Name: subpial adeno-associated virus 9 (aav9) vector delivery in adult mice
Uploaded: 2017-07-13T12:30:00
Description: Watch this Scientific Journal Video about Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice at JoVE.com

This study was supported by the SANPORC and ALSA Foundation grant (Martin Marsala); the National Sustainability Programme, project number LO1609 (Czech Ministry of Education, Youth and Sports); and RVO: 67985904 (Stefan Juhas and Jana Juhasova).

These studies were carried out under a protocol approved by the Institutional Animal Care and Use Committee of the University of California, San Diego and were in compliance with the Association for Assessment of Laboratory Animal Care guidelines for animal use. All studies were performed in such a manner as to minimize group size and animal suffering.
1. General Animal and Surgical Preparation
<ol>
	<li>Before starting the surgical procedure, thaw the virus (AAV9-UBI-GFP; 5 &#181;L ...

<ol>
	<li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravascular+AAV9+preferentially+targets+neonatal+neurons+and+adult+astrocytes.">Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes.</a> Nat Biotechnol. 27 (1), 59-65 (2009).</li><li>Duque, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravenous+administration+of+self-complementary+AAV9+enables+transgene+delivery+to+adult+motor+neurons.">Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons.</a> Mol Ther. 17 (7), 1187-1196 (2009).</li><li>Gray, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+differences+of+intravascular+AAV9+delivery+to+neurons+and+glia:+a+comparative+study+of+adult+mice+and+nonhuman+primates.">Preclinical differences of intravascular AAV9 delivery to neurons and glia: a comparative study of adult mice and nonhuman primates.</a> Mol Ther. 19 (6), 1058-1069 (2011).</li><li>Meyer, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+single+injection+CSF+delivery+of+AAV9-mediated+gene+therapy+for+SMA:+a+dose-response+study+in+mice+and+nonhuman+primates.">Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates.</a> Mol Ther. 23 (3), 477-487 (2015).</li><li>Foust, K. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+AAV9-mediated+suppression+of+mutant+SOD1+slows+disease+progression+and+extends+survival+in+models+of+inherited+ALS.">Therapeutic AAV9-mediated suppression of mutant SOD1 slows disease progression and extends survival in models of inherited ALS.</a> Mol Ther. 21 (12), 2148-2159 (2013).</li><li>Passini, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+fidelity+of+intrathecal+delivery+of+self-complementary+AAV9-survival+motor+neuron+1+for+spinal+muscular+atrophy.">Translational fidelity of intrathecal delivery of self-complementary AAV9-survival motor neuron 1 for spinal muscular atrophy.</a> Hum Gene Ther. 25 (7), 619-630 (2014).</li><li>Bell, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neuron+transduction+after+intracisternal+delivery+of+AAV9+in+a+cynomolgus+macaque.">Motor neuron transduction after intracisternal delivery of AAV9 in a cynomolgus macaque.</a> Hum Gene Ther Methods. 26 (2), 43-44 (2015).</li><li>Miyanohara, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Potent+spinal+parenchymal+AAV9-mediated+gene+delivery+by+subpial+injection+in+adult+rats+and+pigs.">Potent spinal parenchymal AAV9-mediated gene delivery by subpial injection in adult rats and pigs.</a> Mol Ther Methods Clin Dev. 3, 16046 (2016).</li><li>Xu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+gene+knockdown+in+rat+dorsal+root+ganglia+mediated+by+self-complementary+adeno-associated+virus+serotype+5+following+intrathecal+delivery.">In vivo gene knockdown in rat dorsal root ganglia mediated by self-complementary adeno-associated virus serotype 5 following intrathecal delivery.</a> PLoS One. 7 (3), 32581 (2012).</li><li>Xiao, X., Li, J., Samulski, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+high-titer+recombinant+adeno-associated+virus+vectors+in+the+absence+of+helper+adenovirus.">Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.</a> J Virol. 72 (3), 2224-2232 (1998).</li></ol>

The current study describes a technique of subpial vector (AAV9) delivery in adult mice. As demonstrated in the accompanying video, this approach and technique can effectively be used, provided that the required instruments and pia-penetrating needle and subpial injection needle are properly manufactured, according to the established and tested specifications.
Critical Technical Variables in Performing a Consistent and Safe Subpial Injection in Mice: 
As demonstrated, a s...

