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

この研究は、SANPORCおよびALSA財団の助成金（Martin Marsala）によって支持された。国家持続可能性プログラム、プロジェクト番号LO1609（チェコ文部省、青少年スポーツ）。 RVO：67985904（Stefan JuhasとJana Juhasova）。

これらの試験は、カリフォルニア大学サンディエゴ校の動物実験および使用委員会の承認を受けたプロトコールのもとで実施され、動物実験のための動物実験ガイドライン評価協会に準拠しています。すべての研究は、群の大きさおよび動物の苦痛を最小限に抑えるように実施した。 1.一般的な動物および外科的調製物<ol><li> 手術を開始する前に、ウイルスを解?...

<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>

現在の研究では、成体マウスにおける腹側ベクター（AAV9）送達の技術が記載されている。付属のビデオで実証されているように、確立され、試験された仕様に従って、必要な器具および穿刺針および胸骨下注射針が適切に製造されるならば、この手法および技法を効果的に使用することができる。 マウスにおける一貫した安全な副作用注射を行う際の重要な技術?...

多様な脊髄およびCNS神経変性疾患を治療するためのAAVベクターの使用は、関心のある遺伝子の発現を効果的にアップレギュレートまたはサイレンシングするための十分に容認されたプラットフォームとなっている。 CNS /脊髄障害を治療するためのこの技術のより有効な利用に対する重要な制限の1つは、成人哺乳動物の深部脳または脊髄実質にAAVベクターを送達する能力が限られていることである。 それは大人の齧歯類、ネコ、または非ヒト霊長類におけるAAV9の全身送達は、脳や脊髄の1、2、3のニューロンに導入遺伝子発現を誘導することでのみ適度に有効であること、例えば、実証されました。 AAV9ベクターのより効果的な髄腔内送達は、解剖学的に定義されたニューロンのプールにおいて限られたトランスジーン発現しかもたらさないことも示されている。より具体的には、それは悪魔非ヒト霊長類、ブタ、またはげっ歯類におけるcisternalまたはlumbo-sacralの髄腔内髄腔内AAV9送達が、脊髄α-運動ニューロンおよび部分的な背根神経節ニューロンにおいて高レベルのトランスジーン発現をもたらすことを示している。しかし、脊髄介在ニューロン又は昇順または白質における下降軸索における最小または全く発現は、4、5、6、7を見ています。まとめると、これらのデータは、髄腔内に送達されたAAVのより深い脊柱実質への拡散を防止する、非常に有効な生物学的 - 解剖学的障壁が存在することを示す。 成体ラットおよびブタを用いた以前の研究では、新規な小児ベクター送達技術が開発された8 。このアプローチを使用して、単一ボーラスAAV9送達後に、非常に強力で多セグメントのトランスジーン発現が実証された。激しいGFP発現が一貫して見られた神経細胞、グリア細胞、および下垂/上行軸索において、注入された脊髄分節を通過する。この研究は、髄膜腔からの脊髄実質への効果的なAAV9の拡散を制限する主な障壁であることを初めて実証した。この以前に開発された技術および小児注射デバイスは、大きなげっ歯類（ラットのような）または成人のブタで使用するのが比較的容易であるが、成体マウスのような小型動物での使用には適していない。様々な神経変性障害の利用可能なトランスジェニックマウスモデルの数が多いため、マウスにおける効果的な脊柱 - 実質細胞ベクター送達技術の開発が明らかに必要とされている。そのような技術の利用可能性は、細胞非特異的（ 例えば、サイトメガロウイルス-CMVまたはユビキチン）または細胞特異的（ 例えば、シナプシンまたはグリア）を用いた特異的遺伝子サイレンシング（ 例えば、 shRNAを用いる）またはアップレギュレーションの研究を可能にする線維性酸性プロテイン（GFAP））プロモーターの活性を阻害する可能性がある。 したがって、本研究では、成体マウスにおいて効果的に使用され得る小型の小児ベクター送達システムを開発し、検証した。同様に、以前のラットおよびブタの研究の場合と同様に、この研究は、マウスにおける単球ボーラスsubpial AAV9送達後の脊柱実質全体にわたる有力なトランスジーン発現を実証する。このアプローチの単純さ、腹腔内AAV9送達に対する注射されたマウスの非常に良好な忍容性、および脊髄実質における導入遺伝子発現の高い効力は、この技術が任意の実験室環境において効果的に実施され得、脊髄遺伝子発現を標的とする実験において使用され得ることを示唆する。

