A User's Guide to the Inverted Terminal Repeats of Adeno-Associated Virus.

Evidence for the Failure of Adeno-associated Virus Serotype 5 to Package a Viral Genome ≥8.2 kb

Inverted terminal repeat (ITR) integrity is critical for the replication, packaging, and transduction of recombinant adeno-associated virus (rAAV), a promising gene therapy vector. Because AAV ITRs possess 70% GC content and are palindromic, they are notoriously difficult to sequence. The purpose of this work was to develop a reliable ITR sequencing method. The ITRs of two molecular clones of AAV2, pTZAAV and pAV2, were (1) sequenced directly from plasmid DNA in the presence of denaturant (direct sequencing method, DSM) or (2) first amplified in a reaction in which 7-deaza-dGTP was substituted for dGTP and the resultant amplification product sequenced (amplification sequencing method, ASM). The DSM and ASM techniques yielded clear chromatograms, read through the ITR hairpin, and revealed hitherto unreported mutations in each ITR. pTZAAV and pAV2 possess identical mutations at the upstream MscI site of the 5' ITR (T>G, nt 2) and the downstream MscI site of the 3' ITR (del. nt 4672-4679). The chromatograms for pAV2 also revealed that the ITRs of this construct were arranged in a FLOP/FLOP orientation. In addition, the DSM was successfully employed to recover ITR-chromosomal junction sequences from a variety of rAAV-transduced tissue types. Both the DSM and ASM can be employed to sequence through the AAV ITR hairpin, and both techniques reliably detect mutations in the ITR. Because the DSM and ASM offer a way to verify ITR integrity, they constitute powerful tools for the process development of rAAV gene therapy.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

Asymmetric deletion of the junction between the short unique region and the inverted repeat does not affect viral growth in culture and vaccine-induced immunity against Marek's disease

Self-complementary AAV Vectors; Advances and Applications

BackgroundThe Tc1/mariner superfamily might represent the most diverse and widely distributed group of DNA transposons. Several families have been identified; however, exploring the diversity of this superfamily and updating its classification is still ongoing in the life sciences.ResultsHere we identified a new family of Tc1/mariner transposons, named Incomer (IC), which is close to, but distinct from the known family DD34E/Tc1. ICs have a total length of about 1.2 kb, and harbor a single open reading frame encoding a ~ 346 amino acid transposase with a DD36E motif and flanked by short terminal inverted repeats (TIRs) (22–32 base pairs, bp). This family is absent from prokaryotes, and is mainly distributed among vertebrates (141 species of four classes), including Agnatha (one species of jawless fish), Actinopterygii (132 species of ray-finned fish), Amphibia (four species of frogs), and Mammalia (four species of bats), but have a restricted distribution in invertebrates (four species in Insecta and nine in Arachnida). All ICs in bats (Myotis lucifugus, Eptesicus fuscus, Myotis davidii, and Myotis brandtii) are present as truncated copies in these genomes, and most of them are flanked by relatively long TIRs (51–126 bp). High copy numbers of miniature inverted-repeat transposable elements (MITEs) derived from ICs were also identified in bat genomes. Phylogenetic analysis revealed that ICs are more closely related to DD34E/Tc1 than to other families of Tc1/mariner (e.g., DD34D/mariner and DD × D/pogo), and can be classified into four distinct clusters. The host and IC phylogenies and pairwise distance comparisons between RAG1 genes and all consensus sequences of ICs support the idea that multiple episodes of horizontal transfer (HT) of ICs have occurred in vertebrates. In addition, the discovery of intact transposases, perfect TIRs and target site duplications of ICs suggests that this family may still be active in Insecta, Arachnida, frogs, and fish.ConclusionsExploring the diversity of Tc1/mariner transposons and revealing their evolutionary profiles will help provide a better understanding of the evolution of DNA transposons and their impact on genomic evolution. Here, a newly discovered family (DD36E/Incomer) of Tc1/mariner transposons is described in animals. It displays a similar structural organization and close relationship with the known DD34E/Tc1 elements, but has a relatively narrow distribution, indicating that DD36E/IC might have originated from the DD34E/Tc1 family. Our data also support the hypothesis of horizontal transfer of IC in vertebrates, even invading one lineage of mammals (bats). This study expands our understanding of the diversity of Tc1/mariner transposons and updates the classification of this superfamily.

