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Find video protocols related to scientific articles indexed in Pubmed.
Selective in vivo targeting of human liver tumors by optimized AAV3 vectors in a murine xenograft model.
Hum. Gene Ther.
PUBLISHED: 10-09-2014
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Current challenges for recombinant adeno-associated virus (rAAV) vector-based cancer treatment include the low-efficiency and the lack of specificity in vivo. rAAV serotype 3 (rAAV3) vectors have previously been shown to be ineffective in normal mouse tissues following systemic administration. In the present study, we report that rAAV3 vectors can efficiently target and transduce various human liver cancer cells in vivo. Elimination of specific surface-exposed serine and threonine residues on rAAV3 capsids results in further augmentation in the transduction efficiency of these vectors, without any change in the viral tropism and cellular receptor interactions. In addition, we have identified a potential chemotherapy drug, shikonin, as a multifunctional compound to inhibit liver tumor growth as well as to significantly enhance the efficacy of rAAV vector-based gene therapy in vivo. Furthermore, we also document that suppression of tumorigenesis in a human liver cancer xenograft model can be achieved through systemic administration of the optimized rAAV3 vectors carrying a therapeutic gene, and shikonin at a dose that does not lead to severe liver damage. Our research provides a novel means to achieve not only targeted delivery, but also the potential for gene therapy of human liver cancer.
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Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.
Hum. Gene Ther.
PUBLISHED: 10-03-2014
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Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
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State-of-the-art human gene therapy: part II. Gene therapy strategies and clinical applications.
Discov Med
PUBLISHED: 09-18-2014
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In Part I of this Review (Wang and Gao, 2014), we introduced recent advances in gene delivery technologies and explained how they have powered some of the current human gene therapy applications. In Part II, we expand the discussion on gene therapy applications, focusing on some of the most exciting clinical uses. To help readers to grasp the essence and to better organize the diverse applications, we categorize them under four gene therapy strategies: (1) gene replacement therapy for monogenic diseases, (2) gene addition for complex disorders and infectious diseases, (3) gene expression alteration targeting RNA, and (4) gene editing to introduce targeted changes in host genome. Human gene therapy started with the simple idea that replacing a faulty gene with a functional copy can cure a disease. It has been a long and bumpy road to finally translate this seemingly straightforward concept into reality. As many disease mechanisms unraveled, gene therapists have employed a gene addition strategy backed by a deep knowledge of what goes wrong in diseases and how to harness host cellular machinery to battle against diseases. Breakthroughs in other biotechnologies, such as RNA interference and genome editing by chimeric nucleases, have the potential to be integrated into gene therapy. Although clinical trials utilizing these new technologies are currently sparse, these innovations are expected to greatly broaden the scope of gene therapy in the near future.
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State-of-the-art human gene therapy: part I. Gene delivery technologies.
Discov Med
PUBLISHED: 08-06-2014
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Safe and effective gene delivery is a prerequisite for successful gene therapy. In the early age of human gene therapy, setbacks due to problematic gene delivery vehicles plagued the exciting therapeutic outcome. However, gene delivery technologies rapidly evolved ever since. With the advancement of gene delivery techniques, gene therapy clinical trials surged during the past decade. As the first gene therapy product (Glybera) has obtained regulatory approval and reached clinic, human gene therapy finally realized the promise that genes can be medicines. The diverse gene delivery techniques available today have laid the foundation for gene therapy applications in treating a wide range of human diseases. Some of the most urgent unmet medical needs, such as cancer and pandemic infectious diseases, have been tackled by gene therapy strategies with promising results. Furthermore, combining gene transfer with other breakthroughs in biomedical research and novel biotechnologies opened new avenues for gene therapy. Such innovative therapeutic strategies are unthinkable until now, and are expected to be revolutionary. In part I of this review, we introduced recent development of non-viral and viral gene delivery technology platforms. As cell-based gene therapy blossomed, we also summarized the diverse types of cells and vectors employed in ex vivo gene transfer. Finally, challenges in current gene delivery technologies for human use were discussed.
