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1.  Dicer expression is essential for adult midbrain dopaminergic neuron maintenance and survival 
The type III RNAse, Dicer, is responsible for the processing of microRNA (miRNA) precursors into functional miRNA molecules, non-coding RNAs that bind to and target messenger RNAs for repression. Dicer expression is essential for mouse midbrain development and dopaminergic (DAergic) neuron maintenance and survival during the early post-natal period. However, the role of Dicer in adult mouse DAergic neuron maintenance and survival is unknown. To bridge this gap in knowledge, we selectively knocked-down Dicer expression in individual DAergic midbrain areas, including the ventral tegmental area (VTA) and substantia nigra pars compacta (SNpc) via viral-mediated expression of Cre in adult floxed Dicer knock-in mice (Dicerflox/flox). Bilateral Dicer loss in the VTA resulted in progressive hyperactivity that was significantly reduced by the dopamine agonist, amphetamine. In contrast, decreased Dicer expression in the SNpc did not affect locomotor activity but did induce motor-learning impairment on an accelerating rotarod. Knock-down of Dicer in both midbrain regions of adult Dicerflox/flox mice resulted in preferential, progressive loss of DAergic neurons likely explaining motor behavior phenotypes. In addition, knock-down of Dicer in midbrain areas triggered neuronal death via apoptosis. Together, these data indicate that Dicer expression and, as a consequence, miRNA function, is essential for DAergic neuronal maintenance and survival in adult midbrain DAergic neuron brain areas.
doi:10.1016/j.mcn.2013.10.009
PMCID: PMC3944994  PMID: 24184162
2.  Empty Virions In AAV8 Vector Preparations Reduce Transduction Efficiency And May Cause Total Viral Particle Dose-Limiting Side-Effects 
Empty virions are inadvertent by-products of recombinant adeno-associated virus (rAAV) packaging process, resulting in vector lots with mixtures of full and empty virions at variable ratios. Impact of empty virions on the efficiency and side-effects of rAAV transduction has not been well characterized. Here, we generated partially and completely empty AAV8 virions, fully packaged rAAV8 lots as well as mixtures of empty and fully packaged virions with variable ratios of empty virions (REVs). The aforementioned dosing formulations of rAAV8 expressing either cellular (EGFP or nuclear-targeted (n) LacZ) or secreted (human α1-antitrypsin, hA1AT) reporter genes were intravenously injected into two different mouse strains, followed by analyses of transgene expressions and serum alanine aminotransferase (ALT) levels at different time points. We found that addition of empty particles to the fixed doses of rAAV8 preparations repressed liver transduction up to 64% (serum hA1AT) and 44% (nLacZ) in C57BL/6 mice, respectively. The similar trend in inhibiting EGFP expression together with concurrent elevations of serum ATL levels were observed in the BALB/c mice, indicating that empty particles may also exacerbate side-effects of rAAV8EGFP transduction. Our results suggest that removal of empty particles from rAAV preparations may improve efficacy and safety of AAV in clinical applications.
doi:10.1038/mtm.2013.9
PMCID: PMC4255953  PMID: 25485285
rAAV; empty virions; liver gene transfer; side-effect
3.  Prefrontal Cortical Dysfunction After Overexpression of Histone Deacetylase 1 
Biological psychiatry  2013;74(9):696-705.
Background
Postmortem brain studies have shown that HDAC1—a lysine deacetylase with broad activity against histones and nonhistone proteins—is frequently expressed at increased levels in prefrontal cortex (PFC) of subjects diagnosed with schizophrenia and related disease. However, it remains unclear whether upregulated expression of Hdac1 in the PFC could affect cognition and behavior.
Methods
Using adeno-associated virus, an Hdac1 transgene was expressed in young adult mouse PFC, followed by behavioral assays for working and long-term memory, repetitive activity, and response to novelty. Prefrontal cortex transcriptomes were profiled by microarray. Antipsychotic drug effects were explored in mice treated for 21 days with haloperidol or clozapine.
Results
Hdac1 overexpression in PFC neurons and astrocytes resulted in robust impairments in working memory, increased repetitive behaviors, and abnormal locomotor response profiles in novel environments. Long-term memory remained intact. Over 300 transcripts showed subtle but significant changes in Hdac1-overexpressing PFC. Major histocompatibility complex class II (MHC II)-related transcripts, including HLA-DQA1/H2-Aa, HLA-DQB1/H2-Ab1, and HLA-DRB1/H2-Eb1, located in the chromosome 6p21.3-22.1 schizophrenia and bipolar disorder risk locus, were among the subset of genes with a more robust (>1.5-fold) downregulation in expression. Hdac1 levels declined during the course of normal PFC development. Antipsychotic drug treatment, including the atypical clozapine, did not affect Hdac1 levels in PFC but induced expression of multiple MHC II transcripts.
