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Chemical Reviews Sep 2022Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery... (Review)
Review
Adeno-associated virus (AAV) has a single-stranded DNA genome encapsidated in a small icosahedrally symmetric protein shell with 60 subunits. AAV is the leading delivery vector in emerging gene therapy treatments for inherited disorders, so its structure and molecular interactions with human hosts are of intense interest. A wide array of electron microscopic approaches have been used to visualize the virus and its complexes, depending on the scientific question, technology available, and amenability of the sample. Approaches range from subvolume tomographic analyses of complexes with large and flexible host proteins to detailed analysis of atomic interactions within the virus and with small ligands at resolutions as high as 1.6 Å. Analyses have led to the reclassification of glycan receptors as attachment factors, to structures with a new-found receptor protein, to identification of the epitopes of antibodies, and a new understanding of possible neutralization mechanisms. AAV is now well-enough characterized that it has also become a model system for EM methods development. Heralding a new era, cryo-EM is now also being deployed as an analytic tool in the process development and production quality control of high value pharmaceutical biologics, namely AAV vectors.
Topics: Cryoelectron Microscopy; Dependovirus; Epitopes; Genetic Therapy; Humans
PubMed: 35575684
DOI: 10.1021/acs.chemrev.1c00936 -
Quantification of adeno-associated virus particles and empty capsids by optical density measurement.Molecular Therapy : the Journal of the... Jan 2003We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid...
We show here that UV absorbance of denatured adeno-associated virus (AAV) vector provides a simple, rapid, and direct method for quantifying vector genomes and capsid proteins in solution. We determined the molar extinction coefficients of capsid protein to be 3.72 x 10(6) M(-1) cm(-1) at 260 nm and 6.61 x 10(6) M(-1) cm(-1) at 280 nm. For recombinant AAV vectors, extinction coefficients can be calculated by including the predicted absorbance of the vector DNA. Since the amount of empty capsids in purified vector preparations lowers the A(260)/A(280) ratio in a predictable manner, the vector genome (vg) and capsid particle (cp) titers in purified AAV vector preparations can be calculated from the absorbance at 260 nm and the A(260)/A(280) ratio. To validate this method, the vg and cp titers calculated by UV absorbance were compared with titers determined by quantitative (Q)-PCR and capsid ELISA. The vg titers determined by absorbance agreed well with titers determined by Q-PCR. The cp/vg ratio determined by the A(260)/A(280) method also correlated well with those determined by AAV capsid ELISA in conjunction with Q-PCR. This new method provides a simple and rapid means to determine AAV vg titers and the ratio of empty to full particles in purified virus preparations.
Topics: Capsid; Cell Line; Dependovirus; Genetic Vectors; Humans; Optics and Photonics; Polymerase Chain Reaction; Sensitivity and Specificity; Virion
PubMed: 12573625
DOI: 10.1016/s1525-0016(02)00019-9 -
Journal of Virology Jun 2008Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy....
Adeno-associated virus (AAV) serotypes differ broadly in transduction efficacies and tissue tropisms and thus hold enormous potential as vectors for human gene therapy. In reality, however, their use in patients is restricted by prevalent anti-AAV immunity or by their inadequate performance in specific targets, exemplified by the AAV type 2 (AAV-2) prototype in the liver. Here, we attempted to merge desirable qualities of multiple natural AAV isolates by an adapted DNA family shuffling technology to create a complex library of hybrid capsids from eight different wild-type viruses. Selection on primary or transformed human hepatocytes yielded pools of hybrids from five of the starting serotypes: 2, 4, 5, 8, and 9. More stringent selection with pooled human antisera (intravenous immunoglobulin [IVIG]) then led to the selection of a single type 2/type 8/type 9 chimera, AAV-DJ, distinguished from its closest natural relative (AAV-2) by 60 capsid amino acids. Recombinant AAV-DJ vectors outperformed eight standard AAV serotypes in culture and greatly surpassed AAV-2 in livers of naïve and IVIG-immunized mice. A heparin binding domain in AAV-DJ was found to limit biodistribution to the liver (and a few other tissues) and to affect vector dose response and antibody neutralization. Moreover, we report the first successful in vivo biopanning of AAV capsids by using a new AAV-DJ-derived viral peptide display library. Two peptides enriched after serial passaging in mouse lungs mediated the retargeting of AAV-DJ vectors to distinct alveolar cells. Our study validates DNA family shuffling and viral peptide display as two powerful and compatible approaches to the molecular evolution of novel AAV vectors for human gene therapy applications.