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Global CNS transduction of adult mice by intravenously delivered rAAVrh.8 and rAAVrh.10 and nonhuman primates by rAAVrh.10.
Mol. Ther.
PUBLISHED: 04-15-2014
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Some recombinant adeno-associated viruses (rAAVs) can cross the neonatal blood-brain barrier (BBB) and efficiently transduce cells of the central nervous system (CNS). However, in the adult CNS, transduction levels by systemically delivered rAAVs are significantly reduced, limiting their potential for CNS gene therapy. Here, we characterized 12 different rAAVEGFPs in the adult mouse CNS following intravenous delivery. We show that the capability of crossing the adult BBB and achieving widespread CNS transduction is a common character of AAV serotypes tested. Of note, rAAVrh.8 is the leading vector for robust global transduction of glial and neuronal cell types in regions of clinical importance such as cortex, caudate-putamen, hippocampus, corpus callosum, and substantia nigra. It also displays reduced peripheral tissue tropism compared to other leading vectors. Additionally, we evaluated rAAVrh.10 with and without microRNA (miRNA)-regulated expressional detargeting from peripheral tissues for systemic gene delivery to the CNS in marmosets. Our results indicate that rAAVrh.8, along with rh.10 and 9, hold the best promise for developing novel therapeutic strategies to treat neurological diseases in the adult patient population. Additionally, systemically delivered rAAVrh.10 can transduce the CNS efficiently, and its transgene expression can be limited in the periphery by endogenous miRNAs in adult marmosets.
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Comparison of the efficacy of five adeno-associated virus vectors for transducing dorsal raphé nucleus cells in the mouse.
J. Neurosci. Methods
PUBLISHED: 04-02-2014
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Delivery of genes to various brain regions can be accomplished using serotype 2 of the adeno-associated virus (AAV). Pseudotype AAV2 vectors, composed of the AAV2 genome packaged in the capsid of an alternative serotype, have increased efficiency of viral transduction. Transduction of pseudotype AAV2 vectors depends on cell type, brain region and stage of development. The dorsal raphé nucleus (DRN) and median raphé provides the majority of serotonin to forebrain regions and are implicated in the pathology and treatment of depression and anxiety. Viral vector technology in combination with stereotaxic surgery in mice provides a means to differentiate gene function in the DRN compared to the median raphé nucleus.
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The PPAR?-FGF21 hormone axis contributes to metabolic regulation by the hepatic JNK signaling pathway.
Cell Metab.
PUBLISHED: 03-03-2014
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The cJun NH2-terminal kinase (JNK) stress signaling pathway is implicated in the metabolic response to the consumption of a high-fat diet, including the development of obesity and insulin resistance. These metabolic adaptations involve altered liver function. Here, we demonstrate that hepatic JNK potently represses the nuclear hormone receptor peroxisome proliferator-activated receptor ? (PPAR?). Therefore, JNK causes decreased expression of PPAR? target genes that increase fatty acid oxidation and ketogenesis and promote the development of insulin resistance. We show that the PPAR? target gene fibroblast growth factor 21 (Fgf21) plays a key role in this response because disruption of the hepatic PPAR?-FGF21 hormone axis suppresses the metabolic effects of JNK deficiency. This analysis identifies the hepatokine FGF21 as a critical mediator of JNK signaling in the liver.
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Subretinal delivery of AAV2-mediated human erythropoietin gene is protective and safe in experimental diabetic retinopathy.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 02-11-2014
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We studied and developed a gene-based intraocular erythropoietin (EPO) therapy for diabetic retinopathy (DR), by which the applicability of neuroprotective therapy with favorable safety profile is attempted.
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Nutrient sensing by the mitochondrial transcription machinery dictates oxidative phosphorylation.
