AAV Inverted Terminal Repeats Research Articles

Targeted long-read sequencing captures CRISPR editing and AAV integration outcomes in brain

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing is an emerging therapeutic modality that shows promise in Huntington's disease and spinocerebellar ataxia (SCA) mouse models. However, advancing CRISPR-based therapies requires methods to fully define invivo editing outcomes. Here, we use polymerase-free, targeted long-read nanopore sequencing and evaluate single- and dual-gRNA AAV-CRISPR editing of human ATXN2 in transgenic mouse models of SCA type 2 (SCA2). Unbiased high sequencing coverage showed 10%-25% editing. Along with intended edits there was AAV integration, 1%-2% of which contained the entire AAV genome and were largely unmethylated. More than 150 kb deletions at target loci and rearrangements of the transgenic allele (1%) were also found. In contrast, PCR-based nanopore sequencing showed bias for partial AAV fragments and inverted terminal repeats (ITRs) and failed to detect full-length AAV. Cumulatively this work defines the spectrum of outcomes of CRISPR editing in mouse brain after AAV gene transfer using an unbiased long-read sequencing approach.

Development of an AAV DNA-based synthetic vector for the potential gene therapy of hemophilia in children.

Recombinant AAV serotype vectors and their variants have been or are currently being used for gene therapy for hemophilia in several phase I/II/III clinical trials in humans. However, none of these trials have included children with hemophilia since the traditional liver-directed AAV gene therapy will not work in these patients because of the following reasons: (i) Up until age 10–12, the liver is still growing and dividing, and with every cell division, the AAV vector genomes will be diluted out due to their episomal nature; and (ii) Repeated gene delivery will be needed, but repeat dosing, even with an ideal AAV vector is not an option because of pre-existing antibodies to AAV vectors following the first administration. Here we describe the development of an optimized human Factor IX (hF.IX) gene expression cassette under the control of a human liver-specific transthyretin promoter covalently flanked by AAV inverted terminal repeats (ITRs) with no open ends (optNE-TTR-hF.IX), which mediated ~sixfold higher hF.IX levels than that from a linear TTR-hF.IX DNA construct in human hepatoma cells up to four-weeks post-transfection. In future studies, encapsidation of the optNE-TTR-hF.IX DNA in liver-targeted synthetic liposomes, may provide a viable approach for the potential gene therapy for hemophilia in children.

AAV ablates neurogenesis in the adult murine hippocampus.

Recombinant adeno-associated virus (rAAV) has been widely used as a viral vector across mammalian biology and has been shown to be safe and effective in human gene therapy. We demonstrate that neural progenitor cells (NPCs) and immature dentate granule cells (DGCs) within the adult murine hippocampus are particularly sensitive to rAAV-induced cell death. Cell loss is dose dependent and nearly complete at experimentally relevant viral titers. rAAV-induced cell death is rapid and persistent, with loss of BrdU-labeled cells within 18 hr post-injection and no evidence of recovery of adult neurogenesis at 3 months post-injection. The remaining mature DGCs appear hyperactive 4 weeks post-injection based on immediate early gene expression, consistent with previous studies investigating the effects of attenuating adult neurogenesis. In vitro application of AAV or electroporation of AAV2 inverted terminal repeats (ITRs) is sufficient to induce cell death. Efficient transduction of the dentategyrus (DG)- without ablating adult neurogenesis- can be achieved by injection of rAAV2-retro serotyped virus into CA3. rAAV2-retro results in efficient retrograde labeling of mature DGCs and permits in vivo two-photon calcium imaging of dentate activity while leaving adult neurogenesis intact. These findings expand on recent reports implicating rAAV-linked toxicity in stem cells and other cell types and suggest that future work using rAAV as an experimental tool in the DG and as a gene therapy for diseases of the central nervous system should be carefully evaluated.

The Unusual Properties of the AAV Inverted Terminal Repeat.

Although the sequence of the AAV inverted terminal repeat has been known for 40 years, there are still unanswered questions about functions attributable to the terminal 125 nucleotides.