Conclusions
Excessive HDAC1 activity, due to developmental defects or other factors, is associated with behavioral alterations and dysregulated expression of MHC II and other gene transcripts in the PFC.
doi:10.1016/j.biopsych.2013.03.020
PMCID: PMC3797203  PMID: 23664640
Bipolar disorder; gene expression; major histocompatibility complex II; prefrontal cortex; protein deacetylase; schizophrenia
4.  Gene transfer in the liver using recombinant adeno-associated virus 
Current protocols in microbiology  2013;0 14:Unit14D.6.
Liver-directed gene transfer and gene therapy are rapidly gaining attention primarily because the liver is centrally involved in a variety of metabolic functions that are affected in various inherited disorders. Recombinant adeno-associated virus (rAAV) is a popular gene delivery vehicle for gene therapy and intravenous delivery of some rAAV serotypes results in very efficient transduction of the liver. rAAV-mediated and liver-directed gene transfer can help in creating somatic transgenic animals or disease models and studying the function of various genes and miRNAs. The liver is the target tissue for gene therapy of many inborn metabolic diseases and may also be exploited as a “bio-factory” for the production of coagulation factors, insulin and growth hormones and other non-hepatic proteins. Hence efficient delivery of transgenes and small RNAs to the liver by rAAV vectors has been of long-standing interest to research scientists and clinicians alike.
doi:10.1002/9780471729259.mc14d06s29
PMCID: PMC4161158  PMID: 23686826
Adeno-associated Virus; Liver; Gene Transfer; Gene therapy
5.  Vector Sequences Are Not Detected in Tumor Tissue from Research Subjects with Ornithine Transcarbamylase Deficiency Who Previously Received Adenovirus Gene Transfer 
Human Gene Therapy  2013;24(9):814-819.
Abstract
A 66-year-old woman heterozygous for a mutation in the ornithine transcarbamylase gene (Otc) participated in a phase I gene therapy trial for OTC deficiency. She received an adenovirus (Ad) vector expressing the functional OTC gene by intraportal perfusion. Fourteen years later she developed and subsequently died of hepatocellular carcinoma. A second subject, a 45-year-old woman, enrolled in the same trial presented with colon cancer 15 years later. We sought to investigate a possible association between the development of a tumor and prior adenoviral gene transfer in these two subjects. We developed and validated a sensitive nested polymerase chain reaction assay for recovering recombinant Ad sequences from host tissues. Using this method, we could not detect any Ad vector DNA in either tumor or normal tissue from the two patients. Our results are informative in ruling out the possibility that the adenoviral vector might have contributed to the development of cancer in those two subjects.
Zhong and colleagues use a nested PCR assay to examine whether adenoviral gene transfer led to the development of cancer in two clinical research subjects with ornithine transcarbamylase deficiency. Using this approach, they were unable to detect adenovirus vector genomes in normal or tumor tissues from the subjects more than a decade after vector infusion. These results suggest that adenoviral gene transfer was an unlikely contributor to the colon cancer or hepatocellular carcinoma observed in the two research subjects.
doi:10.1089/hum.2013.118
PMCID: PMC3768231  PMID: 24010702
6.  AAV-mediated gene transfer to the mouse CNS 
Current protocols in microbiology  2013;0 14:Unit14D.5.
Recombinant adeno associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. Recombinant AAVs have been used at various ages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the CNS, depending on the stage of development of the mouse. In neonates, intravascular injections into the facial vein are often used. In adults, direct injections into target regions of the brain are achieved with great spatiotemporal control through stereotaxic surgeries. Recently, discoveries of new AAV vectors with the ability to cross the blood brain barrier have made it possible to also target the adult CNS by intravascular injections. rAAVs have been successfully used as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway.
doi:10.1002/9780471729259.mc14d05s29
PMCID: PMC4135440  PMID: 23686825
AAV; intracranial; intravascular; neonate; sterotaxic
7.  The potential of adeno-associated viral vectors for gene delivery to muscle tissue 
Expert opinion on drug delivery  2014;11(3):345-364.
