Basic biology of adenoassociated virus aav vectors used in. We recently developed adeno associated virus aav capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. Please read through the entire kit instructions before performing virus infection and purification. These studies have shown outstanding safety and efficacy of the aav vector for gene therapy.
The virus causes a mild immune response, lending further support to its apparent lack of pathogenicity. Methods and prools, 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. Determination of the cellular tropism of viral vectors is imperative for designing precise gene therapy. 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. More than 100 serotypes, aav2 well studied and frequently used as viral vector. For these diseases, an effective gene therapy will require systemic delivery of the therapeutic vector to all affected locations. 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.
Here, we set up a scalable process for aav production, using orbitally shaken bioreactors and a fully characterized suspensionadapted cell. Jan 21, 2020 adeno associated virus aav vectors are currently considered to be one of the most promising platforms for in vivo gene transfer 1. 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. Adenoassociated virus biology and trafficking in host cells. Storage buffers provided virus is provided ready for injection in biocompatible f68pbs buffer.
Adenoassociated virus aav protocol 9601 medical center. Us10294452b2 lysis, extraction and purification of adeno. The present disclosure provides compositions and methods for extracting viral particles of a recombinant adenoassociated virus aav or an adenovirus adv from a. Adenoassociated viral aav vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Pdf adenoassociated virus aav vectors have evolved over the past decade as a particularly useful gene. Infectious entry pathway of adenoassociated virus and adenoassociated virus vectors.
Purification of adenoassociated virus aav vectors using. A simplified purification protocol for recombinant adeno. 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. The method takes advantage of two general biochemical properties of all characterized aav serotypes.
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. Recombinant adenoassociated virus aavbased gene therapy has been promising, but several hostrelated transduction or immune challenges remain. Adeno associated viral aav vectors are becoming an important tool for gene therapy of numerous genetic and other disorders. Adeno associated virus aav and recombinant adeno associated virus raav at the university of lethbridge. Adeno associated virus is a small single stranded dna virus and is a member of the parvoviridae family.
Adenoassociated virus aav vectors are currently among the most commonly applied for in vivo gene therapy approaches. In order to develop pcr protocols which discriminate between the different serotypes of aav, the dna of aav5 was sequenced. Media in category adenoassociated virus the following 7 files are in this category, out of 7 total. We recently developed adenoassociated virus aav capsids to facilitate efficient and noninvasive gene transfer to the central and peripheral nervous systems. Adeno associated virus biology and trafficking in host cells. The protocol is validated for raav serotypes 2, 8, and 9. Aav stocks are supplied in liquid form at indicated tite 80 0c. In this commentary, my objective is to highlight a potential critical quality attribute of adenoassociated virus aav vectors that is not well characterised, but that may seriously affect the potential for longterm durable transgene. Adeno associated 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. As mentioned above, aav infection is initiated by binding to cell surface receptors that are serotypespecific. A role for adenoassociated viral vectors in gene therapy. Cre activated and inactivated recombinant adeno associated viral vectors for neuronal anatomical tracing or activity manipulation.
Pdf adenoassociated virus aav as a vector for gene therapy. Adenoassociated virus aav is a small virus which infects humans and some other primate species. Cre activated and inactivated recombinant adenoassociated viral vectors for neuronal anatomical tracing or activity manipulation. Inexpensive, serotypeindependent protocol for native and. Methods and protocols, methods in molecular biology, vol. 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. Adeno associated virus aav has been used as a gene therapy vector for decades in preclinical studies and human trials. Pdf adenoassociated virus aav vectors have evolved over the past decade as a particularly useful gene vector for in vivo applications. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Discrimination between different types of human adeno. Video articles in jove about recombinant adeno associated virus include a quantitative dot blot assay for aav titration and its use for functional assessment of the adenoassociated virus assemblyactivating proteins, adenoassociated virusmediated transgene expression in genetically defined neurons of the spinal cord, engineering and evolution of synthetic adenoassociated virus aav. Vector characterization methods for quality control. Kevin bowes1, melanie vargas2, suzanne podmore1, rebecca boffey1, caroline lester1, sarah davies1, liam good2, john ridden2 and daniel smith1.