Comparison of AAV Serotypes for Gene Delivery to Dorsal Root Ganglion Neurons

In airway epithelia, the kinetics of recombinant adeno-associated virus (AAV) transgene expression is slow. This has negative practical implications for research, as well as for translation into therapy. The DNA minor groove-binding agent Hoechst-33342 has been shown to enhance AAV transgene expression. In the present study, we investigated the mechanism of Hoechst-related augmentation of AAV-mediated transgene expression. We investigated the effect of Hoechst-33342 on HT1080, COS-7, mouse and human airway epithelia transduced with different AAV serotypes encoding enhanced green fluorescent protein (eGFP). We exposed cells to increasing concentrations of Hoechst-33342 at different time points. We evaluated the effect on second-strand DNA synthesis using AAV with a self-complementary genome. We also investigated the effect on expression from transfected plasmids with and without AAV2 inverted terminal repeats (ITRs). We found that Hoechst-33342 significantly accelerated AAV transgene expression for all serotypes tested. Hoechst-33342 only had an effect when the treatment was given during or after transduction, even 120 days post-transduction, suggesting an effect on transgene expression regulation. Hoechst-33342 increased transgene expression when cells were transduced with a self-complementary AAV with the cytomegalovirus promoter, although there was no effect on cells transduced with conventional single-stranded AAV encoding the Rous sarcoma virus promoter. Finally, Hoechst-33342 increases gene expression from transfected plasmids regardless of the presence of AAV2 ITRs. Hoechst dramatically augments and accelerates AAV-mediated transgene expression in airway epithelia without altering AAV-mediated gene transfer. Hoechst activation of the cytomegalovirus promoter is seen in plasmids, although it is drastically enhanced in the context of AAV.

In this work the structural variations of Terminal Inverted Repeats (TIR) of Bari like transposons in Drosophila species has been studied. The aim is to try and assess the relevance of different variants in the evolutionary distribution of Bari elements. Bari is a member of the widespread Tc1 superfamily of transposable elements that has colonized most species of the Drosophila genus. We previously reported the structure of two related elements that differ in their TIR organization: Bari1 harbouring 26-bp TIR (short TIRs) and Bari2 with about 250-bp TIR (long TIIR). While elements with short TIRs are complete and potentially autonomous, long ones are invariably composed of defective copies. The results show that in D. pseudobscura, D. persimilis and D. mojavensis, there is a third class of Bari elements, Bari3, that exhibit a long TIR structure and are not defective. Phylogenetic relationships among reconstructed transposases are consistent with the three subfamilies sharing a common origin. However, the final TIR organization into long or short structure is not related by descent but appears to be lineage-specific. Furthermore, we show that, independently of origin and organization, within the 250-bp terminal sequences there are three regions that are conserved in both sequence and position suggesting they are under functional constraint.

Background Recombinant Adeno-associated virus (rAAV) based vectors recently emerged as very promising candidates for viral gene therapy due to a large toolbox available including twelve different AAV serotypes, natural isolates, designer capsids and library technologies [2]. Furthermore, rAAV vectors have favourable properties such as non-pathogenicity of AAV, low B-/T-cell immunogenicity against transgenes delivered and long-term transgene expression from a non-integrating vector [5,9]. Promising data from clinical trials using rAAVbased vectors for the treatment of e.g. haemophilia or retinal diseases as well as the recent approval of the first gene therapy drug in the European Union, Glybera to treat lipoprotein lipase deficiency, emphasise the potential of rAAV vectors for gene therapy approaches in a wide variety of indications [8,7,15]. Thereby, the demand for robust and cost-effective manufacturing of those vectors for market supply rose steadily. Standard production systems comprise transient transfectionand/or infection-based approaches using mammalian cells [3], or insect cells [16]. However, high production costs combined with considerable regulatory effort and safety concerns gave rise to the development of producer cell lines enabling stable rAAV production [3]. AAVs are parvoviruses whose productive infection is depending on the presence of helper viruses like e.g. adenovirus (AdV). Their single-stranded DNA genome carries two genes. The rep gene encodes proteins responsible for site-specific integration, viral genome replication as well as packging. The cap gene is translated into three structural proteins building the capsid shelf. Furthermore, cap encodes a protein required for capsid assembly (AAP or assembly-activating protein) that has been described recently [13]. The AAV genes are flanked by inverted terminal repeat (ITR) sequences constituting the replication, integration and packaging signal. In a stable producer cell line with integral helper functions, all required genetic elements are stably integrated into the genome of the host cell as independent expression constructs: the recombinant vector implying a transgene flanked by AAV ITRs, the AAV genes rep and cap required for replication and encapsidation, as well as adenoviral helper function delivered by sequences encoding genes E1a, E1b, E2a, E4orf6 and viral associated (VA) I/II RNA [9]. In a timely regulated fashion, viral proteins are expressed and the AAV genome is replicated and encapsidated. As some of the gene products arising during rAAV production are toxic, an inducible expression of the gene products is indispensable for generation of stable production cells. The aim of the underlying study is to provide all tools necessary to generate a stable and versatile producer cell line In order to circumvent the problems triggered by toxic proteins inevitably arising during rAAV formation, one objective of the project is to establish stable producer cells where rAAV production can be induced by temperature shift at the final production scale. To begin with, we first performed some general feasability studies to investigate whether the generation of stable and inducible producer cell lines using proprietary constructs is a viable approach. For this purpose, experiments for rAAV manufacturing based on a transient packaging approach were conducted. Infection of rep, cap and rAAV vector plasmid transfected cells with wildtype Adenovirus was compared with co-tranfection of the cells with additional plasmids carrying the Adenoviral * Correspondence: Verena.Emmerling@rentschler.de Rentschler Biotechnologie GmbH, Erwin-Rentschler-Strase 21, 88471 Laupheim, Germany Full list of author information is available at the end of the article Emmerling et al. BMC Proceedings 2013, 7(Suppl 6):P12 http://www.biomedcentral.com/1753-6561/7/S6/P12

