Itrseq, a nextgeneration sequencing assay, identifies. 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. We have compiled the key pieces of information from hundreds of published papers for education and convenience. The first fdaapproved aavbased gene therapies are luxturna by spark therapeutics and zolgensma by novartis for treating a rare in. Given the variety of applications and tissue types toward which these. Aav vector integration sites in mouse hepatocellular carcinoma. Largescale analysis of adenoassociated virus vector integration. Adenoassociated viruses aav are small viruses that affect humans and other members of the primate species fig. Aav vector integration after crispr intervention aav used for gene therapy lacks dedicated machinery to integrate into the genome. A key element is hence to investigate if and how parts of the vector genome are incorporated into a host. Portions of aav vector sequences are shown in uppercase. Adenoassociated vector toxicityto be or not to be molecular. Aav vector integration into crisprinduced dna breaks. 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.
To map the individual integration events in human chromosomal dna, the program bowtie. 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. Aav vector integration sites in mouse hepatocellular. Although aav vector genomes can persist within cells as episomes, vector integration has been observed in various. Here we have performed a largescale analysis of aav vector integration. Guidance for working with adenoassociated virus aav vectors. Integration of adenoassociated virus aav and recombinant aav. Adenoassociated virus aav cloning and expression vectors. Considerations for preclinical safety assessment of adeno.
Genetics computer group program, using fasta with default parameters and. Up to 62 nucleotides of flanking sequences are shown for each junction in lowercase and are aligned. 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. Analytical approaches to characterize aav vector production. Aav vectors are quickly moving forward in the clinic and repdependent vector targeting strategies are being actively pursued. 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. Gene therapy adenoassociated virus aav vectors explained. 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. Aav used for gene therapy lacks dedicated machinery to integrate into the genome. 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. Mar 24, 2011 gene transfer using adenoassociated virus aav vectors has great potential for treating human disease. To prepare particles, cotransfect hek293t cells with the three plasmids. The early observation that aav can establish a latent infection by integrating into the host chromosome has been central to this interest.
The overall structure of the mouse 45s rdna unit and the location of the region used in the rdna vector are shown. They belong to the parvovirus family and require a helper virus, such as adenovirus, herpes simplex virus, or vaccina virus, to replicate 1. Targeted integration of a raav vector into the aavs1 region. 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. Adenoassociated viruses aav are promising gene therapy vectors that have little or no acute toxicity. 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. Next, we analyzed aav vector integration into crisprinduced breaks in vivo in three different organs brain, cochlea, and muscle.
Nd not determined aav helper free vector technologies are available in the following formats. New studies refocus attention on the genotoxicity of aav vectors in gene therapy. Recombinant adenoassociated virus transduction and integration. Figure 1 aav rdnahfah yields about 30 times higher integration frequency. There are a few disadvantages to using aav, including the small amount of dna it ca. Adenoassociated virus aav as a vector for gene therapy michael f. 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. Instead, the raav genome is typically processed into a doublestranded circular episome through double stranded synthesis. The unc vector core offers an extensive inventory of premade aav vectors available in multiple serotypes. Adenoassociated virus vector integration junctions journal of. 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. 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. Pdf adenoassociated virus vector integration junctions. Unit 4, 7074 industry dr, tweed heads south, nsw 2486 qld location.
To accomplish integration into aavs1 with raav vectors rep must. Because aav integrates in between bases, the bases indicated in this file are those immediately 3 to the integration. Integration preferences of wildtype aav2 for consensus rep. Chromosomal effects of adenoassociated virus vector integration. Therefore therapeutic aav integrates at a very low frequency of. Therefore therapeutic aav integrates at a very low frequency of 0. In comparison to the widespread use of aav vectors in clinical, preclinical, and. Dickinson facs canto flow cytometer using facs diva software. 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. Each kit includes three plasmids encoding the factors necessary to prepare recombinant aav particles by transfection of hek293t cells. Sep 30, 2019 vector integration into therapeutically relevant genes in vivo. Comparison of gene delivery to the kidney by adenovirus.