Introduction
Muscle-directed gene therapy is rapidly gaining attention primarily because muscle is an easily accessible target tissue and is also associated with various severe genetic disorders. Localized and systemic delivery of recombinant adeno-associated virus (rAAV) vectors of several serotypes results in very efficient transduction of skeletal and cardiac muscles, which has been achieved in both small and large animals, as well as in humans. Muscle is the target tissue in gene therapy for many muscular dystrophy diseases, and may also be exploited as a biofactory to produce secretory factors for systemic disorders. Current limitations of using rAAVs for muscle gene transfer include vector size restriction, potential safety concerns such as off-target toxicity and the immunological barrier composing of pre-existing neutralizing antibodies and CD8+ T-cell response against AAV capsid in humans.
Areas covered
In this article, we will discuss basic AAV vector biology and its application in muscle-directed gene delivery, as well as potential strategies to overcome the aforementioned limitations of rAAV for further clinical application.
Expert opinion
Delivering therapeutic genes to large muscle mass in humans is arguably the most urgent unmet demand in treating diseases affecting muscle tissues throughout the whole body. Muscle-directed, rAAV-mediated gene transfer for expressing antibodies is a promising strategy to combat deadly infectious diseases. Developing strategies to circumvent the immune response following rAAV administration in humans will facilitate clinical application.
doi:10.1517/17425247.2014.871258
PMCID: PMC4098646  PMID: 24386892
adeno-associated virus; gene therapy; gene transfer; muscle
8.  Recombinant Adeno-Associated Virus Integration Sites in Murine Liver After Ornithine Transcarbamylase Gene Correction 
Human Gene Therapy  2013;24(5):520-525.
Abstract
Recombinant adeno-associated viruses (rAAVs) have been tested in humans and other large mammals without adverse events. However, one study of mucopolysaccharidosis VII correction in mice showed repeated integration of rAAV in cells from hepatocellular carcinoma (HCC) in the Dlk1–Dio3 locus, suggesting possible insertional mutagenesis. In contrast, another study found no association of rAAV integration with HCC, raising questions about the generality of associations between liver transformation and integration at Dlk1–Dio3. Here we report that in rAAV-treated ornithine transcarbamylase (Otc)–deficient mice, four examples of integration sites in Dlk1–Dio3 could be detected in specimens from liver nodule/tumors, confirming previous studies of rAAV integration in the Dlk1–Dio3 locus in the setting of another murine model of metabolic disease. In one case, the integrated vector was verified to be present at about one copy per cell, consistent with clonal expansion. Another verified integration site in liver nodule/tumor tissue near the Tax1bp1 gene was also detected at about one copy per cell. The Dlk1–Dio3 region has also been implicated in human HCC and so warrants careful monitoring in ongoing human clinical trials with rAAV vectors.
Zhong and colleagues use deep sequencing to examine the distribution of integrated AAV genomes in nodules/tumors from the livers of ornithine transcarbamylace (Otc) deficient mice. Using this approach, they report four examples of integration sites in the Dlk1-Dio3 locus; this site has previously been implicated in human hepatocellular carcinoma and may warrant careful monitoring in human clinical trials using AAV vectors.
doi:10.1089/hum.2012.112
PMCID: PMC3655627  PMID: 23621841
9.  Targeted Intraceptor Nanoparticle Therapy Reduces Angiogenesis and Fibrosis in Primate & Murine Macular Degeneration 
ACS nano  2013;7(4):3264-3275.
Monthly intraocular injections are widely used to deliver protein-based drugs that cannot cross the blood-retina barrier for the treatment of leading blinding diseases such as age-related macular degeneration (AMD). This invasive treatment carries significant risks, including bleeding, pain, infection, and retinal detachment. Further, current therapies are associated with a rate of retinal fibrosis and geographic atrophy significantly higher than that which occurs in the described natural history of AMD. A novel therapeutic strategy which improves outcomes in a less invasive manner, reduces risk, and provides long-term inhibition of angiogenesis and fibrosis is a felt medical need. Here we show that a single intravenous injection of targeted, biodegradable nanoparticles delivering a recombinant Flt23k intraceptor plasmid homes to neovascular lesions in the retina and regresses CNV in primate and murine AMD models. Moreover, this treatment suppressed subretinal fibrosis, which is currently not addressed by clinical therapies. Murine vision, as tested by OptoMotry©, significantly improved with nearly 40% restoration of visual loss induced by CNV. We found no evidence of ocular or systemic toxicity from nanoparticle treatment. These findings offer a nanoparticle-based platform for targeted, vitreous-sparing, extended-release, nonviral gene therapy.
doi:10.1021/nn305958y
PMCID: PMC3634882  PMID: 23464925
nanoparticles; gene delivery; choroidal neovascularization models; anti-VEGF therapy; angiogenesis
10.  Gene Transfer in Skeletal and Cardiac Muscle Using Recombinant Adeno-Associated Virus 
Current protocols in microbiology  2013;0 14:Unit-14D.3.