The use of AAV vectors to treat a variety of spinal cord and CNS neurodegenerative disorders is becoming a well-accepted platform to effectively upregulate or silence the expression of gene(s) of interest. One of the key limitations to the more effective utilization of this technology to treat CNS/spinal cord disorders is the limited ability to deliver AAV vector(s) to the deep brain or spinal cord parenchyma in adult mammals.
It was demonstrated, for example, that the systemic delivery of AAV9 in adult rodents, cats, or non-human primates is only moderately effective at inducing transgene expression in neurons in the brain and spinal cord1,2,3. The more effective intrathecal delivery of AAV9 vectors has also been shown to lead to only limited transgene expression in anatomically-defined pools of neurons. More specifically, it has been demonstrated that cisternal or lumbo-sacral intrathecal AAV9 delivery in non-human primates, pigs, or rodents leads to a high level of transgene expression in spinal &#945;-motoneurons and segmental dorsal root ganglion neurons. However, minimal or no expression in spinal interneurons or ascending or descending axons in the white matter is seen4,5,6,7. Collectively, these data show that a highly effective biological-anatomical barrier exists, which prevents the diffusion of intrathecally delivered AAV into deeper spinal parenchyma.
In a previous study using adult rats and pigs, a novel subpial vector delivery technique was developed8. Using this approach, highly potent and multi-segmental transgene expression was demonstrated after a single-bolus subpial AAV9 delivery. Intense GFP expression was consistently seen in neurons, glial cells, and descending/ascending axons through the injected spinal segments. This study demonstrated for the first time that the pia mater represents the primary barrier limiting effective AAV9 diffusion into the spinal parenchyma from the intrathecal space. While this previously developed technique and subpial injection device is relatively easy to use in large rodents (like rats) or adult pigs, the system is not suitable for use in small animals, such as adult mice. Because of the high number of available transgenic mouse models of a variety of neurodegenerative disorders, there is a clear need for the development of an effective spinal-parenchymal vector delivery technique in mice. The availability of such a technique would permit the study of the effect of specific gene silencing (e.g., using shRNA) or upregulation using cell-non-specific (e.g., cytomegalovirus-CMV or Ubiquitin) or cell-specific (e.g., synapsin or glial fibrillary acidic protein (GFAP)) promoters during early postnatal development or under diseased conditions.
Accordingly, in the present study, we have developed and validated a miniature subpial vector delivery system that can effectively be used in adult mice. Similarly, as in previous rat and pig studies, this work demonstrates potent transgene expression throughout the spinal parenchyma after a single-bolus subpial AAV9 delivery in mice. The simplicity of this approach, the very good tolerability of injected mice to subpial AAV9 delivery, and the high potency of transgene expression in the spinal parenchyma suggest that this technique can effectively be implemented in any laboratory setting and used in experiments targeting spinal gene expression.

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subpial adeno-associated virus 9 (aav9) vector delivery in adult mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Potent Transgene Expression in Subpially AAV9-injected Segments: 
The analysis of transgene (GFP) expression in spinal cord sections at 14 days after AAV9 delivery showed AAV9-dose dependent GFP expression throughout the spinal parenchyma. First, two bilateral 3 &#181;L injections of AAV9-UBI-GFP injected into the upper lumbar subpial space were associated with the near-complete infection of the white and gray matter in the whole lumbar spinal cord, extending to the ...

Watch this Scientific Journal Video about Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice at JoVE.com

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

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