We show that normal mice and mice with mucopolysaccharidosis vii mps vii develop hepatocellular carcinoma hcc after neonatal injection of an aav vector expressing bglucuronidase. Corroborating this are studies of aav vector integration sites is in mice, nonhuman primates nhp and muscle or liver biopsies from vector treated human patients 49,50,51,52, which revealed a. The first fdaapproved aavbased gene therapies are luxturna by spark therapeutics and zolgensma by novartis for treating a rare in. Our helperfree aav systems are designed for the preparation of hightiter aav particles without the use of a helper virus. The early observation that aav can establish a latent infection by integrating into the host chromosome has been central to this interest. Aav vector integration into crisprinduced dna breaks. High levels of aav vector integration into crisprinduced. Adenoassociated virus aav as a vector for gene therapy.
Gene therapy adenoassociated virus aav vectors explained. However, as aav vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Adenoassociated virus aav cloning and expression vectors. Ubisap is a firstofitskind software available for is analysis to. This article is an open access publication abstract there has been a resurgence in gene therapy efforts that is partly fueled by the identi. New studies refocus attention on the genotoxicity of aav vectors in gene therapy.
Guidance for working with adenoassociated virus aav vectors. We previously demonstrated that after administering adenoassociated viral aav vectormediated genomeediting strategies in vivo, vector sequences integrated into the host organisms genomic dna at doublestranded breaks. Aav used for gene therapy lacks dedicated machinery to integrate into the genome. Adenoassociated virus aav as a vector for gene therapy michael f. Each kit includes three plasmids encoding the factors necessary to prepare recombinant aav particles by transfection of hek293t cells. They belong to the parvovirus family and require a helper virus, such as adenovirus, herpes simplex virus, or vaccina virus, to replicate 1. Due to poor in vitro to in vivo and animal to human translatability associated with developing novel aav vector capsids and transgene expression constructs lisowski et al.
Largescale analysis of adenoassociated virus vector integration. The integration of wildtype aav aav is a helperdependent parvovirus with an approximately 4. The adenoassociated virus aav is an attractive delivery vehicle in gene therapy1,2 attributed to its mild immune response and ability to deliver its genetic payload into a wide range of host cells. Here we have performed a largescale analysis of aav vector integration. Because aav integrates in between bases, the bases indicated in this file are those immediately 3 to the integration. These viruses can insert genetic material at a specific site on chromosome 19 with near 100% certainty. The analysis of viral vector genomic integration sites is an important. Quantifying quality attributes of aav gene therapy. Current viral vectors are designed to be less mutagenetic, as in the case of sin. Adenoassociated vector toxicityto be or not to be molecular.
New studies refocus attention on the genotoxicity of aav. Adenoassociated virus vector as a platform for gene. However, an extensive evaluation of adenoassociated virus aav vector integration sites has not been completed, despite the ongoing use of aav vectors in clinical trials. Aav vector integration sites in mouse hepatocellular carcinoma. Adenoassociated virus vector integration junctions journal of. Gt is a therapeutic intervention to genetically alter or modify living cells by means of gene delivery achieved using either viral vectors or nonviral vectors, with adenoassociated virus aav vectors constituting marketshare majority. Recombinant adenoassociated viral aav vectors have been shown to be one of the most promising vectors for therapeutic gene delivery because they can induce efficient and longterm transduction in nondividing cells with negligible sideeffects. Chromosomal effects of adenoassociated virus vector. Recombinant adenoassociated virus raav holds promise as a gene therapy vector for a multitude of genetic disorders such as hemophilia, cystic fibrosis, and the muscular dystrophies. However, gene delivery to kidney cells is inefficient. We have compiled the key pieces of information from hundreds of published papers for education and convenience. By eliminating the requirement for live helper virus the aav helperfree system provides a safer, purer and more convenient alternative to retroviral and adenoviral gene delivery systems.
