Adenoassociated virus biology and trafficking in host cells. Protocols for the generation of raav can be classified. Design and delivery aims to enhance the utility of aav vectors for targeted gene transfer to living animals and continue the ongoing development of novel aavbased gene therapies for human disease. Chapter 1 adenoassociated virus biology matthew eitzman w. A simplified purification protocol for recombinant adenoassociated. Images were created from pdb coordinates from viperdb. A simplified baculovirusaav expression vector system coupled. The methods of the present invention may express a therapeutically. A simplified purification protocol for recombinant adeno.
Aav is not associated with any known diseases in humans. Molecular design for recombinant adenoassociated virus. Figure 5schematic flowchart of adenoassociated virus aav purification protocol. A virus that infects primates in the genus dependoparvovirus. Frontiers adenoassociated virus technologies and methods. Considerable effort is currently being focused on aav capsid engineering strategies with the aim of developing novel variants with enhanced tropism for specific human cell types, decreased human seroreactivity, and increased manufacturability. B see the materials and methods section for details. Purity analysis of adenoassociated virus aav capsid proteins using cesds method tingting li, mukesh malik, handy yowanto, sahana mollah sciex, brea, ca introduction adenoassociated virus aav is one of the most widely used. Replication of the virus can also vary in one cell type, depending on the cells current cell cycle phase.
Adenoassociated aav guide to components, serotypes, and safe use in the lab. Kevin bowes1, melanie vargas2, suzanne podmore1, rebecca boffey1, caroline lester1, sarah davies1, liam good2, john ridden2 and daniel smith1. Adenoassociated virus protocol adenoassociated virus protocol storage instructions and infe this product is for laboratory research only and not for diagnostic use contents and storage. Read optimization of packaging of adenoassociated virus gene therapy vectors using plasmid transfections, journal of virological methods on deepdyve, the largest online rental service for scholarly research with thousands of academic publications available at your fingertips. The initial discovery of adenoassociated virus aav mixed with adenovirus particles was not a fortuitous one but rather an expression of aav biology. A simplified baculovirusaav expression vector system. This volume provides a complete and timely guide to the use of adenoassociated virus aav vectors for genetic manipulation of mammalian tissues. Chapters are divided into four parts, and cover topics such as design, construction, and application of transcription activationlike effectors.
Adenoassociated virus aav vector production and purification protocol. Produce and titer recombinant adenoassociated viruses. Adenoassociated virus how is adenoassociated virus abbreviated. Production, purification, and quality control for adeno. A role for adenoassociated viral vectors in gene therapy. The recent successes seen in phase iii clinical trials have also highlighted the issues related to the host and vectorrelated immune response that preclude. Wo2018035457a1 methods and compositions for treating. Visualization of gene therapy with a liver cancertargeted adeno associated virus 3 vector. Strategies for targeted manipulation include combinations of classical genetic tools such as creloxp and flpfrt, use of cisregulatory elements, targeted knockin transgenic mice, and gene delivery by aav and. The method takes advantage of two general biochemical properties of all characterized aav serotypes. Current protocols in microbiology is a comprehensive source of practical methods for designing and implementing successful experiments in microbial research across viruses as well as unicellular and cellcluster organisms including prokaryotes bacteria, archaea and eukaryotes fungi, protists, microalgae to study their genetic, biochemical, and biophysical properties.
Our previous studies have demonstrated that haploid aav vectors made from capsids of two different serotypes induced high transduction and prevented serotypespecific antibody binding. Aav stocks are supplied in liquid form at indicated tite 80 0c. Adenoassociated virus a member of the parvovirus family, which is composed of small viruses with a genome of a singlestranded dna. Wo2014172669a1 recombinant adenoassociated virus delivery. Liu x, huang h, gao y, zhou l, yang j, li x, li y, zhao h, su s, ke c, pei z. Adeno associated virus aav vectors have become one of the most widely used gene transfer tools in human gene therapy. The complexity of gene transfer agents in the context of their clinical use. Pdf adenoassociated virus aav vector production and. Aug, 2014 scalable purification of adenoassociated virus type 2, 4, or 5 using ionexchange chromatography. Bat adenoassociated viruses as gene therapy vectors with. Jan 05, 2014 images used as slide screen saver during the positive strand rna virus 2010 in atlanta. One of the most promising viral vector systems is based on the adenoassociated virus aav.