J. Clin. Invest.
PUBLISHED: 01-16-2014
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Sirtuin 3 (SIRT3), an important regulator of energy metabolism and lipid oxidation, is induced in fasted liver mitochondria and implicated in metabolic syndrome. In fasted liver, SIRT3-mediated increases in substrate flux depend on oxidative phosphorylation (OXPHOS), but precisely how OXPHOS meets the challenge of increased substrate oxidation in fasted liver remains unclear. Here, we show that liver mitochondria in fasting mice adapt to the demand of increased substrate oxidation by increasing their OXPHOS efficiency. In response to cAMP signaling, SIRT3 deacetylated and activated leucine-rich protein 130 (LRP130; official symbol, LRPPRC), promoting a mitochondrial transcriptional program that enhanced hepatic OXPHOS. Using mass spectrometry, we identified SIRT3-regulated lysine residues in LRP130 that generated a lysine-to-arginine (KR) mutant of LRP130 that mimics deacetylated protein. Compared with wild-type LRP130 protein, expression of the KR mutant increased mitochondrial transcription and OXPHOS in vitro. Indeed, even when SIRT3 activity was abolished, activation of mitochondrial transcription and OXPHOS by the KR mutant remained robust, further highlighting the contribution of LRP130 deacetylation to increased OXPHOS in fasted liver. These data establish a link between nutrient sensing and mitochondrial transcription that regulates OXPHOS in fasted liver and may explain how fasted liver adapts to increased substrate oxidation.
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The potential of adeno-associated viral vectors for gene delivery to muscle tissue.
Expert Opin Drug Deliv
PUBLISHED: 01-03-2014
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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.
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Widespread spinal cord transduction by intrathecal injection of rAAV delivers efficacious RNAi therapy for amyotrophic lateral sclerosis.
Hum. Mol. Genet.
PUBLISHED: 09-18-2013
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Amyotrophic lateral sclerosis (ALS) causes motor neuron degeneration and paralysis. No treatment can significantly slow or arrest the disease progression. Mutations in the SOD1 gene cause a subset of familial ALS by a gain of toxicity. In principle, these cases could be treated with RNAi that destroys the mutant mRNA, thereby abolishing the toxic protein. However, no system is available to efficiently deliver the RNAi therapy. Recombinant adenoassociated virus (rAAV) is a promising vehicle due to its long-lasting gene expression and low toxicity. However, ALS afflicts broad areas of the central nervous system (CNS). A lack of practical means to spread rAAV broadly has hindered its application in treatment of ALS. To overcome this barrier, we injected several rAAV serotypes into the cerebrospinal fluid. We found that some rAAV serotypes such as rAAVrh10 and rAAV9 transduced cells throughout the length of the spinal cord following a single intrathecal injection and in the broad forebrain following a single injection into the third ventricle. Furthermore, a single intrathecal injection of rAAVrh10 robustly transduced motor neurons throughout the spinal cord in a non-human primate. These results suggested a therapeutic potential of this vector for ALS. To test this, we injected a rAAVrh10 vector that expressed an artificial miRNA targeting SOD1 into the SOD1G93A mice. This treatment knocked down the mutant SOD1 expression and slowed the disease progression. Our results demonstrate the potential of rAAVs for delivering gene therapy to treat ALS and other diseases that afflict broad areas of the CNS.
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Vector sequences are not detected in tumor tissue from research subjects with ornithine transcarbamylase deficiency who previously received adenovirus gene transfer.
Hum. Gene Ther.
PUBLISHED: 09-10-2013
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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.
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Gene transfer in the liver using recombinant adeno-associated virus.
Curr Protoc Microbiol
PUBLISHED: 05-21-2013
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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 in the liver. rAAV-mediated gene transfer to the liver can be used to create somatic transgenic animals or disease models for 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 "biofactory" for production of coagulation factors, insulin, 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. This unit describes methods for delivery of rAAV vectors by several injection routes, followed by a range of analytical methods for assessing the expression, activity, and effects of the transgene and its product.
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Gene transfer to the CNS using recombinant adeno-associated virus.