Adeno-associated virus (AAV) is a DNA virus with a small (~4.7kb) single-stranded genome. It is a naturally replication-defective parvovirus of the dependovirus group. Recombinant AAV (rAAV), for use as a gene transfer vector, is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. Only the viral inverted terminal repeats (ITRs) are required in cis for replication and packaging during production. The ITRs are also necessary and sufficient for vector genome processing and persistence during transduction. The tissue tropism of the rAAV vector is determined by the AAV capsid. In this unit we will discuss several methods to deliver rAAV in order to transduce cardiac and/or skeletal muscle, including: intravenous delivery, intramuscular delivery, isolated limb infusion, intrapericardial injection in neonatal mice, and left ventricular wall injection in adult rats.
doi:10.1002/9780471729259.mc14d03s28
PMCID: PMC3641885  PMID: 23408131
Adeno-associated virus; gene therapy; heart; skeletal muscle; vector delivery
11.  Retinal gene delivery by rAAV and DNA electroporation 
Current protocols in microbiology  2013;0 14:Unit-14D.4.
Ocular gene therapy is a fast growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged, easily accessible, and direct viral delivery results primarily in local infection. Because the eye is not a vital organ, mutations in eye specific genes tend to be more common. To date, over 40 eye specific genes have been identified which harbor mutations that lead to blindness. Gene therapy with recombinant Adeno Associated Virus (rAAV) holds the promise to treat patients with such mutations. However, proof-of-concept and safety evaluation for gene therapy remains to be established for most of these diseases. This unit describes the in vivo delivery of genes to the mouse eye by rAAV-mediated gene transfer and plasmid DNA electroporation. Advantages and limitations of these methods are discussed, and detailed protocols for gene delivery, required materials, as well as subsequent tissue processing methods are described.
doi:10.1002/9780471729259.mc14d04s28
PMCID: PMC3643812  PMID: 23408132
Retina; eye; gene therapy; gene transfer
12.  Gene Transfer in the Lung Using Recombinant Adeno-Associated Virus 
Current protocols in microbiology  2012;CHAPTER:Unit14D.2.
Adeno-associated virus (AAV) in a small replication deficient DNA virus belonging to the Parvovirinae family. It has a single-stranded approximately 4.7kb genome. Recombinant AAV (rAAV) is created by replacing the viral rep and cap genes with the transgene of interest along with promoter and polyadenylation sequences. The short viral inverted terminal repeats must remain intact for replication and packaging in production as well as vector genome processing and persistence in the transduction process. The AAV capsid (serotype) determines the tissue tropism of the rAAV vector. In this unit we will discuss serotype selection for lung targeting along with the factors effecting efficient delivery of rAAV vectors to the murine lung. Detailed procedures for lung delivery (intranasal, orotracheal, and surgical tracheal injection), sample collection and post-mortem tissue processing will be described.
doi:10.1002/9780471729259.mc14d02s26
PMCID: PMC3486738  PMID: 22875566
Adeno-associated virus; vector delivery; lung; gene therapy
13.  SU9518 Inhibits Proliferative Vitreoretinopathy in Fibroblast and Genetically Modified Müller Cell–Induced Rabbit Models 
Purpose.
Proliferative vitreoretinopathy (PVR) is a complication of retinal detachment that can lead to surgical failure and vision loss. Previous studies suggest that a variety of retinal cells, including RPE and Müller glia, may be responsible. Platelet-derived growth factor receptor alpha (PDGFRα) has been strongly implicated in the pathogenesis, and found to be intrinsic to the development of PVR in rabbit models. We examine whether SU9518, a tyrosine kinase inhibitor with PDGFRα specificity, can inhibit the development of PVR in fibroblast and Müller cell rabbit models of PVR.
Methods.
SU9518 was injected in rabbit eyes along with fibroblasts, Müller cells (MIO-M1), or Müller cells transfected to increase their expression of PDGFRα (MIO-M1α). Indirect ophthalmoscopy and histopathology were used to assess efficacy and toxicity.
Results.
SU9518 was an effective inhibitor of PVR in both fibroblast and Müller cell models of PVR. No toxic effects were identified by indirect ophthalmoscopy or histopathology.
Conclusions.