Adenoassociated virus methods and protocols richard o. Methods and protocols, experts in the field present a collection of their knowledge and experience featuring methodologies that involve virus production, transferring protocols, and evaluating the efficacy of gene products. A supernatant is subjected to a one concentrated using tangential flow filtration. Aavs insert genomic material at a specific site on human chromosome 19 with nearly 100% certainty, and are used to. The methods may feature direct intraarticular injection of a recombinant selfcomplementary adenoassociated virus scraav with a vector adapted to express a modified il1ra peptide. Optimization of packaging of adenoassociated virus gene. Jan 27, 2016 an adeno associated virus aav receptor protein essential for aav2 entry into cells is identified. Adenoassociated virus technologies and methods for.
Gene therapy is a promising modality for the potential treatment of rare mendelian diseases. Recombinant adeno associated virus raav vectors have emerged as one of the most. Gene therapy in skeletal muscle mediated by adenoassociated virus vectors. While thoroughly covering the most popular viral vector systems of adenovirus. Gene therapy using adenoassociated virus vectors ncbi. The present invention relates to recombinant adenoassociated virus raav delivery of polynucleotides for treating duchenne muscular dystrophy resulting from the duplication of dmd exon 2.
One disadvantage of adeno associated viruses is their integration into the host genome, preferentially in a noncoding region in human chromosome 19. Protocol for efficient generation and characterization of adeno. To avoid this problem, recombinant adenoassociated virus vectors have been developed that lack the inverted terminal repeat itr sequences and the rep gene necessary for integration. The production of recombinant aav raav vectors is possible because the rep and cap genes can be deleted. We describe how raavs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced. Methods and protocols serves as a valuable resource for scientists looking to bring new methods and procedures into their lab in order to make exciting discoveries that will continue to deepen our understanding of this fascinating virus family. Aavs insert genomic material at a specific site on human chromosome 19 with nearly 100% certainty, and are used to construct vectors that introduce genes into cultured cells. The development of recombinant aav vectors raav was facilitated by early studies that generated infectious molecular clones, determined the sequence of the genome, and defined the genetic elements of the virus.
The invention provides raav products and methods of using the raav in the treatment of duchenne muscular dystrophy. Recombinant adenoassociated viruses raav have gained increasing attention in the viral gene therapy field. Aav, raav, adenoassociated virus, recombinant aav, gene delivery. Most of these series of images were created with molecular graphics software qutemol, vmd or chimera. A read is counted each time someone views a publication summary such as the title, abstract, and list of authors, clicks on a figure, or views or downloads the fulltext. Aav receptor binds directly to the virus, and its ablation renders a. Adeno associated virus is a small single stranded dna virus and is a member of the parvoviridae family. To evaluate the feasibility of a selfcomplementing recombinant adenoassociated virus 3 scraav3 vector targeting liver cancer and noninvasively monitor gene therapy of liver cancer materials and methods. An adenoassociated virus aav receptor protein essential for aav2 entry into cells is identified. Visualization of gene therapy with a liver cancertargeted.
To avoid this problem, recombinant adeno associated virus vectors have been developed that lack the inverted terminal repeat itr sequences and the rep gene necessary for integration. Combining reported techniques, we optimized our protocol to obtain aavs and purify the viral particles from producer cells and medium efficiently. Codonoptimization of wildtype adenoassociated virus capsid. It allows the user to obtain a vector preparation with hightiter.
A crude cell lysate is acidified by the addition of citric acid. Another virus, such as adenovirus or herpes simplex virus, or a genotoxic agent such as uv radiation or hydroxyurea, is necessary for lytic cycle activation. Google scholar zolotukhin s, potter m, zolotukhin i, sakai y, loiler s, fraites tj. Protocol for efficient generation and characterization of adenoassociated viral aav vectors. The protocol is validated for raav serotypes 2, 8, and 9.
Adenoassociated virusmediated delivery of genes to mouse. Basic biology of adenoassociated virus aav vectors used. Codonoptimization of wildtype adenoassociated virus. Current methods for purification of aav entail the use of.
Adenoassociated virus definition of adenoassociated virus. In this study, we explored the transduction efficiency of several haploid viruses, which were made from the vp1vp2 of one serotype and vp3 of another compatible serotype. Recombinant adenoassociated virus raav vectors have emerged as one of. Methods and protocols, experts from the united states and europe have contributed current knowledge of this multidimensional field relating to the biology of aav, raav vector design, vector manufacturing and product testing, performance of raav vectors in major organs. Purity analysis of adenoassociated virusaav capsid. Adenoassociated virus aav vectors have become one of the most widely used gene transfer tools in human gene therapy. The characteristic feature of the adenoassociated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. Gene therapy section, department of molecular cell biology, leiden university medical center, wassenaarseweg 72, 2333 al leiden, the netherlands the initial discovery of adenoassociated virus aav mixed with adenovirus particles was not a fortuitous one but rather an expression.