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

成体ラットおよびブタにおける亜麻痺性アデノ随伴ウイルス9（AAV9）ベクター送達技術の成功した開発は以前に報告されている。 AAV9送達のために下位に配置されたポリエチレンカテーテル（PE-10またはPE-5）を使用して、脊髄実質を介した潜在的なトランスジーン発現（白人および灰白質）が、浅部注射された脊髄セグメントにおいて実証されている。神経変性疾患のトランスジェニックマウスモデルの広い範囲のため、成体マウスにおける強力な中枢神経系（CNS）標的化ベクター送達技術の開発に対する強い要望がある。したがって、本研究は、成体C57BL / 6Jマウスにおいて安全かつ有効な脊髄AAV9送達を可能にする脊髄小脳ベクター送達装置および技術の開発を記載する。脊椎固定化および麻酔したマウスでは、XYZマニピュレータを用いて、脊髄（頚部1および腰部1-2の脊髄分節レベル）を鋭利な34Gの針で切開した。第2のXYZma次いで、鈍い36G針を腰部および/または頸部の胸部空間に進めるために、ニプレーターを使用した。次いで、緑色蛍光タンパク質（GFP）をコードするAAV9ベクター（3-5μL; 1.2×10 13ゲノムコピー（gc））を皮下注射した。注射後、神経機能（運動および感覚）を定期的に評価し、4％パラホルムアルデヒドでAAV9送達してから14日後に動物を灌流固定した。水平または横断脊髄切片の分析は、灰白質および白質の両方において、脊髄全体にわたって導入遺伝子発現を示した。さらに、強く逆行的に媒介されるGFP発現は、運動皮質、核破裂部および網様体における下行運動軸およびニューロンにおいて見られた。いずれの動物においても神経学的機能障害は認められなかった。これらのデータは、小児ベクター送達技術が、処置関連の脊髄損傷を引き起こすことなく、成体マウスにおいて首尾よく使用され得、非常に強力な導入遺伝子発現に関連することを示す脊髄神経叢全体にわたって

<html> 亜型AAV9注射部位における有力なトランスジーン発現： AAV9送達後14日目の脊髄切片における導入遺伝子（GFP）発現の分析は、脊髄実質全体にわたってAAV9用量依存性GFP発現を示した。第1に、上部腰椎腔に注入されたAAV9-UBI-GFPの2回の両側3μL注射は、腰部脊髄全体における白色および灰白質のほぼ完全な感染と関連し、上部胸部セグメントに及んだ...

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

本研究の目的は、成体マウスにおける新規な遺伝子サブ送達技術を用いて、脊髄アデノ随伴ウイルス9（AAV9）媒介遺伝子送達の効力および安全性を開発および検証することであった。

