A pilot study to determine the optimal dose of scAAVIL-1ra in a large animal model of post-traumatic osteoarthritis.

Gene therapy approaches using adeno-associated viral vectors have been successfully tested in the equine post-traumatic osteoarthritis (PTOA) model. Owing to differences in the levels of transgene expression and adverse tissue reactions observed in published studies, we sought to identify a safe therapeutic dose of scAAVIL-1ra in an inflamed and injured joint that would result in improved functional outcomes without any adverse events. scAAVIL-1ra was delivered intra-articularly over a 100-fold range, and horses were evaluated throughout and at the end of the 10-week study. A dose-related increase in IL-1ra levels with a decrease in PGE2 levels was observed, with the peak IL-1ra concentration being observed 7 days post-treatment in all groups. Perivascular infiltration with mononuclear cells was observed within the synovial membrane of the joint treated with the highest viral dose of 5 × 1012 vg, but this was absent in the lower-dosed joints. The second-highest dose of scAAVeqIL-1ra 5 × 1011 vg demonstrated elevated IL-1ra levels without any cellular response in the synovium. Taken together, the data suggest that the 10-fold lower dose of 5 × 1011vg scAAVIL-1ra would be a safe therapeutic dose in an equine model of PTOA.

Direct interaction of human serum proteins with AAV virions to enhance AAV transduction: immediate impact on clinical applications.

Recent hemophilia B clinical trials using adeno-associated virus (AAV) gene delivery have demonstrated much lower coagulation factor IX (FIX) production in patients compared with the high levels observed in animal models and AAV capsid-specific cytotoxic T lymphocyte response elicited at high doses of AAV vectors. These results emphasize the necessity to explore effective approaches for enhancement of AAV transduction. Initially, we found that incubation of all AAV vectors with human serum enhanced AAV transduction. Complementary analytical experiments demonstrated that human serum albumin (HSA) directly interacted with the AAV capsid and augmented AAV transduction. The enhanced transduction was observed with clinical grade HSA. Mechanistic studies suggest that HSA increases AAV binding to target cells, and that the interaction of HSA with AAV does not interfere with the AAV infection pathway. Importantly, HSA incubation during vector dialysis also increased transduction. Finally, HSA enhancement of AAV transduction in a model of hemophilia B displayed greater than a fivefold increase in vector-derived circulating FIX, which improved the bleeding phenotype correction. In conclusion, incubation of HSA with AAV vectors supports a universal augmentation of AAV transduction and, more importantly, this approach can be immediately transitioned to the clinic for the treatment of hemophilia and other diseases.

Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism.

No adeno-associated virus (AAV) capsid has been described in the literature to exhibit a primary oligodendrocyte tropism when a constitutive promoter drives gene expression, which is a significant barrier for efficient in vivo oligodendrocyte gene transfer. The vast majority of AAV vectors, such as AAV1, 2, 5, 6, 8 or 9, exhibit a dominant neuronal tropism in the central nervous system. However, a novel AAV capsid (Olig001) generated using capsid shuffling and directed evolution was recovered after rat intravenous delivery and subsequent capsid clone rescue, which exhibited a >95% tropism for striatal oligodendrocytes after rat intracranial infusion where a constitutive promoter drove gene expression. Olig001 contains a chimeric mixture of AAV1, 2, 6, 8 and 9, but unlike these parental serotypes after intravenous administration Olig001 has very low affinity for peripheral organs, especially the liver. Furthermore, in mixed glial cell cultures, Olig001 exhibits a 9-fold greater binding when compared with AAV8. This novel oligodendrocyte-preferring AAV vector exhibits characteristics that are a marked departure from previously described AAV serotypes.

Adeno-associated virus gene therapy vector scAAVIGF-I for transduction of equine articular chondrocytes and RNA-seq analysis.