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. Recombinant adenoassociated virus aav vectors are highly promising gene delivery tools. 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. Adenoassociated virus spreads by coinfecting a cell with a helper virus. Adenoassociated virus aav was first discovered as a contaminant of adenovirus stocks in the 1960s. Adenoassociated virus aav is a small virus with a singlestranded dna. Aav2, for instance, has the heparan sulfate proteoglycan hspg as one of its primary attachment receptors summerford and samulski, 1998. Aav2, for instance, has the heparan sulfate proteoglycan hspg as one of its primary attachment receptors summerford and. Several recombinant aav vectors raav have the ability to transduce striated muscles in a variety of animals following intramuscular and intravascular administration, and have attracted widespread interest for therapy of muscle disorders such as the muscular. The present disclosure provides compositions and methods for extracting viral particles of a recombinant adeno associated virus aav or an adenovirus adv from a sample comprising cells enclosing the viral particles. Adenoassociated virus methods and protocols richard o.
Aav is not associated with any known diseases in humans. Of the available dna virus systems, adenovirus is the best studied, and probably the most accessible and generally useful, although its applicability for longterm gene therapy is still very much unclear. 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. Mouse models are often used as a preliminary preclinical model in order to. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. One disadvantage of adenoassociated viruses is their integration into the host genome, preferentially in a noncoding region in human chromosome 19. Positive features of adenovi rus include the capacity for expression of relatively large dna inserts up to. Binding of adenoassociated virus type 5 to 2,3linked sialic acid is required for gene transfer. 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. 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.
Adeno associated virus aav was first discovered as a contaminant of adenovirus stocks in the 1960s. If desired, aliquot viral stock upon arrival, and do not freeze and thaw repeatedly. Adenoassociated virus an overview sciencedirect topics. Adeno associated virus aavbased vectors expressing therapeutic gene products have shown great promise for human gene therapy. Inexpensive, serotypeindependent protocol for native and bioengineered recombinant adenoassociated virus purification recombinant adenoassociated virus aav is a. 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.
Guidelines for use of adenoassociated viral vectors aav. Pdf adenoassociated virus aav vector production and. Video articles in jove about recombinant adeno associated virus include a quantitative dot blot assay for aav titration and its use for functional assessment of the adeno associated virus assemblyactivating proteins, adeno associated virus mediated transgene expression in genetically defined neurons of the spinal cord, engineering and evolution of synthetic adeno associated virus aav. Scalable production of aav vectors in orbitally shaken hek293. Gene therapy in skeletal muscle mediated by adenoassociated virus. Adenoassociated virus aav has been used as a gene therapy vector for decades in preclinical studies and human trials. Adeno associated virus spreads by coinfecting a cell with a helper virus. Adenoassociated virus is a small single stranded dna virus and is a member of the parvoviridae family. 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. Adenoassociated virus aav is a nonenveloped virus that can be engineered to deliver dna to target cells, and has attracted a significant amount of attention in the field, especially in. Adeno associated virus aav is a small virus which infects humans and some other primate species.
Cre activated and inactivated recombinant adenoassociated. Adeno associated virus aav vectors are currently among the most commonly applied for in vivo gene therapy approaches. The initial discovery of adenoassociated virus aav mixed with adenovirus particles was not a fortuitous one but rather an expression of aav biology. Recombinant adeno associated virus aav vectors are highly promising gene delivery tools. Adenoassociated virus how is adenoassociated virus. Recombinant adenoassociated virus vectors, though replication deficient, transduce mammalian cells and should be handled with bsl2 standards. The first helper virus that was described as providing successful generation of new aav particles, was the adenovirus, from which the aav name originated. Aug, 2014 scalable purification of adenoassociated virus type 2, 4, or 5 using ionexchange chromatography. Since the aav life cycle revolves around another unrelated virus it was dubbed a satellite.