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

Ongoing development of recombinant vectors based on adeno-associated virus (rAAV) is providing an increasingly powerful and widely used toolkit for gene transfer and genome editing applications. While conceptually simple, the system harbors considerable complexity that presents many potential pitfalls for the inexperienced user. The short inverted terminal repeats (ITRs) can prove to be particularly problematic during vector engineering due to inherent instability necessitating diligent quality control measures during vector manufacture. This is especially important from a clinical standpoint when consistent purity and potency are paramount, and all components of the system are rigorously scrutinized by regulatory agencies. Despite the discovery over 30 years ago that the AAV ITRs are the only cis-acting elements of the virus required for vector production, there is a scarcity of reviews specifically focused on these complex elements. This review provides an overview of the ITR with the dual purpose of acting as a user's guide in the application of AAV vector technology and as a roadmap for ongoing vector development and optimization.

Highly Efficient and Marker-free Genome Editing of Human Pluripotent Stem Cells by CRISPR-Cas9 RNP and AAV6 Donor-Mediated Homologous Recombination.

Genome editing of human pluripotent stem cells (hPSCs) provides powerful opportunities for invitro disease modeling, drug discovery, and personalized stem cell-based therapeutics. Currently, only small edits can be engineered with high frequency, while larger modifications suffer from low efficiency and a resultant need for selection markers. Here, we describe marker-free genome editing in hPSCs using Cas9 ribonucleoproteins (RNPs) in combination with AAV6-mediated DNA repair template delivery. We report highly efficient and bi-allelic integration frequencies across multiple loci and hPSC lines, achieving mono-allelic editing frequencies of up to 94% at the HBB locus. Using this method, we show robust bi-allelic correction of homozygous sickle cell mutations in a patient-derived induced PSC (iPSC) line. Thus, this strategy shows significant utility for generating hPSCs with large gene integrations and/or single-nucleotide changes at high frequency and without the need for introducing selection genes, enhancing the applicability of hPSC editing for research and translational uses.

87. Rationally Designed Inverted Terminal Repeats Enhance Transduction of Embryonic Stem Cells

Despite the prevalence of AAV vectors in gene therapy, treatments combining AAV vectors and cell therapy have not progressed into the clinic despite previous reports of successful transduction of stem cells by AAV vectors. One potential explanation is AAV vector toxicity in stem and stem-like cells, as demonstrated in previous studies. In particular, the AAV inverted terminal repeat (ITR) sequence was shown to induce apoptosis in human embryonic stem cells (hESCs) in a p53 dependent manner. A bioinformatic analysis of putative transcription factor binding sites in the AAV2 ITR sequence revealed six putative p53 binding sites. A panel of rationally designed AAV2 ITRs was generated which abrogated putative p53 binding sites and demonstrated reduced p53 binding in an in vitro binding assay. The synthetic ITRs were capable of all of the vector production requirements and were verified by sequencing directly from vector preparations. AAV vector transduction and the hESC DNA damage response was altered by the synthetic ITRs. Importantly, our ITRs demonstrated reduced toxicity in hESCs compared to an AAV vector having the AAV2 ITR sequence. The collective results allude to modulation of the host's DNA damage response using rationally designed ITRs, which may enhance the relevancy of AAV vectors in stem cell therapies.

Strategies to generate high-titer, high-potency recombinant AAV3 serotype vectors