OBJECTIVE: IGF-I is one of several anabolic factors being investigated for the treatment of osteoarthritis (OA). Due to the short biological half-life, extended administration is required for more robust cartilage healing. Here we create a self-complimentary adeno-associated virus (AAV) gene therapy vector utilizing the transgene for IGF-I. DESIGN: Various biochemical assays were performed to investigate the cellular response to scAAVIGF-I treatment vs an scAAVGFP positive transduction control and a negative for transduction control culture. RNA-sequencing analysis was also performed to establish a differential regulation profile of scAAVIGF-I transduced chondrocytes. RESULTS: Biochemical analyses indicated an average media IGF-I concentration of 608 ng/ml in the scAAVIGF-I transduced chondrocytes. This increase in IGF-I led to increased expression of collagen type II and aggrecan and increased protein concentrations of cellular collagen type II and media glycosaminoglycan vs both controls. RNA-seq revealed a global regulatory pattern consisting of 113 differentially regulated GO categories including those for chondrocyte and cartilage development and regulation of apoptosis. CONCLUSIONS: This research substantiates that scAAVIGF-I gene therapy vector increased production of IGF-I to clinically relevant levels with a biological response by chondrocytes conducive to increased cartilage healing. The RNA-seq further established a set of differentially expressed genes and gene ontologies induced by the scAAVIGF-I vector while controlling for AAV infection. This dataset provides a static representation of the cellular transcriptome that, while only consisting of one time point, will allow for further gene expression analyses to compare additional cartilage healing therapeutics or a transient cellular response.

Prediction of adeno-associated virus neutralizing antibody activity for clinical application.

Patients with neutralizing antibodies (Nabs) against adeno-associated virus (AAV) are usually excluded from the treatment with AAV vectors. To develop a standard assay for detecting Nab inhibition activity, we systematically studied current AAV Nab assays in vitro and in vivo. Several factors were found that influence the Nab titers based on the in vitro assay, including sera volume, AAV dose per cell, cell number and choice of transgenes. When the Nab titer assay was performed in vivo via intramuscular (IM) or systemic administration, a fourfold increase in sensitivity for measurement of Nab titers was observed compared with an identical in vitro test. To better mimic the clinical setting, after passively transferring human Nabs into mice, blood was collected before systemic injection of AAV vector and used for Nab titer analysis in vitro or via IM injection. The results showed that AAV delivered via IM injection had a similar inhibition pattern to systemic administration. These studies indicate critical parameters necessary for optimizing Nab sensitivity and that an in vivo Nab assay is more sensitive than an in vitro assay for inclusion/exclusion criteria. The variables identified by this study may explain some of the compounding clinical data seen to date with respect to efficiency of AAV transduction in various phase I clinical trials.

scAAVIL-1ra dosing trial in a large animal model and validation of long-term expression with repeat administration for osteoarthritis therapy.

A gene therapeutic approach to treat osteoarthritis (OA) appears to be on the horizon for millions of people who suffer from this disease. Previously we described optimization of a scAAVIL-1ra gene therapeutic vector and initially tested this in an equine model verifying long-term intrasynovial IL-1ra protein at therapeutic levels. Using this vector, we carried out a dosing trial in six horses to verify protein levels and establish a dose that would express relevant levels of therapeutic protein for extended periods of time (8 months). A novel arthroscopic procedure used to detect green fluorescence protein (GFP) fluorescence intrasynovially confirmed successful transduction of the scAAVGFP vector in both the synovial and cartilage tissues. No evidence of intra-articular toxicity was detected. Immune responses to vector revealed development of neutralizing antibodies (Nabs) within 2 weeks of administration, which persisted for the duration of the study but did not lower protein expression intra-articularly. Re-dosing with a different serotype to attain therapeutic levels of protein confirmed establishment of successful transduction. This is the first study in an equine model to establish a dosing/redosing protocol, as well as examine the Nab response to capsid and supports further clinical investigation to determine the clinical efficacy of scAAVIL-1ra to treat OA.

Zinc-finger nuclease-mediated gene correction using single AAV vector transduction and enhancement by Food and Drug Administration-approved drugs.

An emerging strategy for the treatment of monogenic diseases uses genetic engineering to precisely correct the mutation(s) at the genome level. Recent advancements in this technology have demonstrated therapeutic levels of gene correction using a zinc-finger nuclease (ZFN)-induced DNA double-strand break in conjunction with an exogenous DNA donor substrate. This strategy requires efficient nucleic acid delivery and among viral vectors, recombinant adeno-associated virus (rAAV) has demonstrated clinical success without pathology. However, a major limitation of rAAV is the small DNA packaging capacity and to date, the use of rAAV for ZFN gene delivery has yet to be reported. Theoretically, an ideal situation is to deliver both ZFNs and the repair substrate in a single vector to avoid inefficient gene targeting and unwanted mutagenesis, both complications of a rAAV co-transduction strategy. Therefore, a rAAV format was generated in which a single polypeptide encodes the ZFN monomers connected by a ribosome skipping 2A peptide and furin cleavage sequence. On the basis of this arrangement, a DNA repair substrate of 750 nucleotides was also included in this vector. Efficient polypeptide processing to discrete ZFNs is demonstrated, as well as the ability of this single vector format to stimulate efficient gene targeting in a human cell line and mouse model derived fibroblasts. Additionally, we increased rAAV-mediated gene correction up to sixfold using a combination of Food and Drug Administration-approved drugs, which act at the level of AAV vector transduction. Collectively, these experiments demonstrate the ability to deliver ZFNs and a repair substrate by a single AAV vector and offer insights for the optimization of rAAV-mediated gene correction using drug therapy.