Produce and titer recombinant adenoassociated viruses. Recombinant adeno associated virus aavbased gene therapy has been promising, but several hostrelated transduction or immune challenges remain. Aavs insert genomic material at a specific site on human chromosome 19 with nearly 100% certainty, and are used to. One disadvantage of adeno associated viruses is their integration into the host genome, preferentially in a noncoding region in human chromosome 19. Recombinant adeno associated virus raavs vectors are becoming increasingly valuable for in vivo studies in animals. Aav raav crelox credependent virus viral tracer how to cite this article. Systemic delivery of adenoassociated viral vectors in mice. Vector characterization methods for quality control testing. Production, purification, and quality control for adenoassociated. Methods and protocols methods in molecular biology snyder, richard o.
Adenoassociated virus aavbased vectors expressing therapeutic gene products have shown great promise for human gene therapy. Adenoassociated viral vectors do not efficiently target. 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. Adeno associated virus aav is a nonenveloped virus that can be engineered to deliver dna to target cells, and has attracted a significant amount of attention in the field, especially in. Recombinant adenoassociated virus raavs vectors are becoming increasingly valuable for in vivo studies in animals. A generic method for fast and sensitive detection of adeno. Small scale production of recombinant adenoassociated viral. Adenoassociated virus aav and recombinant adenoassociated virus raav at the university of lethbridge. Adenoassociated viruses aav belong to a family of small singlestranded dna viruses parvoviruses that include the b19 parvovirus that causes a rash in children known as fifth disease measles, mumps, rubella, and varicella make up the first four. They may be used for a variety of gene transfer applications, both in vivo and in.
In contrast to oncoretro and lentiviral vectors, this vector stays essentially episomal after gene transfer, making it. Pdf discusses aav purification focusing on iodixanol gradient. The lack of pathogenicity of the wildtype virus, the ability to. This volume provides a complete and timely guide to the use of adeno associated virus aav vectors for genetic manipulation of mammalian tissues. Adeno associated virus an overview sciencedirect topics. 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. Storage and transduction instructions for aav vectors. The eluted virus may then be further concentrated by either centrifugal spin devices or tangential flow filtration yielding material of high titer and good manufacturing practice gmp grade biochemical purity. Because glycosylation of capsid proteins is known to be rate limiting in the life.
Scalable purification of adenoassociated virus type 2, 4, or 5 using ionexchange chromatography. Production of adenoassociated virus vectors for in vitro and in vivo. Persistent infection of human tissues with the helper virusdependent parvovirus, adenoassociated virus aav was detected by polymerase chain reaction pcr using primer pairs detecting aav types 2, 3 or 5. A major challenge for translation of promising research to clinical development is the establishment of appropriate quality control qc test methods to characterize clinical grade aav vectors. Molecular engineering of adenoassociated virus capsid. 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.
This volume provides a complete and timely guide to the use of adenoassociated virus aav vectors for genetic manipulation of mammalian tissues. Please contact the biosafety officer before beginning any work with avvraav vectors. The journal of virological methods focuses on original, high quality research papers that describe novel and comprehensively tested methods which enhance human, animal, plant, bacterial or environmental virology and prions research and discovery. Inexpensive, serotypeindependent protocol for native and bioengineered recombinant adeno associated virus purification recombinant adeno associated virus aav is a valuable and often used gene therapy vector. This protocol can be executed by investigators with experience in cell culture and molecular biological techniques. Adenoassociated virus aav vectors have evolved over the past decade as a particularly useful gene vector for in vivo applications. Google scholar zolotukhin s, potter m, zolotukhin i, sakai y, loiler s, fraites tj.
Adenoassociated virus a member of the parvovirus family, which is composed of small viruses with a genome of a singlestranded dna. 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. 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. Novel approaches to augment adenoassociated virus type2. Use of recombinant adenovirus for metabolic engineering. Produce and titer recombinant adenoassociated viruses protocol. Combining reported techniques, we optimized our protocol to obtain aavs and purify the viral particles from producer cells and medium efficiently. Adenoassociated virus and aav vectors fact sheet pdf. Gene delivery tools based on adenoassociated viruses aavs are a popular choice for the delivery of. Recombinant adeno associated virus vectors, though replication deficient, transduce mammalian cells and should be handled with bsl2 standards. Capsidspecific removal of circulating antibodies to adeno. Aav, raav, adenoassociated virus, recombinant aav, gene delivery, dependovirus. Pdf gene therapy in skeletal muscle mediated by adenoassociated virus vectors.
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