Although recombinant adeno-associated virus serotype 3 (AAV3) vectors were largely ignored previously, owing to their poor transduction efficiency in most cells and tissues examined, our initial observation of the selective tropism of AAV3 serotype vectors for human liver cancer cell lines and primary human hepatocytes has led to renewed interest in this serotype. AAV3 vectors and their variants have recently proven to be extremely efficient in targeting human and nonhuman primate hepatocytes in vitro as well as in vivo. In the present studies, we wished to evaluate the relative contributions of the cis-acting inverted terminal repeats (ITRs) from AAV3 (ITR3), as well as the trans-acting Rep proteins from AAV3 (Rep3) in the AAV3 vector production and transduction. To this end, we utilized two helper plasmids: pAAVr2c3, which carries rep2 and cap3 genes, and pAAVr3c3, which carries rep3 and cap3 genes. The combined use of AAV3 ITRs, AAV3 Rep proteins, and AAV3 capsids led to the production of recombinant vectors, AAV3-Rep3/ITR3, with up to approximately two to fourfold higher titers than AAV3-Rep2/ITR2 vectors produced using AAV2 ITRs, AAV2 Rep proteins, and AAV3 capsids. We also observed that the transduction efficiency of Rep3/ITR3 AAV3 vectors was approximately fourfold higher than that of Rep2/ITR2 AAV3 vectors in human hepatocellular carcinoma cell lines in vitro. The transduction efficiency of Rep3/ITR3 vectors was increased by ~10-fold, when AAV3 capsids containing mutations in two surface-exposed residues (serine 663 and threonine 492) were used to generate a S663V+T492V double-mutant AAV3 vector. The Rep3/ITR3 AAV3 vectors also transduced human liver tumors in vivo approximately twofold more efficiently than those generated with Rep2/ITR2. Our data suggest that the transduction efficiency of AAV3 vectors can be significantly improved both using homologous Rep proteins and ITRs as well as by capsid optimization. Thus, the combined use of homologous Rep proteins, ITRs, and capsids should also lead to more efficacious other AAV serotype vectors for their optimal use in human gene therapy.

94. AAV Integration Site Determination Using Illumina Mate Pair Sequencing

Integration of AAV into genomic loci has been well studied using low throughput techniques, which has revealed clear integration hotspots. Recent studies using high throughput sequencing have uncovered new genomic loci where AAV may integrate. However, the low integration frequency combined with the AAV inverted terminal repeats (ITR) makes amplification technologically challenging and prone to artifacts that may mis-identify or miss some integration sites. Our lab has utilized a modified Illumina Mate Pair sequencing technique to uncover genomic integration sites and bypass the need to amplify through the AAV ITR. We believe that this technique can accurately establish bona-fide integration sites, uncover novel integration sites, and provide further evidence for existing known hotspots. At its core, mate pair sequencing involves self-circularization of fractured genomic fragments followed by recovery and sequencing of the junction. In essence, it bridges the low throughput shuttle vector systems used in the past with high throughput sequencing. Here we have utilized biotinylated loxP linkers added to the ends of genomic fragments and at unique sites in AAV vectors followed by Cre circularization (Van Nieuwerburgh, 2012). The circularized fragments are enriched by exonuclease treatment to remove linear fragments then fractured to ~400 bp. The fragments containing the loxP junction are purified using magnetic strepavadin coated beads and Illumina sequencing adapters are added before sequencing. Using this technique, we have successfully recovered control sequences of plasmids mixed with genomic DNA. Importantly, the Cre recombination reaction allows us to accurately distinguish self-circularized fragments from cross-recombined fragments and eliminate apparent false positive integration sites. We are working on applying this technique to DNA samples from mice injected with AAV. Continued study of AAV integration will be extremely important for establishing the safety profile of AAV used in clinical applications.

Differential contribution of adeno-associated virus type 2 Rep protein expression and nucleic acid elements to inhibition of adenoviral replication in cis and in trans.

The helper-dependent adeno-associated virus type 2 (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses and when it is present in cis, as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is due predominantly to the expression of the AAV-2 Rep proteins was recently challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep open reading frame (ORF). We closely analyzed the relative contributions of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis, a sequence element in the 3' part of the rep gene, comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed the RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation, and by inactivation of the RIS-Ad, it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2-Ad coinfection. In this case, expression of the Rep proteins is required, as well as the presence of an amplifiable inverted terminal repeat (ITR)-containing template. Thus, very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad-AAV coinfection. This is the first study to systematically compare the contributions of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector generation and in AAV-2-adenovirus coinfection. This study shows that the two inhibitory processes are very different with regard to AAV-2 functions and the mechanisms involved. Whereas inhibition of rAd/AAV-2 hybrid vector propagation mostly involves a 3' nucleic acid element in the rep gene, inhibition of an adenoviral genome in trans requires the Rep proteins and the AAV ITRs. These findings have important implications both for a basic understanding of the AAV replication cycle and for generation of rAd/AAV-2 hybrid vectors expressing the nonstructural and structural proteins of AAV-2.