Three-dimensional multipotent progenitor cell aggregates for expansion, osteogenic differentiation and 'in vivo' tracing with AAV vector serotype 6.

Multipotent adult progenitor cells (MAPCs) are bone marrow-derived stem cells with a high growth rate suitable for therapeutical applications as three-dimensional (3D) aggregates. Combined applications of osteogenically differentiated MAPC (OD-MAPC) aggregates and adeno-associated viral vectors (AAV) in bone bioengineering are still deferred until information with regard to expansion technologies, osteogenic potential, and AAV cytotoxicity and transduction efficiency is better understood. In this study, we tested whether self-complementary AAV (scAAV) can potentially be used as a gene delivery system in an OD-MAPC-based 'in vivo' bone formation model in the craniofacial region. Both expansion of rat MAPC (rMAPC) and osteogenic differentiation with dexamethasone were also tested in 3D aggregate culture systems 'in vitro' and 'vivo'. rMAPCs grew as undifferentiated aggregates for 4 days, with a population doubling time of 37 h. After expansion, constant levels of Oct4 transcripts, and Oct4 and CD31 surface markers were observed, which constitute a hallmark of undifferentiated stage of rMAPCs. Dexamethasone effectively mediated rMAPC osteogenic differentiation by inducing the formation of a mineralized collagen type I network, and facilitated the activation of the wnt/ß-catenin, a crucial pathway in skeletal development. To investigate the genetic modification of rMAPCs grown as 3D aggregates before implantation, scAAV serotypes 2, 3 and 6 were evaluated. scAAV6 packaged with the enhanced green fluorescent protein expression cassette efficiently mediated long-term transduction (10 days) 'in vitro' and 'vivo'. The reporter transduction event allowed the tracing of OD-rMAPC (induced by dexamethasone) aggregates following OD-rMAPC transfer into a macro-porous hydroxyapatite scaffold implanted in a rat calvaria model. Furthermore, the scAAV6-transduced OD-rMAPCs generated a bone-like matrix with a collagenous matrix rich in bone-specific proteins (osteocalcin and osteopontin) in the scaffold macro-pores 10 days post-implantation. Newly formed bone was also observed in the interface between native bone and scaffold. The collective work supports future bone tissue engineering applications of 3D MAPC cultures for expansion, bone formation and the ability to alter genetically these cells using scAAV vectors.

Efficient transduction of vascular smooth muscle cells with a translational AAV2.5 vector: a new perspective for in-stent restenosis gene therapy.

Coronary artery disease represents the leading cause of mortality in the developed world. Percutaneous coronary intervention involving stent placement remains disadvantaged by restenosis or thrombosis. Vascular gene therapy-based methods may be approached, but lack a vascular gene delivery vector. We report a safe and efficient long-term transduction of rat carotid vessels after balloon injury intervention with a translational optimized AAV2.5 vector. Compared with other known adeno-associated virus (AAV) serotypes, AAV2.5 demonstrated the highest transduction efficiency of human coronary artery vascular smooth muscle cells (VSMCs) in vitro. Local delivery of AAV2.5-driven transgenes in injured carotid arteries resulted in transduction as soon as day 2 after surgery and persisted for at least 30 days. In contrast to adenovirus 5 vector, inflammation was not detected in AAV2.5-transduced vessels. The functional effects of AAV2.5-mediated gene transfer on neointimal thickening were assessed using the sarco/endoplasmic reticulum Ca(2+) ATPase isoform 2a (SERCA2a) human gene, known to inhibit VSMC proliferation. At 30 days, human SERCA2a messenger RNA was detected in transduced arteries. Morphometric analysis revealed a significant decrease in neointimal hyperplasia in AAV2.5-SERCA2a-transduced arteries: 28.36±11.30 (n=8) vs 77.96±24.60 (n=10) µm(2), in AAV2.5-green fluorescent protein-infected, P<0.05. In conclusion, AAV2.5 vector can be considered as a promising safe and effective vector for vascular gene therapy.