The characteristic feature of the adeno associated virus is a deficiency in replication and thus its inability to multiply in unaffected cells. Recombinant adenoassociated virus raavs vectors are becoming increasingly valuable for in vivo studies in animals. In particular, the production of raav vectors in insect cells via the use of recombinant baculovirus technology has proven to be an efficient and scalable means of raav production. Methods and protocols, experts from the united states and europe have contributed current knowledge of this multidimensional field relating to the biology of aav, raav vector design, vector manufacturing and product testing, performance of raav vectors in major organs, raavrelated immunological issues, design of animal and clinical studies, and clinical experience. Production of adenoassociated virus vectors for in vitro and in vivo. Molecular process optimization by jacek lubelski, wim hermens, and harald petry abstracta n increasing number of clinical trials, and the recent approval of the first gene therapy in europe, alipogene tiparvovec glybera, uniqure, holds promise. Adeno associated virus a member of the parvovirus family, which is composed of small viruses with a genome of a singlestranded dna. Methods and protocols methods in molecular biology snyder, richard o. Beginning with methods for the design and characterization of aav vectors, the book continues with protocols for aav delivery to various components of the central nervous system, to a number of. To date a number of high profile proofofconcept studies within the industry have demonstrated the significant diseasecorrecting promise of this therapeutic strategy.
It has been widely accepted that transduction of hepatocytes using adenoassociated virus serotype 8 aav8 is a promising approach to correct inborn errors in neonates, but the type of neonatal hepatic cells transduced by aav8 has not been thoroughly investigated. The recent successes seen in phase iii clinical trials have also highlighted the issues related to the host and vectorrelated immune response that preclude the universal application of this promising vector system. Purification of adenoassociated virus aav vectors using. Adeno associated virus an overview sciencedirect topics. Interest in improving vector performance has in turn driven studies that have. Adenoassociated virus aav is a member of the parvovirus family of singlestranded small dna viruses that require a helper virus such as adenovirus or herpes simplex virus for replication siegl et al. Adenoassociated virus aav protocol 9601 medical center. Adenoassociated virus an overview sciencedirect topics. Pdf design and construction of functional aav vectors. Methods and protocols, methods in molecular biology, vol. The unique life cycle of adenoassociated virus aav and its ability.
Adenoassociated virus aav based vectors have emerged as important tools for gene therapy in humans. Aav receptor binds directly to the virus, and its ablation renders a diverse range of mammalian. Adenoassociated virus is a small single stranded dna virus and is a member of the parvoviridae family. Bat adenoassociated viruses as gene therapy vectors with the.
Pdf discusses aav purification focusing on iodixanol gradient. The present invention relates to recombinant adeno associated virus raav delivery of polynucleotides for treating duchenne muscular dystrophy resulting from the duplication of dmd exon 2. Insect cellbased recombinant adenoassociated virus. Methods and compositions for treating symptoms of conditions such as but not limited to osteoarthritis and rheumatoid arthritis in canines. The invention provides raav products and methods of using the raav in. Adeno associated virus protocol adeno associated virus protocol storage instructions and infe this product is for laboratory research only and not for diagnostic use contents and storage.
Recombinant adenoassociated virus aav vectors are highly promising gene delivery tools. They may be used for a variety of gene transfer applications, both in vivo and in. Nov 26, 2019 celltypespecific expression of molecular tools and sensors is critical to construct circuit diagrams and to investigate the activity and function of neurons within the nervous system. Determination of the cellular tropism of viral vectors is imperative for designing precise gene therapy. Adenoassociated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Adenoassociated virus aav is unique among virus vectors because of its target specificity and low toxicity in somatic cells, and clinical trials have shown that it has promise for gene therapy. Basic biology of adenoassociated virus aav vectors used in. Insect cellbased recombinant adenoassociated virus production. Adenoassociated virus vectors for gene therapy flotte berns elsevier 2005. Recombinant adeno associated virus raavs vectors are becoming increasingly valuable for in vivo studies in animals. The refinement of methods and protocols for the production and. Authoritative and comprehensive, adenoassociated virus vectors. Woodard k, cepko c, guerin ki, rego ma, ersing i, bachle sm, kamens j and fan m 2019 adenoassociated virus technologies and methods for targeted neuronal manipulation. Indeed, as it came to be known, in addition to the unavoidable host cell, aav typically needs a socalled helper virus such as adenovirus to replicate.