SU9518 is an effective and safe inhibitor of PVR in rabbit models, and could potentially be used in humans for the treatment of this and other proliferative diseases of the retina involving fibrosis and gliosis. Further animal studies need to be performed to examine retinal toxicity and sustained delivery mechanisms.
Proliferative vitreoretinopathy is a challenging complication of retinal detachment that often leads to surgical failure and vision loss. Tyrosine kinase inhibitors appear promising for the treatment of this disease and other diseases of the retina involving fibrosis.
doi:10.1167/iovs.12-10320
PMCID: PMC3597189  PMID: 23341018
14.  Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1 
eLife  2013;2:e00324.
Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life.
DOI: http://dx.doi.org/10.7554/eLife.00324.001
eLife digest
The inner surface of the vertebrate eye is lined with a multilayered structure known as the retina. The bottom layer of the retina is composed of rods and cones—neurons that are directly sensitive to light—and is called the photoreceptor layer. Rods function primarily in dim light and provide black-and-white vision, while cones support daytime vision and are responsible for colour perception. Unlike the upper layers of the retina, the photoreceptor layer does not contain blood vessels: oxygen and nutrients are instead provided by a structure just underneath the retina called the choroid.
The eye relies on the rods and cones converting light into electrical signals, and the photoreceptor layer must remain free of blood vessels for this process to work properly. If blood vessels extend into the photoreceptor layer from rest of the retina (which is above it) or the choroid (below), they can disrupt the retina and give rise to a condition called age-related macular degeneration, which is a leading cause of irreversible blindness in adults.
Within the eye, the development of new blood vessels from pre-existing vessels is stimulated by a protein known as vascular endothelial growth factor A (VEGF-A), while an inhibitor protein called sFLT-1 prevents the growth of new blood vessels in the other tissues of the eye like the cornea. However, it has not been clear what keeps the photoreceptor layer (and also the cells that support the photoreceptor layer) free of blood vessels, and what happens to disrupt this process of vascular demarcation in age-related macular degeneration.
Now, Luo et al. reveal that cells in the photoreceptor layer produce sFLT-1, and that the levels of this protein are indeed reduced in people with age-related macular degeneration. Using genetic and pharmacological methods, they show that a reduction in sFLT-1 triggers blood vessels to grow into the photoreceptor layer from above or below. Luo et al. also report two new genetic mouse models in which blood vessels form spontaneously in the photoreceptor layer at an early age, which should prove useful for further research into age-related macular degeneration.
DOI: http://dx.doi.org/10.7554/eLife.00324.002
doi:10.7554/eLife.00324
PMCID: PMC3687373  PMID: 23795287
age-related macular degeneration; photoreceptor metabolism; retinal vasculature; soluble VEGF receptor-1; vascular demarcation; transgenic model; Human; Mouse
15.  High-Efficiency Transduction of Primary Human Hematopoietic Stem Cells and Erythroid Lineage-Restricted Expression by Optimized AAV6 Serotype Vectors In Vitro and in a Murine Xenograft Model In Vivo 
PLoS ONE  2013;8(3):e58757.
We have observed that of the 10 AAV serotypes, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs), and that the transduction efficiency can be further increased by specifically mutating single surface-exposed tyrosine (Y) residues on AAV6 capsids. In the present studies, we combined the two mutations to generate a tyrosine double-mutant (Y705+731F) AAV6 vector, with which >70% of CD34+ cells could be transduced. With the long-term objective of developing recombinant AAV vectors for the potential gene therapy of human hemoglobinopathies, we generated the wild-type (WT) and tyrosine-mutant AAV6 vectors containing the following erythroid cell-specific promoters: β-globin promoter (βp) with the upstream hyper-sensitive site 2 (HS2) enhancer from the β-globin locus control region (HS2-βbp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-βp, and increased up to 30-fold and up to 20-fold, respectively, following erythropoietin (Epo)-induced differentiation of CD34+ cells in vitro. Transgene expression from the B19p6 or the HS2-βp was also evaluated in an immuno-deficient xenograft mouse model in vivo. Whereas low levels of expression were detected from the B19p6 in the WT AAV6 capsid, and that from the HS2-βp in the Y705+731F AAV6 capsid, transgene expression from the B19p6 promoter in the Y705+731F AAV6 capsid was significantly higher than that from the HS2-βp, and was detectable up to 12 weeks post-transplantation in primary recipients, and up to 6 additional weeks in secondary transplanted animals. These data demonstrate the feasibility of the use of the novel Y705+731F AAV6-B19p6 vectors for high-efficiency transduction of HSCs as well as expression of the b-globin gene in erythroid progenitor cells for the potential gene therapy of human hemoglobinopathies such as β-thalassemia and sickle cell disease.
doi:10.1371/journal.pone.0058757
PMCID: PMC3597592  PMID: 23516552
17.  Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates 
Molecular Genetics and Metabolism  2011;104(3):395-403.