Curr Protoc Microbiol
PUBLISHED: 05-21-2013
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Recombinant adeno-associated virus (rAAV) vectors are great tools for gene transfer due to their ability to mediate long-term gene expression. rAAVs have been used successfully as gene transfer vehicles in multiple animal models of CNS disorders, and several clinical trials are currently underway. rAAV vectors have been used at various stages of development with no apparent toxicity. There are multiple ways of delivering AAV vectors to the mouse CNS, depending on the stage of development. 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 target the adult CNS by intravascular injections.
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Recombinant adeno-associated virus integration sites in murine liver after ornithine transcarbamylase gene correction.
Hum. Gene Ther.
PUBLISHED: 04-30-2013
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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.
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Prefrontal cortical dysfunction after overexpression of histone deacetylase 1.
Biol. Psychiatry
PUBLISHED: 03-20-2013
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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.
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Targeted intraceptor nanoparticle therapy reduces angiogenesis and fibrosis in primate and murine macular degeneration.
ACS Nano
PUBLISHED: 03-20-2013
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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.
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A Single Intravenous rAAV Injection as Late as P20 Achieves Efficacious and Sustained CNS Gene Therapy in Canavan Mice.
Mol. Ther.
PUBLISHED: 03-10-2013
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Canavans disease (CD) is a fatal pediatric leukodystrophy caused by mutations in aspartoacylase (AspA) gene. Currently, there is no effective treatment for CD; however, gene therapy is an attractive approach to ameliorate the disease. Here, we studied progressive neuropathology and gene therapy in short-lived (?1 month) AspA(-/-) mice, a bona-fide animal model for the severest form of CD. Single intravenous (IV) injections of several primate-derived recombinant adeno-associated viruses (rAAVs) as late as postnatal day 20 (P20) completely rescued their early lethality and alleviated the major disease symptoms, extending survival in P0-injected rAAV9 and rAAVrh8 groups to as long as 2 years thus far. We successfully used microRNA (miRNA)-mediated post-transcriptional detargeting for the first time to restrict therapeutic rAAV expression in the central nervous system (CNS) and minimize potentially deleterious effects of transgene overexpression in peripheral tissues. rAAV treatment globally improved CNS myelination, although some abnormalities persisted in the content and distribution of myelin-specific and -enriched lipids. We demonstrate that systemically delivered and CNS-restricted rAAVs can serve as efficacious and sustained gene therapeutics in a model of a severe neurodegenerative disorder even when administered as late as P20.Molecular Therapy (2013); 21 12, 2136-2147. doi:10.1038/mt.2013.138.
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Dicer expression is essential for adult midbrain dopaminergic neuron maintenance and survival.
Mol. Cell. Neurosci.
PUBLISHED: 03-05-2013
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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 (Dicer(flox/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 Dicer(flox/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, are essential for DAergic neuronal maintenance and survival in adult midbrain DAergic neuron brain areas.
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Retinal gene delivery by rAAV and DNA electroporation.
Curr Protoc Microbiol
PUBLISHED: 02-15-2013
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Ocular gene therapy is a fast-growing area of research. The eye is an ideal organ for gene therapy since it is immune privileged and 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 harboring 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, and subsequent tissue processing methods are described.
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Gene transfer in skeletal and cardiac muscle using recombinant adeno-associated virus.
Curr Protoc Microbiol
PUBLISHED: 02-15-2013
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Adeno-associated virus (AAV) is a DNA virus with a small (?4.7 kb) 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.
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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
PUBLISHED: 02-06-2013
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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: ?-globin promoter (?p) with the upstream hyper-sensitive site 2 (HS2) enhancer from the ?-globin locus control region (HS2-?bp), and the human parvovirus B19 promoter at map unit 6 (B19p6). Transgene expression from the B19p6 was significantly higher than that from the HS2-?p, 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-?p 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-?p 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-?p, 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 ?-thalassemia and sickle cell disease.
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SU9518 inhibits proliferative vitreoretinopathy in fibroblast and genetically modified Müller cell-induced rabbit models.