Long-term gene expression and phenotypic correction using adeno-associated virus vectors in the mammalian brain.

Adeno-associated viral (AAV) vectors are non-pathogenic, integrating DNA vectors in which all viral genes are removed and helper virus is completely eliminated. To evaluate this system in the post-mitotic cells of the brain, we found that an AAV vector containing the lacZ gene (AAVlac) resulted in expression of beta-galactosidase up to three months post-injection in vivo. A second vector expressing human tyrosine hydroxylase (AAVth) was injected into the denervated striatum of unilateral 6-hydroxydopamine-lesioned rats. Tyrosine hydroxylase (TH) immunoreactivity was detectable in striatal neurons and glia for up to four months and we also found significant behavioural recovery in lesioned rats treated with AAVth versus AAVlac controls. Safe and stable TH gene transfer into the denervated striatum may have potential for the genetic therapy of Parkinson's disease.

Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia.

Recombinant adeno-associated virus (rAAV) is a promising gene delivery vector and has recently been used in patients with hemophilia. One limitation of AAV application is that most humans have experienced wild-type AAV serotype 2 exposure, which frequently generates neutralizing antibodies (NAbs) that may inhibit rAAV2 vector transduction. Employing alternative serotypes of rAAV vectors may circumvent this problem. We investigated the development of NAbs in early childhood by examining sera gathered prospectively from 62 children with hemophilia A, participating in a multi-institutional hemophilia clinical trial (the Joint Outcome Study). Clinical applications in hemophilia therapy have been suggested for serotypes AAV2, AAV5 and AAV8, therefore NAbs against these serotypes were serially assayed over a median follow-up of 4 years. NAbs prevalence increased during early childhood for all serotypes. NAbs against AAV2 (43.5%) were observed more frequently and at higher titers compared with both AAV5 (25.8%) and AAV8 (22.6%). NAbs against AAV5 or AAV8 were rarely observed in the absence of co-prevalent and higher titer AAV2 NAbs, suggesting that NAbs to AAV5 and AAV8 were detected following AAV2 exposure due to partial cross-reactivity of AAV2-directed NAbs. The results may guide rational design of clinical trials using alternative AAV serotypes and suggest that younger patients who are given AAV gene therapy will benefit from the lower prevalence of NAbs.

Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs.

Adeno-associated viral vector 9 (AAV9) has recently been shown to penetrate the blood-brain barrier via intravascular administration, making it a good candidate for diffuse gene delivery. However, the potential side effects of systemic delivery are unknown. Intrathecal viral vector administration may be more invasive than intravenous injections, but it requires far less vector and it can be performed on an outpatient basis, making it an ideal route of delivery for clinical translation. A total of 12 domestic farm pigs (<20 kg) underwent a single-level lumbar laminectomy with intrathecal catheter placement for AAV9 delivery. Animals were perfused and the tissue was harvested 30 days after treatment. Gene expression was assessed by anti-green fluorescent protein immunohistochemistry. Although a single lumbar injection resulted in gene expression limited to the lumbar segment of the spinal cord, three consecutive boluses via a temporary catheter resulted in diffuse transduction of motor neurons (MNs) throughout the cervical, thoracic and lumbar spinal cords. We now present the first successful robust transduction of MNs in the spinal cord of a large animal via intrathecal gene delivery using a self-complementary AAV9. These promising results can be translated to many MN diseases requiring diffuse gene delivery.

Preclinical safety evaluation of subretinal AAV2.sFlt-1 in non-human primates.

We report on the long-term safety of AAV2.sFlt-1 (a recombinant adeno-associated virus serotype 2 carrying the soluble form of the Flt-1 receptor) injection into the subretinal space of non-human primates. Levels of sFlt-1 protein were significantly higher (P<0.05) in the vitreous of four out of five AAV2.sFlt-1-injected eyes. There was no evidence of damage to the eyes of animals that received subretinal injections of AAV2.sFlt-1; ocular examination showed no anterior chamber flare, normal fundus and electroretinography responses equivalent to those observed before treatment. Notably, immunological analysis demonstrated that gene therapy involving subretinal injection of AAV2.sFlt-1 does not elicit cell-mediated immunity. Biodistribution analysis showed that AAV2.sFlt-1 could be detected only in the eye and not in the other organs tested. These data indicate that gene therapy with subretinal AAV2.sFlt-1 is safe and well tolerated, and therefore promising for the long-term treatment of neovascular diseases of the eye.