成体マウスにおける子宮頸部アデノ随伴ウイルス9（AAV9）ベクター送達

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

The overall goal of this subpial AAV9 delivery technique is to achieve a potent spinal parenchymal transgene expression throughout the entire length of spinal chord in adult mice. This method can facilitate studies targeted to manipulate spinal gene expression and help to understand the mechanism of several spinal neurodegenerative disorders including amyotrophic lateral sclerosis, spinal traumatic injury, or chronic pain. The main advantage of this technique is that highly potent transgene expression is achieved through the spinal gray and white matter in multiple spinal segments, which are adjacent to subpial AAV9 injected segment.Visual demonstration of this method is critical because the dura-opening step and placement of subpial needle are difficult to learn without visual demonstration. Demonstrating the procedure will be Takahiro Tadokoro, a post-doctorate fellow in our laboratory. Begin by properly anesthetizing the animal for surgery, isoflurane is used here.Ensure the absence of a paw pinch response before proceeding, then cover the eyes with ophthalmic ointment. Next, shave the back of the animal with clippers and clean the skin with 2%chlorhexidine. If lumbar subpial injections are to be performed, cut the skin overlying the T8 to L1 vertebrae with a scalpel and then use scissors to detach the paravertebral muscle from the T10 to T12 spinal vertebrae.Mount the animal into a standard stereotaxic frame, using mouse spinal clamps. Next, use a dental drill to shave both sides of the lamina of the T10 to T12 vertebrae until cracks appear then use forceps to remove cracked bone fragments and expose the dorsal surface of the lumbar spinal chord. The dura is now visible, use a 30 gauge stainless steel needle to make a one to two millimeter incision into the dura.It is important to carefully cut open the dura to avoid injury to the underlying pia membrane. Now, grasp the edge of the cut dura with forceps and extend the dura opening for up to five millimeters. If cervical subpial injections are to be performed, immobilize the head of the mouse by placing the head into a stereotaxic frame using ear bars and use scissors to make a 1.5 to two centimeter incision in the dorsal neck skin to expose the C1 and C2 segments and occipital bone.Then, shave a portion of exposed occipital bone using a dental drill and remove the bone fragments using forceps. Then, use a 23 gauge stainless steel needle and forceps to remove the atlanto occipital membrane of the cisterna magna. Use cotton swabs to clean the incision site of any tissue and bone debris, then cut open the dura, making a two to three millimeter incision.Mount the 34 gauge pia-penetrating needle and 36 gauge injection needle on to the Z arms of two separate X, Y, Z manipulators using glass capillary holders. Next, use a 50 microliter microsyringe connected with PE10 or PE20 tubing to load AAV9-UBI-GFP virus. After the virus is loaded, connect the end of the tubing to the injection needle.Next, while looking through a surgical dissecting scope set to eight to ten X magnification, use the X arm to lower the pia-penetrating needle into the pia to a depth of one millimeter. Keep the angle of the penetrating needle at five to 10 degrees relative to the tissue surface. After opening the pia, use the X arm to remove the pia penetrating needle from the subpial space.Note the penetrated site using a landmark such as a blood vessel. Move the X, Y, and Z arms of the second manipulator to position the tip of the AAV9 virus loaded injection needle into the pia penetrated site. Then, after inserting the tip of the injection needle about 0.5 millimeters in to the subpial space, use the Z arm to lift the pia about 0.3 millimeters, then use the X arm to continue advancing the needle horizontally into the subpial space.Advance the needle until it enters the subpial space at a depth of about two to three millimeters, then use the 50 microliter microsyringe to inject the AAV9-UBI-GFP virus into the subpial space. Remove the injection needle from the subpial space after the AAV9-UBI-GFP injection is complete. Close the muscle and skin using foromonofilament suture and surgical clips.Allow the animal to recover on a heating pad before housing in a clean cage. Two bilateral injections of AAV9-UBI-GFP were delivered into the upper lumbar subpial space and the animals were profusion fixed 14 days after AAV9 delivery. Intense GFP expression is seen in the gray matter located within the dotted area and white matter outside the dotted area.This extends from the lumbar to the upper thoracic segments in animals injected with three plus three microliters of AAV9. Animals injected with a lower volume of AAV9 have less intense expression of GFP. The following images depict potent retrograde and anterograde AAV9-UBI-GFP mediated GFP expression in brain motor and sensory centers.This low-power image clearly shows the presence of intense GFP positivity in the cervical spinal cord, medulla oblongata, cerebellum, and motor cortex. This high-power image taken from a sagittal brain section shows the presence of GFP florescence in neurons in the reticular formation, nucleus ruber, and axons of the spinocerebellar tract. This lower-power image taken from a coronal brain section demonstrates the presence of GFP florescence in pyramidal neurons in the motor cortex and in the terminals of the spinothalamic tract in areas of the reticular thalamic nuclei.Finally, this high-power image demonstrates an intense GFP expression in pyramidal neurons in the motor cortex. Once mastered, this technique can be completed in 30 to 40 minute. The use of this technique by researchers in the field of neuroscience will be an effective tool to study the role of specific genes in the evolution of spinal degenerative disorders.It will also help to explore potential treatment efficacy by suppressing mutated genes known to be linked with progressive spinal neuronal degeneration as in the familiar form of amyotrophic lateral sclerosis.