Invest. Ophthalmol. Vis. Sci.
PUBLISHED: 01-24-2013
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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 (PDGFR?) 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 PDGFR? specificity, can inhibit the development of PVR in fibroblast and Müller cell rabbit models of PVR.
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Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1.
Elife
PUBLISHED: 01-01-2013
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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.
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Exploiting natural diversity of AAV for the design of vectors with novel properties.
Methods Mol. Biol.
PUBLISHED: 10-29-2011
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Twelve AAV serotypes have been described so far in human and nonhuman primate (NHP) populations while surprisingly high diversity of AAV sequences is detected in tissue biopsies. The analysis of these novel AAV sequences has indicated a rapid evolution of the viral genome both by accumulation of mutations and recombination. This chapter describes how this rich resource of naturally evolved sequences is used to derive gene transfer vectors with a wide array of activities depending on the nature of the cap gene used in the packaging system. AAV2-based recombinant genomes have been packaged in dozens of different capsid types, resulting in a wide array of "pseudotyped vectors" that constitute a rich resource for the development of gene therapy clinical trials. We describe a polymerase chain reaction-based molecular rescue method for novel AAV isolation that uses primers designed to recognize the highly conserved regions in known AAV isolates and generate amplicons across the hypervariable regions of novel AAV genomes present in the analyzed sample.
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Transendocardial delivery of AAV6 results in highly efficient and global cardiac gene transfer in rhesus macaques.
Hum. Gene Ther.
PUBLISHED: 06-24-2011
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Heart disease is the leading cause of morbidity and mortality, and cardiac gene transfer has potential as a novel therapeutic approach. We previously demonstrated safe and efficient gene transfer to the canine heart using a percutaneous transendocardial injection procedure to deliver self-complementary (sc) adeno-associated virus 6 (AAV6) vector. In the present study, we proceed with our vertical translation study to evaluate cardiac gene transfer in nonhuman primates (NHPs). We screened approximately 30 adult male rhesus macaques for the presence of neutralizing antibodies against AAV6, AAV8, and AAV9, and then selected seven monkeys whose antibody titers against these three serotypes were lower than 1/5. The animals were then randomized to receive either scAAV6 (n=3), scAAV8 (n=1), or scAAV9 (n=3) vector expressing the enhanced green fluorescent protein (EGFP) reporter gene at a dose of 5.4×10(12) genome copies/kg, which was administered according to a modified version of our previously developed transendocardial injection procedure. One animal treated with scAAV6 died secondary to esophageal intubation. The remaining animals were euthanized 7 days after gene transfer, at which time tissue was collected for analysis of EGFP expression, histopathology, and biodistribution of the vector genome. We found that (i) transendocardial delivery of AAV is safe in the NHP, (ii) AAV6 and AAV8 provide efficient cardiac gene transfer at similar levels and are superior to AAV9, and (iii) AAV6 is more cardiac-specific than AAV8 and AAV9. The results of this NHP study may help guide the development AAV vectors for the treatment of cardiovascular disease in humans.
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Cardiac gene transfer of short hairpin RNA directed against phospholamban effectively knocks down gene expression but causes cellular toxicity in canines.
Hum. Gene Ther.
PUBLISHED: 06-08-2011
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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.
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Several rAAV vectors efficiently cross the blood-brain barrier and transduce neurons and astrocytes in the neonatal mouse central nervous system.
Mol. Ther.
PUBLISHED: 05-24-2011
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Noninvasive systemic gene delivery to the central nervous system (CNS) has largely been impeded by the blood-brain barrier (BBB). Recent studies documented widespread CNS gene transfer after intravascular delivery of recombinant adeno-associated virus 9 (rAAV9). To investigate alternative and possibly more potent rAAV vectors for systemic gene delivery across the BBB, we systematically evaluated the CNS gene transfer properties of nine different rAAVEGFP vectors after intravascular infusion in neonatal mice. Several rAAVs efficiently transduce neurons, motor neurons, astrocytes, and Purkinje cells; among them, rAAVrh.10 is at least as efficient as rAAV9 in many of the regions examined. Importantly, intravenously delivered rAAVs did not cause abnormal microgliosis in the CNS. The rAAVs that achieve stable widespread gene transfer in the CNS are exceptionally useful platforms for the development of therapeutic approaches for neurological disorders affecting large regions of the CNS as well as convenient biological tools for neuroscience research.