Preclinical gene therapy studies both in vitro and in vivo require high purity preparations of adenoassociated virus aav. Binding of adenoassociated virus type 5 to 2,3linked sialic acid is required for gene transfer. Infectious entry pathway of adenoassociated virus and adenoassociated virus vectors. Selfcomplementary recombinant adenoassociated virus scaav vectors promote efficient transduction independently of dna synthesis. Adenoassociated virus and aav vectors fact sheet pdf.
Adeno associated virus aav based vectors have emerged as important tools for gene therapy in humans. Preclinical gene therapy studies both in vitro and in vivo require high purity preparations of adeno associated virus aav. Viruses free fulltext chimeric capsid proteins impact. Pdf gene therapy in skeletal muscle mediated by adenoassociated virus vectors. Adeno associated virus aav was first discovered as a contaminant of adenovirus stocks in the 1960s. Files are available under licenses specified on their description page.
A heavy flocculate is precipitated by a low spin centrifugation. However, there are conflicting reports on the possibility of germline integration of aav into the genome of male germ cells, including sscs. Produce and titer recombinant adenoassociated viruses protocol. This volume discusses protocols, ranging from vector production to delivery methods, used to execute gene therapy applications. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy.
When recombinant aav raav is used for research purposes, the rep protein is supplied in trans, eliminating the ability of raav to integrate into its preferred site of genomic. Adenoassociated virus definition of adenoassociated. This protocol is used for production and purification of aav vectors from plasmid to purified. The prevalence of adenoassociated virus aav has been investigated in bat populations, but little is known about the biological properties of this virus.
Adenoassociated virus aav was discovered as a contaminant of purified adenovirus preparations. Scalable methods of recombinant adeno associated virus raav production have gained much recent interest as the field of raavmediated gene therapy approaches the clinic. Part of the methods in molecular biology book series mimb, volume 807. If desired, aliquot viral stock upon arrival, and do not freeze and thaw repeatedly. May 20, 2019 the prevalence of adeno associated virus aav has been investigated in bat populations, but little is known about the biological properties of this virus. Our study provides an improved protocol for a more economical and efficient purified aav preparation. Since the aav life cycle revolves around another unrelated virus it was dubbed a satellite. The described method makes raav vector technology readily available for the. Recombinant adenoassociated virus raav vectors are increasingly popular tools for gene therapy applications. Molecular process optimization by jacek lubelski, wim hermens, and harald petry. The initial discovery of adeno associated virus aav mixed with adenovirus particles was not a fortuitous one but rather an expression of aav biology. The refinement of methods and protocols for the production and application of raav vectors has come from years of studies that explored the basic biology of this virus and its interaction with host cells. Scalable methods of recombinant adenoassociated virus raav production have gained much recent interest as the field of raavmediated gene therapy approaches the clinic. Methods and protocols, experts from the united states and europe have contributed current knowledge of this multidimensional field relating to the biology of aav, raav vector design, vector manufacturing and product testing, performance of raav vectors in major organs, raavrelated immunological issues, design of.
One disadvantage of adenoassociated viruses is their integration into the host genome, preferentially in a noncoding region in human chromosome 19. Here, we describe an efficient and reproducible strategy to produce, titer, and qualitycontrol batches of adenoassociated virus vectors. Adenoassociated virus spreads by coinfecting a cell with a helper virus. Recombinant adeno associated virus aav vectors are highly promising gene delivery tools. An essential receptor for adenoassociated virus infection. Although the small icosahedral particles diameter 1828 nm were initially considered to be either precursors to the mature adenovirus particle or breakdown products of the virion, they were rapidly demonstrated to represent a distinct virus that was physically, chemically, and. Adeno associated virus spreads by coinfecting a cell with a helper virus. Manufacturing of recombinant adenoassociated viral vectors for. A number of different methods have been suggested and tried with limited.
Adenoassociated virus technologies and methods for targeted neuronal manipulation. Methods and protocols, methods in molecular biology. We describe a new rapid, low cost, and scalable method for purification of various recombinant adenoassociated viruses raavs from the lysates of producer cells of either mammalian or insect origin. Media in category adeno associated virus the following 7 files are in this category, out of 7 total. A variation to this protocol is a twohelper method or triple transfection with aav and ad5 functions provided from. Adenoassociated virus how is adenoassociated virus. Pdf adenoassociated virus aav vectors have evolved over the past decade as a particularly useful gene vector for in vivo applications. Images used as slide screen saver during the positive strand rna virus 2010 in atlanta.