Recombinant adenoassociated viral aav vectors have been shown. Dec 16, 2019 there are many kidney diseases that might be addressed by gene therapy. Sep 30, 2019 vector integration into therapeutically relevant genes in vivo. Nov, 2019 we performed integration target site analysis on liver samples n3dog from six of the aavtreated ha dogs and naive ha dogs n2 by ligationmediated pcr, ilumina pairedend sequencing and analysis using the custom software pipeline, aavenger. Up to 62 nucleotides of flanking sequences are shown for each junction in lowercase and are aligned. 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 integration on human chromosome 19, termed aavs1. Vector integration into therapeutically relevant genes in vivo. In comparison to the widespread use of aav vectors in clinical, preclinical, and. The raav vectors reduced ability to integrate also means that the. Adenoassociated viruses aav are small viruses that affect humans and other members of the primate species fig. Targeted integration of a raav vector into the aavs1 region. Recombinant adenoassociated virus transduction and integration. Analytical approaches to characterize aav vector production. Unit 4, 7074 industry dr, tweed heads south, nsw 2486 qld location.
To accomplish integration into aavs1 with raav vectors rep must. Recently, questions have arisen about the safety of aav vectors, specifically, whether integration of vector dna in transduced cell genomes promotes tumor formation. This section is meant to serve as an introduction for those unfamiliar with aav adenoassociated virus. Mar 17, 2020 identifying nucleaseinduced doublestranded breaks in dna on a genomewide scale is critical for assessing the safety and efficacy of genome editing therapies. In this study, we analyze integration of aav vectors genomewide and. A key element is hence to investigate if and how parts of the vector genome are incorporated into a host. Integration of adenoassociated virus aav and recombinant aav.
Chromosomal effects of adenoassociated virus vector integration. Pdf chromosomal effects of adenoassociated virus vector. Mar 24, 2011 gene transfer using adenoassociated virus aav vectors has great potential for treating human disease. Given the variety of applications and tissue types toward which these. Considerations for preclinical safety assessment of adeno. Cell culture experiments pointed toward viral genome integration with. Dickinson facs canto flow cytometer using facs diva software. Aav vectors containing rdna homology display increased.
In the initial experiments to create and characterize raav transduction vectors, the rep gene was retained in the recombinant vector while the neor gene was. Adenoassociated viruses as gene therapy vectors microbewiki. Comparison of gene delivery to the kidney by adenovirus. Although aav vector genomes can persist within cells as episomes, vector integration has been observed in various. Corroborating this are studies of aav vector integration sites is in mice, nonhuman primates nhp and muscle or liver biopsies from vector. Adenoassociated virus type 2 aav is known to establish latency by preferential integration in human chromosome 19q.
To prepare particles, cotransfect hek293t cells with the three plasmids. Oct 15, 2019 aav vector integration after crispr intervention. Open collection of aav data generously shared by scientists. Digital collection of empty plasmid backbones from publications and commercially available sources. To map the individual integration events in human chromosomal dna, the program bowtie. Hybrid adenoassociated viral vectors utilizing transposase.
Recombinant aav integration is not associated with hepatic genotoxicity in nonhuman primates and patients author links open overlay panel irene gilfarina 1 raffaele fronza 1 christine kaeppel 1 6 esperanza lopezfranco 2 valerie ferreira 3 delia davola 4 5 alberto benito 4 5 jesus prieto 4 5 harald petry 3 gloria gonzalezaseguinolaza 2 5. We constructed ds linear dna vectors with or without adenoassociated virus inverted terminal repeats aav itrs, introduced them into mouse hepatocytes in vivo using a hydrodynamicsbased transfection technique, and analyzed for vector genome integration in various ways. This is due, in part, to the fact that the kidney excludes molecules a. Recombinant adenoassociated viral vectors raav currently. Specifically, instructions on amplification and cloning of cdna into the vector backbone and verification of the final expression constructs are provided in this user manual.
Portions of aav vector sequences are shown in uppercase. The aav nonstructural protein rep appears to target a site called aavs1 by simultaneously binding to repbinding sites rbs present. Longterm aavmediated factor viii expression in nine. Next, we analyzed aav vector integration into crisprinduced breaks in vivo in. Genetics computer group program, using fasta with default parameters and. Pdf adenoassociated virus vector integration junctions. High levels of aav vector integration into crisprinduced dna. Assessing the potential for aav vector genotoxicity in a. Aav vector integration sites in mouse hepatocellular. Aav helper free expression complete systems include packaging vectors, an expression vector, and a gfp control vector aav helper free packaging systems include the two packaging vectors individually not premixed plus a gfp control vector. In addition to reporter genes gfp, luciferase and lacz, we are pleased to stock constructs from dr.