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Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates.
Mol. Genet. Metab.
PUBLISHED: 05-09-2011
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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 ?-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.
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Evaluation of adeno-associated viral vectors for liver-directed gene transfer in dogs.
Hum. Gene Ther.
PUBLISHED: 04-11-2011
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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.
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MicroRNA-regulated, systemically delivered rAAV9: a step closer to CNS-restricted transgene expression.
Mol. Ther.
PUBLISHED: 12-21-2010
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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.
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Molecular characterization of adenoviruses in fecal samples of captively bred rhesus macaques in China.
Vet. Microbiol.
PUBLISHED: 06-10-2010
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In an attempt to study the molecular diversity of simian adenoviruses in nonhuman primate (NHP) populations, we screened a colony of captively bred rhesus macaques (Macaca mulatta) in China for the presence of adenoviral DNA in stool samples. This was done by using the nested PCR method that targeted the adenovirus polymerase gene. Among the 57 animals analyzed, fecal samples from 12 animals were positive for the presence of adenoviral DNA and the PCR fragments were cloned for sequencing and phylogenetic analyses. The results suggested that the viral DNA clones were primarily segregated into two large groups: SAdV-6 (2 non-redundant sequences) and SAdV-7 (9 non-redundant sequences). In addition, there were three clones with more similarity to SAdV-1, SAdV-3 and HAdV-52 respectively. Our data confirmed the prevalence of adenoviral DNA in the feces of NHPs and revealed the heterogeneity and phylogenetics of the adenoviruses in the gastrointestinal tract of the study animals.
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rAAV9--a human-derived adeno-associated virus vector for efficient transgene expression in mouse cingulate cortex.
Cold Spring Harb Protoc
PUBLISHED: 04-03-2010
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The rostro-medial cortex of the mouse and rat, considered the functional homolog to the primate prefrontal cortex (PFC), is of growing importance for preclinical models of schizophrenia and other neurodevelopmental diseases for which symptoms typically emerge in adolescence and early adulthood. Therefore, in order to explore molecular mechanisms operating during these critical stages of PFC development, it will be important to develop an efficient gene delivery system for the PFC of juvenile animals. To this end, adeno-associated virus (AAV)-based systems are increasingly used in mice for targeted gene delivery in specific brain regions such as the hippocampus. Strikingly, there is very little literature on vector-mediated gene expression in the rostro-medial cortex. In addition, multiple AAV serotypes exist based on differences in their envelope capsid proteins. However, to date, the large majority of studies in the central nervous system (CNS) have utilized the AAV2 serotype. This is typically limited to a very focal transduction pattern and therefore is not ideal for the murine PFC, which occupies several square millimeters in the rostral hemisphere. Here, we introduce a protocol for efficient, AAV9-serotype-mediated gene delivery in juvenile (postnatal day 21) and young adult PFC, resulting in long-lasting transgene expression.
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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.
Hum. Gene Ther.
PUBLISHED: 02-02-2010
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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 phi29 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.
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Adenovirus-adeno-associated virus hybrid for large-scale recombinant adeno-associated virus production.
Hum. Gene Ther.
PUBLISHED: 07-22-2009
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Recombinant adeno-associated virus (rAAV) holds promise for applications in gene therapy. Advances in clinical studies of rAAV-based gene therapeutics have generated an encouraging momentum in the field of gene therapy; however, one of the major obstacles to the eventual clinical success of rAAV-mediated gene therapy is the need for large-scale production of clinical-grade vectors. The transfection-based rAAV production method is well suited for preclinical studies in small animal models, but it is difficult to support large-scale clinical studies with this method. In the past decade, several scalable rAAV production methods have emerged from extensive efforts to develop large-scale manufacturing processes. Among those, the recombinant adenovirus-AAV infection method has some unique features in vector quality and yield. This minireview provides an overview of this scaleable rAAV production platform, describing its basic components and biological mechanisms and process.