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

Research

JoVE Journal

Neuroscience

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

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

アデノ随伴ウイルスベクターの頭蓋内注射

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

神経科学

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

メソッドおよびアデノ随伴ウイルス ラットの静脈内投与と中枢神経系の伝達の評価のためのヒント

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Visualization and Live Imaging of Oligodendrocyte Organelles in Organotypic Brain Slices Using Adeno-associated Virus and Confocal Microscopy

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced tools currently used to study neurons, have only partly been taken on by oligodendrocyte researchers. Cell type-specific staining by viral transduction is a useful approach to study live organelle dynamics. This paper describes a protocol for visualizing oligodendrocyte mitochondria in organotypic brain slices by transduction with adeno-associated virus (AAV) carrying genes for mitochondrial targeted fluorescent proteins under the transcriptional control of the myelin basic protein promoter. It includes the protocol for making organotypic coronal mouse brain slices. A procedure for time-lapse imaging of mitochondria then follows. These methods can be transferred to other organelles and may be particularly useful for studying organelles in the myelin sheath. Finally, we describe a readily available technique for visualization of unstained myelin in living slices by Confocal Reflectance microscopy (CoRe). CoRe requires no extra equipment and can be useful to identify the myelin sheath during live imaging.

Myelinating oligodendrocytes promote rapid action potential propagation and neuronal survival. Described here is a protocol for oligodendrocyte-specific expression of fluorescent proteins in organotypic brain slices with subsequent time-lapse imaging. Further, a simple procedure for visualizing unstained myelin is presented.

可視化とアデノ随伴ウイルスと共焦点顕微鏡を用いた脊髄脳スライスにおけるオリゴデンドロ サイト オルガネラのライブ イメージング

Neurons rely on the electric insulation and trophic support of myelinating oligodendrocytes. Despite the importance of oligodendrocytes, the advanced ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

アデノ随伴ウイルスを介する遺伝子発現により脊髄のニューロンを遺伝的に定義されています。

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

成体マウスの遺伝子組換えアデノ随伴ウイルスの直接髄腔内注入

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Production, Purification, and Quality Control for Adeno-associated Virus-based Vectors

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), including gene therapy applications. AAV vectors are non-replicating, able to infect both dividing and non-dividing cells&#160;and provide long-term transgene expression. Importantly, some serotypes, such as the newly described PHP.B, can cross the blood-brain-barrier (BBB) in animal models, following systemic delivery. AAV vectors can be efficiently produced in the laboratory. However, robust and reproducible protocols are required to obtain AAV vectors with sufficient purity levels and titer values high enough for in vivo applications. This protocol describes an efficient and reproducible strategy for AAV vector production, based on an iodixanol gradient purification strategy. The iodixanol purification method is suitable for obtaining batches of high-titer AAV vectors of high purity, when compared to other purification methods. Furthermore, the protocol is generally faster than other methods currently described. In addition, a quantitative polymerase chain reaction (qPCR)-based strategy is described for a fast and accurate determination of the vector titer, as well as a silver staining method to determine the purity of the vector batch. Finally, representative results of gene delivery to the CNS, following systemic administration of AAV-PHP.B, are presented. Such results should be possible in all labs using the protocols described in this article.

Here, we describe an efficient and reproducible strategy to produce, titer, and quality-control batches of adeno-associated virus vectors. It allows the user to obtain a vector preparation with high-titer&#160;(&#8805;1 x 1013 vector genomes/mL) and a high purity, ready for in vitro or in vivo use.

生産・精製・ アデノ随伴ウイルスを用いたベクトルの品質管理

Gene delivery tools based on adeno-associated viruses (AAVs) are a popular choice for the delivery of transgenes to the central nervous system (CNS), ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.

オンラインMRI画像のガイダンスの下でレソスマカクの皮質への光遺伝学アデノ関連ウイルスベクターの対流強化された配信

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we ...

Electrocorticographic Recording of Cerebral Cortex Areas Manipulated Using an Adeno-Associated Virus Targeting Cofilin in Mice

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. Adeno-associated viruses (AAVs) are increasingly used to improve understanding of brain circuits and their functions. The AAV-mediated manipulation of specific cell populations and/or of precise molecular components has been tremendously useful to identify new sleep regulatory circuits/molecules and key proteins contributing to the adverse effects of sleep loss. For instance, inhibiting activity of the filamentous actin-severing protein&#160;cofilin&#160;using AAV prevents sleep deprivation-induced memory impairment. Here, a protocol is&#160;described that combines the manipulation of cofilin function in a cerebral cortex area with the recording of ECoG activity to examine whether cortical cofilin modulates the wakefulness and sleep ECoG signals. AAV injection is performed during the same surgical procedure as the implantation of ECoG and electromyographic (EMG) electrodes in adult male and female mice. Mice are anesthetized, and their heads are shaved. After skin cleaning and incision, stereotaxic coordinates of the motor cortex are determined, and the skull is pierced at this location. A cannula prefilled with an AAV expressing cofilinS3D, an inactive form of cofilin, is slowly positioned in the cortical tissue. After AAV infusion, gold-covered screws (ECoG electrodes) are screwed through the skull and cemented to the skull with gold wires inserted in the neck muscles (EMG electrodes). The animals are allowed three weeks to recover and to ensure sufficient expression of cofilinS3D. The infected area and cell type are verified using immunohistochemistry, and the ECoG is analyzed using visual identification of vigilance states and spectral analysis. In summary, this combined methodological approach allows the investigation of the precise contribution of molecular components regulating neuronal morphology and connectivity to the regulation of synchronized cerebral cortex activity during wakefulness and sleep.