Instead, the raav genome is typically processed into a doublestranded circular episome through double stranded synthesis. Recombinant aav integration is not associated with hepatic. Is chen, s kimmethodology and software to detect viral integration site hot. These findings, as well as the itr deletions and other vector genome modifications noted previously 1,2,3, demonstrate the imprecision and variability of aav vector packaging and integration. Figure 1 aav rdnahfah yields about 30 times higher integration frequency. Aav vectors lack the integration promoting rep gene and therefore only. Mar 15, 2018 in recent years, aav vectors have been increasingly evaluated in various gene therapy clinical trials for treating a plethora of diseases. To allow detailed characterization of integration events we engineer a miniature aav encoding a 465 bp lambda bacteriophage dna aav. Jan 2009 irenedbaav 2dec10 guidance for working with adenoassociated virus aav vectors guidance background to adenoassociated virus aav aav is a small, stable virus that has never been shown to cause disease in humans even though a majority of the population has been exposed to it.
Recent studies employing lmpcr to determine aav integration loci. However, chromosomal integration is a twoedged sword. Detailed information of aav wildtype versus recombinant aav vector integration sites and preferences are needed to evaluate the safety profile of aav vectors in gene therapy. Analysis of the 20 samples identified 2,000 unique aav integration events ie. The overall structure of the mouse 45s rdna unit and the location of the region used in the rdna vector are shown. Although aav vectors are not known to frequently integrate into the host genome, the introduction of doublestranded breaks by crisprcas9 might increase the risk of aav integration. Therefore therapeutic aav integrates at a very low frequency of. Nirao shah university of california at san francisco. The unc vector core offers an extensive inventory of premade aav vectors available in multiple serotypes. Abstract the driving interest in adenoassociated virus aav has been its potential as a gene delivery vector. Integration preferences of wildtype aav2 for consensus rep. Aav vectors are quickly moving forward in the clinic and repdependent vector targeting strategies are being actively pursued. Adenoassociated viruses, from the parvovirus family, are small viruses with a genome of single stranded dna.
Because of aav s specialized gene therapy advantages, researchers have created an altered version of aav termed selfcomplementary adenoassociated virus scaav. The use of an aav system allows for efficient genome modification in a wide variety of mammalian cells in vitro, with the crisprcas9 system using two aav vectors to deliver the larger s. Next, we analyzed aav vector integration into crisprinduced breaks in vivo in three different organs brain, cochlea, and muscle. Aav proviruses were isolated from four tumors and were all located within a 6kilobase region of chromosome 12. Itrseq, a nextgeneration sequencing assay, identifies. Introduction to adenoassociated virus aav vector biolabs. There are a few disadvantages to using aav, including the small amount of dna it ca. Adenoassociated virus aav vectors efficiently transduce various cell types and can produce longterm expression of transgenes in vivo. Aav vector integration after crispr intervention aav used for gene therapy lacks dedicated machinery to integrate into the genome.
In the aav helperfree system, the rep and cap genes have been removed from the viral vector that contains aav 2 itrs and are supplied in trans on the plasmid paavrc. The aav vectors of the present invention are particularly suitable for gene therapy by systemic application, since a transduction of the liver is eliminated or at least drastically reduced and b the transduction efficiency of nonhepatic tissues is increased. Whereas aav packages a single strand of dna and must wait for its second strand to be synthesized, scaav packages two shorter strands that are complementary to each other. Aav vectormediated in vivo reprogramming into pluripotency. Nd not determined aav helper free vector technologies are available in the following formats. This manual does not include information on packaging the constructs into aav viral. Adenoassociated viruses aav are promising gene therapy vectors that have little or no acute toxicity. Helperindependent and aavitrindependent chromosomal. Recombinant adenoassociated viral integration and genotoxicity. In parallel with the production of aav vectors for clinical trials in human diseases, technological progresses have focused on the development of rigorous quality control assays in order to support regulatory applications and favor new product licensing. Vectors derived from adenoassociated virus aav have the potential to stably transduce mammalian cells by integrating into host chromosomes. Therefore therapeutic aav integrates at a very low frequency of 0. The role of integration in the biology of aav, whether repmediated targeted. Despite active research on the use of aav vectors for gene therapy, the structure of integrated vector proviruses has not previously been analyzed at the dna sequence level.
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