New studies refocus attention on the genotoxicity of aav. In this study, we analyze integration of aav vectors genomewide and. The aav nonstructural protein rep appears to target a site called aavs1 by simultaneously binding to repbinding sites rbs present. Recombinant adenoassociated viral aav vectors have been shown. Mar 15, 2018 in recent years, aav vectors have been increasingly evaluated in various gene therapy clinical trials for treating a plethora of diseases. Aav vectormediated in vivo reprogramming into pluripotency.
This manual does not include information on packaging the constructs into aav viral. Is chen, s kimmethodology and software to detect viral integration site hot. 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. 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. High levels of aav vector integration into crisprinduced dna. Ubisap is a firstofitskind software available for is analysis to. Current viral vectors are designed to be less mutagenetic, as in the case of sin. However, gene delivery to kidney cells is inefficient. The role of integration in the biology of aav, whether repmediated targeted.
Aav vectors containing rdna homology display increased. This is due, in part, to the fact that the kidney excludes molecules a. Oct 15, 2019 aav vector integration after crispr intervention. 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. 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. Analysis of the 20 samples identified 2,000 unique aav integration events ie.
The raav vectors reduced ability to integrate also means that the. Adenoassociated viruses as gene therapy vectors microbewiki. 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. Adenoassociated viruses, from the parvovirus family, are small viruses with a genome of single stranded dna. Recombinant adenoassociated viral vectors raav currently. Next, we analyzed aav vector integration into crisprinduced breaks in vivo in. Adenoassociated virus type 2 aav is known to establish latency by preferential integration in human chromosome 19q. The analysis of viral vector genomic integration sites is an important. 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. Pdf chromosomal effects of adenoassociated virus vector. 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.
Chromosomal effects of adenoassociated virus vector. Dec 16, 2019 there are many kidney diseases that might be addressed by gene therapy. Helperindependent and aavitrindependent chromosomal. 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. Aav proviruses were isolated from four tumors and were all located within a 6kilobase region of chromosome 12. Digital collection of empty plasmid backbones from publications and commercially available sources. Adenoassociated virus aav as a vector for gene therapy. This section is meant to serve as an introduction for those unfamiliar with aav adenoassociated virus. Introduction to adenoassociated virus aav vector biolabs.
Quantifying quality attributes of aav gene therapy. 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. Recombinant aav integration is not associated with hepatic. Abstract the driving interest in adenoassociated virus aav has been its potential as a gene delivery vector. Vectors derived from adenoassociated virus aav have the potential to stably transduce mammalian cells by integrating into host chromosomes. Our helperfree aav systems are designed for the preparation of hightiter aav particles without the use of a helper virus.
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. However, as aav vectors mostly remain episomal, vector genomes and transgene expression are lost in dividing cells. Recent studies employing lmpcr to determine aav integration loci. 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. Nirao shah university of california at san francisco. Adenoassociated virus aav vectors efficiently transduce various cell types and can produce longterm expression of transgenes in vivo. These viruses can insert genetic material at a specific site on chromosome 19 with near 100% certainty.
Aav vectors lack the integration promoting rep gene and therefore only. The integration of wildtype aav aav is a helperdependent parvovirus with an approximately 4. Vector integration into therapeutically relevant genes in vivo. Cell culture experiments pointed toward viral genome integration with. To allow detailed characterization of integration events we engineer a miniature aav encoding a 465 bp lambda bacteriophage dna aav. 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. Longterm aavmediated factor viii expression in nine. 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. Corroborating this are studies of aav vector integration sites is in mice, nonhuman primates nhp and muscle or liver biopsies from vector. Recently, questions have arisen about the safety of aav vectors, specifically, whether integration of vector dna in transduced cell genomes promotes tumor formation. Recombinant adenoassociated viral integration and genotoxicity. High levels of aav vector integration into crisprinduced.
This article is an open access publication abstract there has been a resurgence in gene therapy efforts that is partly fueled by the identi. Hybrid adenoassociated viral vectors utilizing transposase. However, chromosomal integration is a twoedged sword. Adenoassociated virus vector as a platform for gene. Assessing the potential for aav vector genotoxicity in a. Because of aav s specialized gene therapy advantages, researchers have created an altered version of aav termed selfcomplementary adenoassociated virus scaav. 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 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.