
                acidic oligopeptide,l-aspartic acid,adeno-associated virus,tropism
                Genetics
                acidic oligopeptide, l-aspartic acid, adeno-associated virus, tropism

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