Novel approaches to render stable producer cell lines viable for the commercial manufacturing of rAAV-based gene therapy vectors

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

Bioengineered Coagulation Factor VIII Enables Long-Term Correction Of Murine Hemophilia A Following Liver-Directed Adeno-Associated Viral Vector Delivery

Preclinical and, more recently, clinical data support the feasibility and safety of recombinant adeno-associated viral vectors (rAAV) in gene therapy applications. Despite several clinical trials of rAAV-based gene transfer for hemophilia B, a unique set of obstacles impede the development of a similar approach for hemophilia A. These include 1) inefficient biosynthesis of human coagulation factor VIII (fVIII), 2) limited packaging capacity of rAAV (4.9kb) for the large B domain-deleted (BDD) fVIII transgene (4.5 kb), 3) humoral immune responses to the transgene product and 4) dose limitations imposed by capsid mediated cytotoxic immunity. Our laboratory has developed and validated bioengineered fVIII molecules that are biosynthesized more efficiently than similar BDD human fVIII molecules due to superior post-translational transit through the ER/golgi/secretory pathway. In the current study, we incorporated one of these constructs, previously designated HP47 (Doering et al. 2004 J Biol Chem. 279:8) and now termed ET-3, into an AAV-based gene transfer approach similar to that previous tested for patients with hemophilia B. Briefly, a rAAV2/8 vector encoding the ET-3 transgene under the control of a liver-specific promoter comprised of an Apo E hepatic control region (HCR) and the α-1 antitrypsin enhancer/promoter (HAAT), flanked by AAV inverted terminal repeats (ITRs), was constructed and designated rAAV-HCR-ET3. In prior studies, we demonstrated that recombinant ET-3 displays i) 100-fold more efficient biosynthesis, ii) 2 – 3-fold higher specific activity, and iii) 3-fold slower decay compared to BDD human fVIII. However, the vector genome size of rAAV-HCR-ET3, 5.9kb, exceeds the packaging capacity of rAAV by 20%. To address this concern, molecular analysis of rAAV-HCR-ET3 was performed and showed packaging primarily of 5' or 3' truncated vector genomes (vg). Further quantitative (q) PCR of vg using a series of primers pairs spanning the vector sequence showed 5-fold fewer copies of the termini than the center, which is indicative of terminally truncated vg. Thus, special consideration must be taken when using qPCR-based methods alone to titer vector preparations. Our data support a model whereby cells are infected by multiple truncated but overlapping vg, which then reassemble at some frequency to form complete, functional fVIII transgenes and/or RNA transcripts. In the present study, adult hemophilia A mice were administered a single peripheral vein injection of rAAV-HCR-ET3 at doses ranging from 3.9e10 vector particles (vp) /kg to 2.0e13 vp/kg as determined by viral protein concentration. These stated doses are 5 – 25-fold higher than would have been assigned using qPCR and expressed as vg/kg as opposed to vp/kg. At the conclusion of the study (11 - 50 weeks post-infusion), correction of fVIII deficiency to curative levels of fVIII (>0.4 U/mL in circulating plasma) was achieved at vector doses as low as 6.25e11 vp/kg, and partial correction (>0.01 U/mL) was seen at doses as low as 1.6e11 vp/kg. Tail transection bleeding assay demonstrated correction of the bleeding phenotype out to as long as 50 weeks after rAAV infusion. While gene transfer and phenotypic correction was achieved in both male and female mice, male mice receiving 2.5e12 vp/kg showed 9-fold greater circulating fVIII levels than female mice receiving the same dose, which is consistent with previous reports. Taken together, these data suggest significant benefit of the bioengineered high expression fVIII transgene (ET-3) in the context of liver-directed rAAV delivery at low viral vector doses, which if based on qPCR, could be stated as low as 1e10 vg/kg and even that includes mostly incomplete vg. Recent data suggest that reducing the size of the rAAV-fVIII vg through the incorporation of shorter DNA control elements and smaller fVIII transgenes can increase the vector potency (McIntosh et al. 2013 Blood 121:17). Despite its large vg size, rAAV-HCR-ET3 achieved curative levels of fVIII activity at vector doses comparable to those recently reported for smaller vectors. Therefore, it appears likely that further enhancement of the rAAV-bioengineered high expression fVIII gene transfer approach will be possible through incorporation of smaller genetic elements that increase the frequency of functional ET-3 gene transfer in the context of hemophilia A gene therapy. Disclosures:Spencer:Expression Therapeutics, LLC: Equity Ownership. Doering:Expression Therapeutics, LLC: Equity Ownership.