Gene transfer vectors based on adeno-associated virus 8 (AAV8) are highly efficient in liver transduction and can be easily administered by intravenous injection. In mice, AAV8 transduces predominantly hepatocytes near central veins and yields lower transduction levels in hepatocytes in periportal regions. This transduction bias has important implications for gene therapy that aims to correct metabolic liver enzymes because metabolic zonation along the porto-central axis requires the expression of therapeutic proteins within the zone where they are normally localized.
In the present study we compared the expression pattern of AAV8 expressing green fluorescent protein (GFP) in liver between mice, dogs, and non-human primates. We confirmed the pericentral dominance in transgene expression in mice with AAV8 when the liver-specific thyroid hormone-binding globulin (TBG) promoter was used but also observed the same expression pattern with the ubiquitous chicken β-actin (CB) and cytomegalovirus (CMV) promoters, suggesting that transduction zonation is not caused by promoter specificity. Predominantly pericentral expression was also found in dogs injected with AAV8. In contrast, in cynomolgus and rhesus macaques the expression pattern from AAV vectors was reversed, i.e. transgene expression was most intense around portal areas and less intense or absent around central veins. Infant rhesus macaques as well as newborn mice injected with AAV8 however showed a random distribution of transgene expression with neither portal nor central transduction bias. Based on the data in monkeys, adult humans treated with AAV vectors are predicted to also express transgenes predominantly in periportal regions whereas infants are likely to show a uniform transduction pattern in liver.
doi:10.1016/j.ymgme.2011.06.002
PMCID: PMC3269907  PMID: 21778099
gene therapy; AAV; liver; animal models
18.  Long-term, efficient inhibition of microRNA function in mice using rAAV vectors 
Nature methods  2012;9(4):403-409.
Understanding the function of individual microRNA (miRNA) species in mice would require the production of hundreds of loss-of-function strains. To accelerate analysis of miRNA biology in mammals, we combined recombinant adeno-associated virus (rAAV) vectors with miRNA `Tough Decoys' (TuDs) to inhibit specific miRNAs. Intravenous injection of rAAV9 expressing anti-miR-122 or anti-let-7 TuD depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122—but not anti-let-7—TuD reduced serum cholesterol by >30% for 25 weeks in wild-type mice. High throughput sequencing of liver miRNAs from the treated mice confirmed that the targeted miRNAs were depleted and revealed that TuD RNAs induce miRNA tailing and trimming in vivo. rAAV-mediated miRNA inhibition thus provides a simple way to study miRNA function in adult mammals and a potential therapy for dyslipidemia and other diseases caused by miRNA deregulation.
doi:10.1038/nmeth.1903
PMCID: PMC3420816  PMID: 22388288
19.  Cardiac Gene Transfer of Short Hairpin RNA Directed Against Phospholamban Effectively Knocks Down Gene Expression but Causes Cellular Toxicity in Canines 
Human Gene Therapy  2011;22(8):969-977.
Abstract
Derangements in calcium cycling have been described in failing hearts, and preclinical studies have suggested that therapies aimed at correcting this defect can lead to improvements in cardiac function and survival. One strategy to improve calcium cycling would be to inhibit phospholamban (PLB), the negative regulator of SERCA2a that is upregulated in failing hearts. The goal of this study was to evaluate the safety and efficacy of using adeno-associated virus (AAV)-mediated cardiac gene transfer of short hairpin RNA (shRNA) to knock down expression of PLB. Six dogs were treated with self-complementary AAV serotype 6 (scAAV6) expressing shRNA against PLB. Three control dogs were treated with empty AAV6 capsid, and two control dogs were treated with scAAV6 expressing dominant negative PLB. Vector was delivered via a percutaneously inserted cardiac injection catheter. PLB mRNA and protein expression were analyzed in three of six shRNA dogs between days 16 and 26. The other three shRNA dogs and five control dogs were monitored long-term to assess cardiac safety. PLB mRNA was reduced 16-fold, and PLB protein was reduced 5-fold, with treatment. Serum troponin elevation and depressed cardiac function were observed in the shRNA group only at 4 weeks. An enzyme-linked immunospot assay failed to detect any T cells reactive to AAV6 capsid in peripheral blood mononuclear cells, heart, or spleen. Microarray analysis revealed alterations in cardiac expression of several microRNAs with shRNA treatment. AAV6-mediated cardiac gene transfer of shRNA effectively knocks down PLB expression but is associated with severe cardiac toxicity. Toxicity may result from dysregulation of endogenous microRNA pathways.