The influence of epileptic neuropathology and prior peripheral immunity on CNS transduction by rAAV2 and rAAV5.

Adeno-associated virus (AAV) provides a promising platform for clinical treatment of neurological disorders owing to its established efficacy and lack of apparent pathogenicity. To use viral vectors in treating neurological disease, however, transduction must occur under neuropathological conditions. Previous studies in rodents have shown that AAV5 more efficiently transduces cells in the hippocampus and piriform cortex than AAV2. Using the kainic acid (KA) model of temporal lobe epilepsy and AAV2 and 5 carrying a hybrid chicken ß-actin promoter driving green fluorescent protein (GFP), we found that limbic seizure activity caused substantial neuropathology and resulted in a significant reduction in subsequent AAV5 transduction. Nonetheless, this reduced transduction still was greater than AAV2 transduction in control rats. Although KA seizures compromise blood-brain barrier function, potentially increasing exposure of target tissue to circulating neutralizing antibodies, we observed no interaction between KA seizure-induced damage and immunization status on AAV transduction. Finally, while we confirmed the near total neuronal-specific transgene expression for both serotypes in control rats, AAV5-GFP expression was increasingly localized to astrocytes in seizure-damaged areas. Thus, the pathological milieu of the injured brain can reduce transduction efficacy and alter viral tropism- both relevant concerns when considering viral vector gene therapy for neurological disorders.

AlphaVbeta5 integrin: a co-receptor for adeno-associated virus type 2 infection.

Understanding the primary steps of viral entry can have important implications for strategies to prevent infection of known viral pathogens as well as determining parameters for efficient gene delivery using viral vectors. Recently, a two-step process for viral infection involving attachment of virus to a primary receptor (coxsackievirus adenovirus receptor and heparan sulfate proteoglycan) and subsequent mediation of virus entry by a co-receptor (alphaV integrins and HVEM) has been determined for both adenovirus and HSV, respectively. Heparan sulfate proteoglycan serves as a primary attachment receptor for adeno-associated virus type 2 (AAV-2)(ref. 5). Here we determined that alphaVbeta5 integrin plays a part in efficient AAV infection. Experiments using the chelating agent EDTA to disrupt integrin function resulted in a corresponding decrease in AAV infection, consistent with the possibility that integrin mediates infection. Viral overlay experiments on purified plasma membrane proteins as well as immunoprecipitated integrin beta5 subunit demonstrated that AAV directly associates with the beta5 subunit of alphaVbeta5 integrin. Genetically defined cells expressing alphaVbeta5 integrin showed increased susceptibility to AAV infection, demonstrating a biological role of this integrin in AAV infection. Finally, viral binding and internalization studies indicate that alphaVbeta5 integrin is not a primary attachment receptor for AAV-2, but is instead involved in facilitating virus internalization. This study supports the idea that alphaVbeta5 integrin serves as a co-receptor for AAV-2 virions, and should have a substantial effect on the use of AAV vectors in human gene therapy.

Self-complementary AAV mediates gene targeting and enhances endonuclease delivery for double-strand break repair.

Adeno-associated virus (AAV) mediates gene targeting in humans by providing exogenous DNA for allelic replacement through homologous recombination. In comparison to other methods of DNA delivery or alternative DNA substrates, AAV gene targeting is reported to be very efficient, perhaps due to its single-stranded DNA genome, the inverted terminal repeats (ITRs), and/or the consequence of induced cellular signals on infection or uncoating. These viral attributes were investigated in the presence and absence of an I-Sce endonuclease-induced double-strand break (DSB) within a chromosomal defective reporter in human embryonic kidney cells. Gene correction was evaluated using self-complementary (sc) AAV, which forms a duplexed DNA molecule and results in earlier and robust transgene expression compared with conventional single-strand (ss) AAV genomes. An scAAV repair substrate was modestly enhanced for reporter correction showing no dependency on ssAAV genomes for this process. The AAV ITR sequences were also investigated in a plasmid repair context. No correction was noted in the absence of a DSB, however, a modest inhibitory effect correlated with the increasing presence of ITR sequences. Similarly, signaling cascades stimulated upon recombinant AAV transduction had no effect on plasmid-mediated DSB repair. Noteworthy, was the 20-fold additional enhancement in reporter correction using scAAV vectors, over ss versions, to deliver both the repair substrate and the endonuclease. In this case, homologous recombination repaired the defective reporter in 4% of cells without any selection. This report provides novel insights regarding the recombination substrates used by AAV vectors in promoting homologous recombination and points to the initial steps in vector optimization that could facilitate their use in gene correction of genetic disorders.