Topics: Animals; DNA Shuffling; Dependovirus; Evolution, Molecular; Female; Gene Transfer Techniques; Genetic Engineering; Genetic Therapy; Genetic Vectors; Humans; In Vitro Techniques; Mice; Mice, Inbred C57BL; Mice, Inbred Strains
PubMed: 18400866
DOI: 10.1128/JVI.00254-08 -
Human Gene Therapy Jan 2022
Topics: Dependovirus; Genetic Therapy; Genetic Vectors
PubMed: 35049369
DOI: 10.1089/hum.2022.29196.hrb -
Viruses May 2023Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications... (Review)
Review
Research on adeno-associated virus (AAV) and its recombinant vectors as well as on fluorescence microscopy imaging is rapidly progressing driven by clinical applications and new technologies, respectively. The topics converge, since high and super-resolution microscopes facilitate the study of spatial and temporal aspects of cellular virus biology. Labeling methods also evolve and diversify. We review these interdisciplinary developments and provide information on the technologies used and the biological knowledge gained. The emphasis lies on the visualization of AAV proteins by chemical fluorophores, protein fusions and antibodies as well as on methods for the detection of adeno-associated viral DNA. We add a short overview of fluorescent microscope techniques and their advantages and challenges in detecting AAV.
Topics: Dependovirus; Genetic Vectors; Viruses; Microscopy, Fluorescence
PubMed: 37243260
DOI: 10.3390/v15051174 -
Methods in Molecular Biology (Clifton,... 2011Adeno-associated virus (AAV) vectors have evolved over the past decade as a particularly useful gene -vector for in vivo applications. In contrast to oncoretro- and...
Adeno-associated virus (AAV) vectors have evolved over the past decade as a particularly useful gene -vector for in vivo applications. In contrast to oncoretro- and lentiviral vectors, this vector stays essentially episomal after gene transfer, making it safer because of the absence of insertional mutagenesis. AAV's non-pathogenicity is a further advantage. For decades, this vector could only be produced at a small scale for research purposes and, eventually, used at very small doses for clinical studies, because only transfection methods were available, which have limited scalability. However, since the development of scalable production methods, this bottleneck is resolved and, from a technical point of view, large quantities of AAV vectors can be produced, opening the possibility of using AAV vectors for whole body treatments in gene therapy trials. This chapter presents the basic principles of small- and large-scale production procedures as well as detailed procedure of small-scale production, purification, and analytical protocols for AAV vectors. In Chapter 10, the reader will find a large-scale production method based on the use of the insect cell/baculovirus system.
Topics: Animals; Cell Line; DNA Probes; Dependovirus; Genes, Viral; Genetic Therapy; Genetic Vectors; Helper Viruses; Humans; Titrimetry; Transduction, Genetic; Transfection; Ultracentrifugation; Virion
PubMed: 21590399
DOI: 10.1007/978-1-61779-095-9_9 -
Advances in Virus Research 1979
Review
Topics: Antigens, Viral; Base Sequence; Cell Transformation, Neoplastic; Cell Transformation, Viral; DNA, Viral; Dependovirus; Helper Viruses; Nucleic Acid Conformation; Protein Biosynthesis; RNA, Viral; Transcription, Genetic; Viral Proteins
PubMed: 230727
DOI: 10.1016/s0065-3527(08)60574-6 -
Molecular Therapy : the Journal of the... Aug 2020
Topics: Antibodies, Neutralizing; Dependovirus; Endopeptidases; Genetic Therapy; Immunoglobulin G
PubMed: 32697940
DOI: 10.1016/j.ymthe.2020.07.015 -
Nature Reviews. Drug Discovery Oct 2022
Topics: Dependovirus; Genetic Vectors; Humans
PubMed: 36056263
DOI: 10.1038/d41573-022-00148-5 -
Current Opinion in Virology Oct 2014Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue... (Review)
Review
Adeno-associated virus (AAV) is a helper-dependent parvovirus which has not been linked with human disease. This aspect, in combination with its broad cell and tissue tropism, and limited viral host response has made it an attractive vector system for gene therapy. The viral protein capsid, the primary interface with the host, is the main determinant for these phenotypes, is highly variable, and is most subject to pressures during replication. Here, we explore the evolutionary path of AAV and other parvoviruses in respect to these phenotypes, as well as directed evolution and engineering strategies that have exploited the lessons learned from natural selection in order to address remaining limitations of AAV as a therapeutic gene transfer platform.
Topics: Biological Evolution; Dependovirus; Directed Molecular Evolution; Genetic Therapy; Genetic Vectors; Humans; Selection, Genetic; Viral Tropism
PubMed: 25128609
DOI: 10.1016/j.coviro.2014.07.008