In this preclinical study, Bish and colleagues report that adeno-associated virus serotype 6 (AAV6)-mediated expression of short hairpin RNA (shRNA) directed against phospholamban (PLB), a regulator of heart failure (HF), is effective at knocking down PLB expression. Yet, safety assessments revealed that healthy canines treated with shRNA, but not empty AAV6 capsid, experienced serum cardiac troponin I elevation, cardiac dysfunction, and alteration of cardiac microRNA expression, suggesting that this approach may not be a feasible therapeutic strategy.
doi:10.1089/hum.2011.035
PMCID: PMC3159526  PMID: 21542669
20.  Evaluation of Adeno-Associated Viral Vectors for Liver-Directed Gene Transfer in Dogs 
Human Gene Therapy  2011;22(8):985-997.
Abstract
This study evaluated six adeno-associated viral (AAV) vectors expressing green fluorescent protein (GFP) from the liver-specific thyroid hormone–binding globulin (TBG) promoter made with novel capsids in canine liver-directed gene transfer. Studies in 1.5-month-old dogs, which were administered vector through a peripheral vein, showed that AAV8 capsid vectors had the most favorable performance profiles. Interestingly, the absolute levels of hepatocyte transduction achieved with AAV8 were lower in dogs compared with what had been achieved in mice and nonhuman primates. Additional studies were performed with AAV8 delivered into the hepatic artery in adult dogs, with higher doses of vector used to assess potential dose-limiting toxicities. These studies showed good transduction on day 7 in one dog that apparently was lost by day 28 in another dog through the generation of GFP-specific T cells. Each adult dog was carefully monitored for any hemodynamic changes associated with vector infusion. Both animals demonstrated mild to moderate hypotension and bradycardia, which appeared to be anesthesia-related, making it difficult to evaluate contributions of the vector.
In this study, six clinically promising AAV serotypes were evaluated in dogs, using the same liver-specific promoter (TBG) and dose (3 × 1012 GC/kg). AAV8 emerged as the best performing serotype in liver, as observed previously in mice and nonhuman primates. However, AAV8 transgene expression levels were lower and less persistent in dogs than in other animal models.
doi:10.1089/hum.2010.194
PMCID: PMC3159528  PMID: 21204705
21.  Development and application of an antigen capture ELISA assay for diagnosis of Japanese encephalitis virus in swine, human and mosquito 
Virology Journal  2012;9:4.
Background
Japanese encephalitis (JE) is a serious zoonosis caused by the Japanese encephalitis virus (JEV) which is a mosquito-borne pathogen of the family Flavivirus. However, the application of several developed laboratory methods for the detection of JEV antigens or antibodies are limited by their requirements of laboratory operations, skilled technicians and special facilities.
Results
To develop a method for detecting JEV antigen in swine, human, mosquito and other clinical specimens specifically, conveniently and effectively, an antigen capture enzyme-linked immunosorbent assay (ELISA) was established in this study. Sensitivity, specificity, repeatability and stability of the developed method were evaluated, and 60 clinical samples were tested in this study. The results demonstrated that the antigen capture ELISA was capable in detecting JEV antigen with high sensitivity and specificity compared with conventional methods. 14 samples showed the positive result with coincidence rate of 70%, and 46 displayed negative result with coincidence rate of 100% as compared to that of reverse transcription-polymerase chain reaction (RT-PCR).
Conclusions
The developed ELISA assay provides a convenient and specific method for the large-scale determination of JEV antigen in infected swine, human and mosquito samples with high sensitivity and specificity.