DNA transposons play a crucial role in determining the size and structure of eukaryotic genomes. In this study, a new family of IS630-Tc1-mariner (ITm) DNA transposons, named Hiker (HK), was identified. HK is characterized by a DD35E catalytic domain and is distinct from all previously known families of the ITm group. Phylogenetic analyses showed that DD35E/Hiker forms a monophyletic clade with DD34E/Gambol, indicating that they may represent a separate superfamily of ITm. A total of 178 Hiker species were identified, with 170 found mainly in Actinopterygii, one in Chondrichthyes, six in Anura and one in Mollusca. Gambol (GM), on the other hand, are found in invertebrates, with 18 in Arthropoda and one in Platyhelminthes. Hiker transposons have a total length ranging from 2.14 to 3.67 kb and contain a single open reading frame that encodes a protein of approximately 370 amino acids (range 311–413 aa). They are flanked by short terminal inverted repeats (TIRs) of 16–30 base pairs and two base pair (TA) target-site duplications. In contrast, most transposons of the Gambol family have a total length of 1.35–5.96 kb, encode a transposase protein of approximately 350 amino acids (range 306–374 aa), and are flanked by TIRs that range from 32 to 1097 bp in length. Both Hiker and Gambol transposases have several conserved motifs, including helix-turn-helix (HTH) motifs and a DDE domain. Our study observed multiple amplification waves and repeated horizontal transfer (HT) events of HK transposons in vertebrate genomes, indicating their role in diversifying and shaping the genomes of Actinopterygii, Chondrichthyes, and Anura. Conversely, GM transposons showed few Horizontal transfer events. According to cell-based transposition assays, most HK transposons are likely inactive due to the truncated DNA binding domains of their transposases. We present an updated classification of the ITm group based on these findings, which will enhance the understanding of both the evolution of ITm transposons and that of their hosts.

Plant MITEs: Useful Tools for Plant Genetics and Genomics

Divergent evolution profiles of DD37D and DD39D families of Tc1/mariner transposons in eukaryotes

The maize transposon Activator (Ac) was the first mobile DNA element to be discovered. Since then, other elements were found that share similarity to Ac, suggesting that it belongs to a transposon superfamily named hAT after hobo from Drosophila, Ac from maize, and Tam3 from snapdragon. We addressed the structure and evolution of hAT elements by developing new tools for transposon mining and searching the public sequence databases for the hallmarks of hAT elements, namely the transposase and short terminal inverted repeats (TIRs) flanked by 8-bp host duplications. We found 147 hAT-related sequences in plants, animals, and fungi. Six conserved blocks could be identified in the transposase of most hAT elements. A total of 41 hAT sequences were flanked by TIRs and 8-bp host duplications and, out of these, 34 sequences had TIRs similar to the consensus determined in this work, suggesting that they are active or recently active transposons. Phylogenetic analysis and clustering of hAT sequences suggest that the hAT superfamily is very ancient, probably predating the plant-fungi-animal separation, and that, unlike previously proposed, there is no evidence that horizontal gene transfer was involved in the evolution of hAT elements.

piggyBac is a short inverted terminal repeat (ITR) transposable element originally discovered in Trichoplusia ni. It is currently the preferred vector of choice for enhancer trapping, gene discovery and identifying gene function in insects and mammals. Many piggyBac-like sequences have been found in the genomes of phylogenetically species from fungi to mammals. We have identified 98 piggyBac-like sequences (BmPBLE1-98) from the genome data of domesticated silkworm (Bombyx mori) and 17 fragments from expressed sequence tags (ESTs). Most of the BmPBLE1-98 probably exist as fossils. A total of 21 BmPBLEs are flanked by ITRs and TTAA host dinucleotides, of which 5 contain a single ORF, implying that they may still be active. Interestingly, 16 BmPBLEs have CAC/GTG not CCC/GGG as the characteristic residues of ITRs, which is a surprising phenomenon first observed in the piggyBac families. Phylogenetic analysis indicates that many BmPBLEs have a close relation to mammals, especially to Homo sapiens, only a few being grouped with the T. ni piggyBac element. In addition, horizontal transfer was probably involved in the evolution of the piggyBac-like elements between B. mori and Daphnia pulicaria. The analysis of the BmPBLEs will contribute to our understanding of the characteristic of the piggyBac family and application of piggyBac in a wide range of insect species.