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Directed evolution of adeno-associated virus for glioma cell transduction.
J. Neurooncol.
PUBLISHED: 06-02-2009
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Glioblastoma multiforme (GBM) is a serious form of brain cancer for which there is currently no effective treatment. Alternative strategies such as adeno-associated virus (AAV) vector mediated-genetic modification of brain tumor cells with genes encoding anti-tumor proteins have shown promising results in preclinical models of GBM, although the transduction efficiency of these tumors is often low. As higher transduction efficiency of tumor cells should lead to enhanced therapeutic efficacy, a means to rapidly engineer AAV vectors with improved transduction efficiency for individual tumors is an attractive strategy. Here we tested the possibility of identifying high-efficiency AAV vectors for human U87 glioma cells by selection in culture of a newly constructed chimeric AAV capsid library generated by DNA shuffling of six different AAV cap genes (AAV1, AAV2, AAV5, AAVrh.8, AAV9, AAVrh.10). After seven rounds of selection, we obtained a chimeric AAV capsid that transduces U87 cells at high efficiency (97% at a dose of 10(4) genome copies/cell), and at low doses it was 1.45-1.6-fold better than AAV2, which proved to be the most efficient parental capsid. Interestingly, the new AAV capsid displayed robust gene delivery properties to all glioma cells tested (including primary glioma cells) with relative fluorescence indices ranging from 1- to 14-fold higher than AAV2. The selected vector should be useful for in vitro glioma research when efficient transduction of several cell lines is required, and provides proof-of-concept that an AAV library can be used to generate AAV vectors with enhanced transduction efficiency of glioma cells.
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Adeno-associated virus-mediated gene transfer to nonhuman primate liver can elicit destructive transgene-specific T cell responses.
Hum. Gene Ther.
PUBLISHED: 05-16-2009
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Gene transfer to murine liver with vectors based on novel adeno-associated virus (AAV) serotypes is efficient, stable, and safe even in the setting of antigenic transgene products. We undertook a study in cynomolgus macaques to evaluate the relevance of these findings to primates. The vectors were based on AAV serotype 7 and expressed green fluorescence protein (GFP) from the cytomegalovirus enhanced beta-actin promoter in both single-stranded and self-complementary genomes. Transduction efficiencies from the single-stranded vectors were similar to those observed in mice, although there was no advantage in primates with the self-complementary vectors. Primates elicited vibrant cytotoxic T cell responses to GFP that correlated with hepatitis and loss of transgene expression. There was no evidence of T cell activation in response to the AAV capsid. These studies indicate that under some conditions primates may activate more robust T cell responses to transgene products than is observed in mice.
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The targeting of 14-succinate triptolide-lysozyme conjugate to proximal renal tubular epithelial cells.
Biomaterials
PUBLISHED: 03-06-2009
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We have synthesized a renal-specific drug carrier, 14-succinyl triptolide-lysozyme (TPS-LZM) conjugate for targeted delivery of TP to the PTECs. TPS-LZM could be taken up by HK-2 cells, free TP would be degraded and released, mainly from basolateral side of the cells. Compared with TP, the overall targeting efficiency (TE) of TPS-LZM was significantly enhanced from 11.74% to 95.54% and its MRT was moderately prolonged from 3.08h to 4.10h. At very low concentration, TPS-LZM could significantly reverse the disease progression in renal ischemia-reperfusion (I/R) injury animal models, while the mixture of free TP and LZM was ineffective. Further, TPS-LZM conjugate presented much lower hepatotoxicity (0.78 folds lower than TP) and no adverse effect on the immune (1.13 folds higher than TP) and genital system. Thus, TPS-LZM represents a very effective drug candidate for specific treatment of immunological renal diseases with low adverse side effect.