doi:10.1186/1743-422X-9-4
PMCID: PMC3275457  PMID: 22221768
Japanese encephalitis; Japanese encephalitis virus; Monoclonal antibody; Polyclonal antibody; Antigen capture ELISA
22.  Percutaneous transendocardial delivery of self-complementary adeno-associated virus 6 achieves global cardiac gene transfer in canines 
Achieving efficient cardiac gene transfer in a large animal model has proven to be technically challenging. Prior strategies have employed cardio-pulmonary bypass or dual catheterization with the aid of vasodilators to deliver vectors, such as adenovirus, adeno-associated virus or plasmid DNA. While single stranded adeno-associated virus vectors have shown the greatest promise, they suffer from delayed expression, which might be circumvented by using self-complementary vectors. We sought to optimize cardiac gene transfer using a percutaneous transendocardial injection catheter to deliver adeno-associated virus vectors to the canine myocardium. Four vectors were evaluated—single stranded adeno-associated virus 9, self-complementary adeno-associated virus 9, self-complementary adeno-associated virus 8, self-complementary adeno-associated virus 6—so that comparison could be made between single stranded and self complementary vectors as well as among serotypes 9, 8, and 6. We demonstrate that self-complementary adeno-associated virus is superior to single stranded adeno-associated virus and that adeno-associated virus 6 is superior to other serotypes evaluated. Biodistribution studies revealed that vector genome copies were 15 to 4000 times more abundant in the heart than in any other organ for self-complementary adeno-associated virus 6. Percutaneous transendocardial injection of self-complementary adeno-associated virus 6 is a safe, effective method for achieving efficient cardiac gene transfer.
doi:10.1038/mt.2008.202
PMCID: PMC3241935  PMID: 18813281
23.  MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression 
Recombinant adeno-associated viruses (rAAVs) that can cross the blood-brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5′ and 3′ mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications.
doi:10.1038/mt.2010.279
PMCID: PMC3048189  PMID: 21179009
rAAV; intravascular delivery; CNS transduction; miRNA-binding site; miRNA regulation
24.  Molecular Analysis of Vector Genome Structures After Liver Transduction by Conventional and Self-Complementary Adeno-Associated Viral Serotype Vectors in Murine and Nonhuman Primate Models 
Human Gene Therapy  2010;21(6):750-761.
The authors report results from a systematic molecular analysis comparing the liver transduction efficiency of single-stranded and self-complementary AAV vectors in mice and nonhuman primates at early and late stages of gene transfer.
Abstract
Vectors based on several new adeno-associated viral (AAV) serotypes demonstrated strong hepatocyte tropism and transduction efficiency in both small- and large-animal models for liver-directed gene transfer. Efficiency of liver transduction by AAV vectors can be further improved in both murine and nonhuman primate (NHP) animals when the vector genomes are packaged in a self-complementary (sc) format. In an attempt to understand potential molecular mechanism(s) responsible for enhanced transduction efficiency of the sc vector in liver, we performed extensive molecular studies of genome structures of conventional single-stranded (ss) and sc AAV vectors from liver after AAV gene transfer in both mice and NHPs. These included treatment with exonucleases with specific substrate preferences, single-cutter restriction enzyme digestion and polarity-specific hybridization-based vector genome mapping, and bacteriophage ϕ29 DNA polymerase-mediated and double-stranded circular template-specific rescue of persisted circular genomes. In mouse liver, vector genomes of both genome formats seemed to persist primarily as episomal circular forms, but sc vectors converted into circular forms more rapidly and efficiently. However, the overall differences in vector genome abundance and structure in the liver between ss and sc vectors could not account for the remarkable differences in transduction. Molecular structures of persistent genomes of both ss and sc vectors were significantly more heterogeneous in macaque liver, with noticeable structural rearrangements that warrant further characterizations.
doi:10.1089/hum.2009.214
PMCID: PMC2938357  PMID: 20113166
25.  MicroRNA-regulated, Systemically Delivered rAAV9: A Step Closer to CNS-restricted Transgene Expression 
Molecular Therapy  2010;19(3):526-535.
Recombinant adeno-associated viruses (rAAVs) that can cross the blood–brain-barrier and achieve efficient and stable transvascular gene transfer to the central nervous system (CNS) hold significant promise for treating CNS disorders. However, following intravascular delivery, these vectors also target liver, heart, skeletal muscle, and other tissues, which may cause untoward effects. To circumvent this, we used tissue-specific, endogenous microRNAs (miRNAs) to repress rAAV expression outside the CNS, by engineering perfectly complementary miRNA-binding sites into the rAAV9 genome. This approach allowed simultaneous multi-tissue regulation and CNS-directed stable transgene expression without detectably perturbing the endogenous miRNA pathway. Regulation of rAAV expression by miRNA was primarily via site-specific cleavage of the transgene mRNA, generating specific 5′ and 3′ mRNA fragments. Our findings promise to facilitate the development of miRNA-regulated rAAV for CNS-targeted gene delivery and other applications.
doi:10.1038/mt.2010.279
PMCID: PMC3048189  PMID: 21179009

Results 1-25 (37)