Delivering multiple gene products in the brain from a single adeno-associated virus vector.

For certain gene therapy applications, the simultaneous delivery of multiple genes would allow for novel therapies. In the case of adeno-associated virus (AAV) vectors, the limited packaging capacity greatly restricts current methods of carrying multiple transgene cassettes. To address this issue, a recombinant AAV (rAAV) vector was designed such that a furin proteolytic cleavage site (RKRRKR) was placed between the coding sequences of two genes (green fluorescent protein (GFP) and galanin), to allow cleavage of the chimeric protein into two fragments. In addition, these constructs contained the fibronectin secretory signal sequence that causes the gene products to be constitutively secreted from transduced cells. In vitro studies show that after transfection of HEK293 cells, the appropriate cleavage and constitutive secretion occurred regardless of the order of the genes in the transgene cassette. In vivo, infusion of rAAV vectors into the piriform cortex resulted in both GFP expression and significant galanin attenuation of kainic acid-induced seizure activity. Thus, the present results establish the utility of a proteolytic approach for the expression and secretion of multiple gene products from a single AAV vector transgene cassette.

A small regulatory element from chromosome 19 enhances liver-specific gene expression.

Tissue-specific promoters for gene therapy are typically too big for adeno-associated virus (AAV) vectors; thus, the exploration of small effective non-viral regulatory elements is of particular interest. Wild-type AAV can specifically integrate into a region on human chromosome 19 termed AAVS1. Earlier work has determined that a 347 bp fragment (Chr19) of AAVS1 has promoter and transcriptional enhancer activities. In this study, we further characterized this genetic regulation and investigated its application to AAV gene therapy in vitro and in vivo. The Chr19 347 bp fragment was dissected into three regulatory elements in human embryonic kidney cells: (i) TATA-independent promoter activity distributed throughout the fragment regardless of orientation, (ii) an orientation-dependent insulator function near the 5' end and (iii) a 107 bp enhancer region near the 3' end. The small enhancer region, coupled to the mini-CMV promoter, was used to drive the expression of several reporters following transduction by AAV2. In vivo data demonstrated enhanced transgene expression from the Chr19-mini-CMV promoter cassette after tail vein injection primarily in the liver at levels comparable to the chicken beta-actin promoter and higher than the liver-specific TTR promoter (>2-fold). However, we did not observe this increase after muscle injection, suggesting tissue-specific enhancement. All of the results support identification of a small DNA fragment (347 bp) from AAV Chr19 integration site capable of providing efficient and enhanced liver-specific transcription when used in recombinant AAV vectors.

Bioluminescent virion shells: new tools for quantitation of AAV vector dynamics in cells and live animals.

Current technologies for visualizing infectious pathways of viruses rely on fluorescent labeling of capsid proteins by chemical conjugation or genetic manipulation. For noninvasive in vivo imaging of such agents in mammalian tissue, we engineered bioluminescent Gaussia luciferase-tagged Adeno-associated viral (gLuc/AAV) vectors. The enzyme was incorporated into recombinant AAV serotypes 1, 2 and 8 capsids by fusing to the N-terminus of the VP2 capsid subunit to yield bioluminescent virion shells. The gLuc/AAV vectors were used to quantify kinetics of cell-surface-binding by AAV2 capsids in vitro. Bioluminescent virion shells displayed an exponential decrease in luminescent signal following cellular uptake in vitro. A similar trend was observed following intramuscular injection in vivo, although the rate of decline in bioluminescent signal varied markedly between AAV serotypes. gLuc/AAV1 and gLuc/AAV8 vectors displayed rapid decrease in bioluminescent signal to background levels within 30 min, whereas the signal from gLuc/AAV2 vectors persisted for over 2 h. Bioluminescent virion shells might be particularly useful in quantifying dynamics of viral vector uptake in cells and peripheral tissues in live animals.

Adeno-associated virus: integration at a specific chromosomal locus.

Recent characterization of integration of the human parvovirus, adeno-associated virus, has uncovered the exciting occurrence of targeted integration. Thus far, such specific integration has been found to be unique among the eukaryotic viruses. The molecular details of the steps involved in virus integration are actively being pursued and should yield significant information for our understanding of the mechanisms of DNA transposition.