eoe sub articles

EoE Sub Articles

1. Treat neonatal mice with rAAV9 at P0.5-P2.5 by subcutaneous injection.
<ol>
	<li>Pre-fill a 29G1/2, 0.33 x 12.7 mm insulin syringe with the recombinant adeno-associated virus 9 (rAAV9) solution. Be careful to remove air bubbles.</li>
	<li>Hold the&#160;cryo-anesthetized pup in one hand with thumb and forefinger. Prior to injection, swipe the back skin of the pup with a swab stick saturated with 70% isopropyl alcohol to maintain the sterile condition. Insert the syringe needle into the anterior-dorsa...

recombinant adeno associated virus based gene delivery: a technique for transgene delivery and cardiac-specific expression in rodent model

Source: Ding, J. et al. <a href="https://www.jove.com/v/54787">Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts.</a> J. Vis. Exp. (2016)
In this video, we demonstrate recombinant adeno associated virus-based gene delivery in the mouse pup. This method helps in studying gene expression in the targeted cells.

Recombinant Adeno Associated Virus Based Gene Delivery: A Technique for Transgene Delivery and Cardiac-Specific Expression in Rodent Model

Source: Ding, J. et al. Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts. J. Vis. Exp. (2016)
In this video, we demonstrate ...

Recombinant adeno-associated virus, rAAV, is a small, single-stranded DNA virus genetically engineered to target a specific cell type and transport its genetic cargo to the host cell nucleus for gene expression.
Begin by restraining a cryo-anesthetized mouse pup. Sterilize the back skin and subcutaneously inject the rAAV vector carrying the desired transgene along a cardiac-specific promoter. The vector gets absorbed into the vascular system and reaches distant organs, including the heart.
Here, the recombinant virus attaches to the surface of the cardiomyocyte and gets internalized via endocytosis into an endosome. Within the endosome, the viral capsid partially disassembles, exposing the viral lytic proteins. This protein induces pores in the endosomal membrane, through which the vector escapes into the cytoplasm.
The vector travels across cytoplasmic microtubules and enters through the nuclear pore. Once in the nucleus, the viral capsid disassembles to release its genome. The single-stranded DNA carrying the target gene is then converted into double-stranded DNA.
Following DNA transcription, the produced mRNA moves into the cytoplasm. Here, the mRNA undergoes translation, expressing the target protein in the cardiomyocytes. Observe tissue samples from different organs under a fluorescence microscope to determine the efficiency and tissue specificity of the gene delivery to the heart.

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Research

JoVE Encyclopedia of Experiments

Biological Techniques

Gene Transfer Techniques

Transduction

Production and Titering of Recombinant Adeno-associated Viral Vectors

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently being tested in human clinical trials. Wild-type AAV is a non-pathogenic member of the parvoviridae family and inherently replication-deficient. The broad transduction profile, low immune response as well as the strong and persistent transgene expression achieved with these vectors has made them a popular and versatile tool for in vitro and in vivo gene delivery. rAAVs can be easily and cheaply produced in the laboratory and, based on their favourable safety profile, are generally given a low safety classification. Here, we describe a method for the production and titering of chimeric rAAVs containing the capsid proteins of both AAV1 and AAV2. The use of these so-called chimeric vectors combines the benefits of both parental serotypes such as high titres stocks (AAV1) and purification by affinity chromatography (AAV2). These AAV serotypes are the best studied of all AAV serotypes, and individually have a broad infectivity pattern. The chimeric vectors described here should have the infectious properties of AAV1 and AAV2 and can thus be expected to infect a large range of tissues, including neurons, skeletal muscle, pancreas, kidney among others. The method described here uses heparin column purification, a method believed to give a higher viral titer and cleaner viral preparation than other purification methods, such as centrifugation through a caesium chloride gradient. Additionally, we describe how these vectors can be quickly and easily titered to give accurate reading of the number of infectious particles produced.

Recombinant adeno-associated virus (rAAVs) vectors are becoming increasingly valuable for in vivo studies in animals. We describe how rAAVs can be produced in the laboratory and how these vectors can be titered to give an accurate reading of the number of infectious particles produced.

In recent years recombinant adeno-associated viral vectors (AAV) have become increasingly valuable for in vivo studies in animals, and are also currently ...

JoVE Journal

Immunology and Infection

Production of Adeno-Associated Virus Vectors in Cell Stacks for Preclinical Studies in Large Animal Models

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. Clinical advancement of novel applications for AAV involves transitioning from small animal models, such as mice, to larger animal models, including dogs, sheep, and nonhuman primates. One of the limitations of administering AAV to larger animals is the requirement for large quantities of high-titer virus. While suspension cell culture is a scalable method for AAV vector production, few research labs have the equipment (e.g., bioreactors) or know how to produce AAV in this manner. Moreover, AAV titers are often significantly lower when produced in suspension HEK 293 cells as compared to adherent HEK293 cells. Described here is a method for producing large quantities of high-titer AAV using cell stacks. A detailed protocol for titering AAV as well as methods for validating vector purity are also described. Finally, representative results of AAV-mediated transgene expression in a sheep model are presented. This optimized protocol for large-scale production of AAV vectors in adherent cells will enable molecular biology laboratories to advance the testing of their novel AAV therapies in larger animal models.

Here we provide a detailed procedure for large-scale production of research-grade AAV vectors using adherent HEK 293 cells grown in cell stacks and affinity chromatography purification. This protocol consistently yields &#62;1 x 1013 vector genomes/mL, providing vector quantities appropriate for large animal studies.

Adeno-associated virus (AAV) vectors are among the most clinically advanced gene therapy vectors, with three AAV gene therapies approved for humans. ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Recombinant Adeno Associated Virus Based Gene Delivery: A Technique for Transgene Delivery and Cardiac-Specific Expression in Rodent Model

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