Birth of a New Therapeutic Platform: 47 Years of Adeno-associated Virus Biology From Virus Discovery to Licensed Gene Therapy

Within American biomedical research, as supported primarily by the National Institutes of Health (NIH) since the post–World War II era, it has been considered axiomatic that investments in discovery research will ultimately lead to the development of novel approaches to diagnostics and therapeutics that will improve the health and well-being of citizens of the United States and the broader world. I reflect here in a historical context on how one such series of investments in discovery has resulted in the emergence of the first licensed human gene therapy product in Europe, namely Glybera.1,2,3 Glybera (alipogene tiparvovec) is a recombinant adeno-associated virus (rAAV) vector with AAV2 inverted terminal repeats (ITRs) encapsidated into AAV1 capsids (so-called rAAV2/1, or, for this purpose, rAAV1), which, when administered intramuscularly, can result in expression of sufficient levels of lipoprotein lipase (LPL) to be considered a safe and effective treatment of LPL deficiency. It was licensed in the European Union in November 2012, 47 years after the near simultaneous discovery of AAV in laboratories at the NIH Bethesda campus and at the University of Pittsburgh.4,5,6,7,8 I trace key milestones in the progress from the discovery of the virus, to its use as a platform for an approved therapeutic product (Figure 1).

Hoechst increases adeno‐associated virus‐mediated transgene expression in airway epithelia by inducing the cytomegalovirus promoter

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.

A Capillary Electrophoresis Sequencing Method for the Identification of Mutations in the Inverted Terminal Repeats of Adeno-Associated Virus

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.

DNA-Binding Activity of Adeno-Associated Virus Rep Is Required for Inverted Terminal Repeat-Dependent Complex Formation with Herpes Simplex Virus ICP8

Herpes simplex virus (HSV) helper functions for (AAV) replication comprise HSV ICP8 and helicase-primase UL5/UL52/UL8. Here we show that N-terminal amino acids of AAV Rep78 that contact the Rep-binding site within the AAV inverted terminal repeat (ITR) are required for ternary-complex formation with infected-cell protein 8 (ICP8) on AAV single-strand DNA (ssDNA) in vitro and for colocalization in nuclear replication domains in vivo. Our data suggest that HSV-dependent AAV replication is initiated by Rep contacting the AAV ITR and by cooperative binding of ICP8 on AAV ssDNA.

Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences

Abstract Viral vectors based on various naturally occurring adeno-associated virus (AAV) serotypes are among the most promising tools in human gene therapy. For the production of recombinant AAV (rAAV) vectors, researchers are focusing predominantly on cross-packaging an artificial AAV genome based on serotype 2 (AAV2) into capsids derived from other serotypes. Within the packaged genome the inverted terminal repeats (ITRs) are the only cis-acting viral elements required for rAAV vector generation and depict the lowest common denominator of all AAV2-derived vector genomes. Up to now, no quantitative PCR (qPCR) for the detection and quantification of AAV2 ITRs could be established because of their extensive secondary hairpin structure formation. Current qPCR-based methods are therefore targeting vector-encoded transgenes or regulatory elements. Herein we establish a molecular biological method that allows accurate and reproducible quantification of AAV2 genomes on the basis of an AAV2 ITR sequence-specific...