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Worldwide epidemiology of neutralizing antibodies to adeno-associated viruses.
J. Infect. Dis.
PUBLISHED: 01-13-2009
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Recombinant adeno-associated viruses (AAVs) have unique gene-transfer properties that speak to their potential as carriers for gene therapy or vaccine applications. However, the presence of neutralizing antibodies to AAV as a result of previous exposure can significantly limit effective gene transfer. In this study, we obtained 888 human serum samples from healthy volunteers in 10 countries around the world. Samples were assayed for neutralizing antibodies to AAV1, AAV2, AAV7, and AAV8, as well as to a novel, structurally distinct AAV vector, rh32.33, in an in vitro transduction inhibition assay. Our data revealed that neutralizing antibodies to AAV2 were the most prevalent antibodies in all regions, followed by antibodies to AAV1. The seroprevalences of antibodies to AAV7 and to AAV8 were lower than that for antibodies to AAV1, and neutralization of AAVrh32.33 was only rarely detected. Our data also indicate a strong linkage of seroreactivity between apparently distinct serotypes that has not been predicted previously in animal models.
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Assessment of a passive immunity mouse model to quantitatively analyze the impact of neutralizing antibodies on adeno-associated virus-mediated gene transfer.
J. Immunol. Methods
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Adeno-associated viruses (AAVs) are common infective agents of primates. As such, healthy primates carry a large pool of AAV-specific neutralizing antibodies (NAbs), which inhibit AAV-mediated gene transfer therapeutic strategies. Thus, a clinical method to screen patient candidates for AAV-specific NAbs prior to treatment, especially with the frequently used AAV8 capsid component, will facilitate individualized treatment design and enhance therapeutic efficacy. In this study, we evaluated the efficacy and sensitivity of a passive immunity mouse model to quantitatively assess anti-AAV8 NAb titers, as compared to an in vitro immunoassay. The passive transfer model was established in C57BL/6 mice by tail vein injection of pre-defined sera from 23 male rhesus monkeys. The mice were then administered low dose (3e10 GC/mouse) self-complementary (sc) AAV8. The in vitro NAb assay indicated that 69.57% of the rhesus donors had pre-existing anti-AAV8 NAb. The in vivo NAb assay, however, was better able to detect low NAb titer (? 1:5), which can mediate neutralization in vivo. Indeed, 17 rhesus donors (74.0%) had pre-existing anti-AAV8 neutralization by in vivo NAb assay. Our findings indicated that the in vivo NAb assay is superior to the in vitro assay for detecting low NAb titers.
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Gene transfer in the lung using recombinant adeno-associated virus.
Curr Protoc Microbiol
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Adeno-associated virus (AAV) is a small replication-deficient DNA virus belonging to the Parvovirinae family. It has a single-stranded ?4.7-kb 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.
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Production and discovery of novel recombinant adeno-associated viral vectors.
Curr Protoc Microbiol
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In this unit, we describe the detailed procedure for a three-plasmid transfection method for rAAV production, and discuss its advantages, limitations, and troubleshooting techniques. We further discuss the rAAV purification process using CsCl gradients, as well as subsequent quality control methods using SDS-PAGE and real-time PCR to assess vector purity, packaging efficiency, and viral titer. Finally, we elaborate on a PCR-based strategy that can be used to discover novel AAV capsid sequences from primate tissue, which can be used to develop newer-generation rAAVs with a greater diversity of tissue tropism for clinical gene therapy.
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Long-term, efficient inhibition of microRNA function in mice using rAAV vectors.
Nat. Methods
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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 TuDs depleted the corresponding miRNA and increased its mRNA targets. rAAV producing anti-miR-122 TuD 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 TuDs induced 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.
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Development and application of an antigen capture ELISA assay for diagnosis of Japanese encephalitis virus in swine, human and mosquito.
Virol. J.
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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.
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