HPV E1 up-regulates replication-related biochemistries of AAV Rep78

Human papillomavirus type 16 (HPV) E1 protein provides helper function for the adeno-associated virus type 2 (AAV) life cycle. E1 is the replication protein of HPV, analogous to AAV Rep78, but without the endonuclease/covalent attachment activity of Rep78. Previously we have shown that E1 and Rep78 interact in vitro. Here we investigated E1's effects on Rep78 interaction with AAV's inverted terminal repeat (ITR) DNA in vitro, using purified Rep78 and E1 proteins from bacteria. E1 enhanced Rep78-ITR binding, ATPase activity, Rep78-ITR-covalent linkage and Rep78-ITR-endonuclease activity (central to AAV replication). These enhancements occurred in a dose-dependent manner whenever assayed. However, overall Rep78-plus-E1 helicase activity was lower than Rep78's helicase activity. These data suggest that E1's broad-based helper function for the AAV life cycle (AAV DNA, mRNA, and protein levels are up-regulated by E1) is likely through its ability to enhance Rep78's critical replication-required biochemistries on ITR DNA.

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

For many experiments in the study of the peripheral nervous system, it would be useful to genetically manipulate primary sensory neurons. We have compared vectors based on adeno-associated virus (AAV) serotypes 1, 2, 3, 4, 5, 6, and 8, and lentivirus (LV), all expressing green fluorescent protein (GFP), for efficiency of transduction of sensory neurons, expression level, cellular tropism, and persistence of transgene expression following direct injection into the dorsal root ganglia (DRG), using histological quantification and qPCR. Two weeks after injection, AAV1, AAV5, and AAV6 had transduced the most neurons. The time course of GFP expression from these three vectors was studied from 1 to 12 weeks after injection. AAV5 was the most effective serotype overall, followed by AAV1. Both these serotypes showed increasing neuronal transduction rates at later time points, with some injections of AAV5 yielding over 90% of DRG neurons GFP(+) at 12 weeks. AAV6 performed well initially, but transduction rates declined dramatically between 4 and 12 weeks. AAV1 and AAV5 both transduced large-diameter neurons, IB4(+) neurons, and CGRP(+) neurons. In conclusion, AAV5 is a highly effective gene therapy vector for primary sensory neurons following direct injection into the DRG.

Differential Transduction Following Basal Ganglia Administration of Distinct Pseudotyped AAV Capsid Serotypes in Nonhuman Primates

We examined the transduction efficiency of different adeno-associated virus (AAV) capsid serotypes encoding for green fluorescent protein (GFP) flanked by AAV2 inverted terminal repeats in the nonhuman primate basal ganglia as a prelude to translational studies, as well as clinical trials in patients with Parkinson's disease (PD). Six intact young adult cynomolgus monkeys received a single 10 microl injection of AAV2/1-GFP, AAV2/5-GFP, or AAV2/8-GFP pseudotyped vectors into the caudate nucleus and putamen bilaterally in a pattern that resulted in each capsid serotype being injected into at least four striatal sites. GFP immunohistochemistry revealed excellent transduction rates for each AAV pseudotype. Stereological estimates of GFP+ cells within the striatum revealed that AAV2/5-GFP transduces significantly higher number of cells than AAV2/8-GFP (P < 0.05) and there was no significant difference between AAV2/5-GFP and AAV2/1-GFP (P = 0.348). Consistent with this result, Cavalieri estimates revealed that AAV2/5-GFP resulted in a significantly larger transduction volume than AAV2/8-GFP (P < 0.05). Each pseudotype transduced striatal neurons effectively [>95% GFP+ cells colocalized neuron-specific nuclear protein (NeuN)]. The current data suggest that AAV2/5 and AAV2/1 are superior to AAV2/8 for gene delivery to the nonhuman primate striatum and therefore better candidates for therapeutic applications targeting this structure.