This article describes a protocol for the manipulation of molecular targets in the cerebral cortex using adeno-associated viruses and for monitoring the effects of this manipulation during wakefulness and sleep using electrocorticographic recordings.

マウスにおけるコフィリンを標的とするアデノ関連ウイルスを用いて操作した大脳皮質領域の電気コルチコチコ

The use of electrocorticographic (ECoG) recordings in rodents is relevant to sleep research and to the study of a wide range of neurological conditions. ...

Time-Lapse Imaging of Neuronal Arborization using Sparse Adeno-Associated Virus Labeling of Genetically Targeted Retinal Cell Populations

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a powerful model system for the investigation of cell type-specific mechanisms of neuronal morphogenesis and connectivity. The organization and composition of retinal subtypes are well-defined, and genetic tools are available to access specific types during development. Many retinal cell types also constrain their dendrites and/or axons to narrow layers, which facilitates time-lapse imaging. Mouse retina explant cultures are well suited for live-cell imaging using confocal or multiphoton microscopy, but methods optimized for imaging dendrite dynamics with temporal and structural resolution are lacking. Presented here is a method to sparsely label and image the development of specific retinal populations marked by the Cre-Lox system. Commercially available adeno-associated viruses (AAVs) used here expressed membrane-targeted fluorescent proteins in a Cre-dependent manner. Intraocular delivery of AAVs in neonatal mice produces fluorescent labeling of targeted cell types by 4-5 days post-injection (dpi). The membrane fluorescent signals are detectable by confocal imaging and resolve fine branch structures and dynamics. High-quality videos spanning 2-4 h are acquired from imaging retinal flat-mounts perfused with oxygenated artificial cerebrospinal fluid (aCSF). Also provided is an image postprocessing pipeline for deconvolution and three-dimensional (3D) drift correction. This protocol can be used to capture several cellular behaviors in the intact retina and to identify novel factors controlling neurite morphogenesis. Many developmental strategies learned in the retina will be relevant for understanding the formation of neural circuits elsewhere in the central nervous system.

Here, we present a method for investigating neurite morphogenesis in postnatal mouse retinal explants by time-lapse confocal microscopy. We describe an approach for sparse labeling and acquisition of retinal cell types and their fine processes using recombinant adeno-associated virus vectors that express membrane-targeted fluorescent proteins in a Cre-dependent manner.

遺伝子標的網膜細胞集団のスパースアデノ随伴ウイルス標識を用いた神経樹木化のタイムラプスイメージング

Discovering mechanisms that pattern dendritic arbors requires methods to visualize, image, and analyze dendrites during development. The mouse retina is a ...

CNS遺伝子治療のための幼若ラットにおけるAAV9の腰椎穿刺送達

Intrathecal Vector Delivery in Juvenile Rats via Lumbar Cistern Injection

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a toxic gene, or provide a gene whose product can modulate the biology of the disease. The delivery method for the therapeutic vector can take many forms, ranging from intravenous infusion for systemic delivery to direct injection into the target tissue. For neurodegenerative disorders, it is often desirable to skew transduction towards the brain and/or spinal cord. The least invasive approach to target the entire central nervous system involves injection into the cerebrospinal fluid (CSF), allowing the therapeutic to reach a large fraction of the central nervous system. The safest approach to deliver a vector into the CSF is the lumbar intrathecal injection, where a needle is introduced into the lumbar cistern of the spinal cord. This technique, also known as a lumbar puncture, has been widely used in neonatal and adult rodents and in large animal models. While the technique is similar across species and developmental stages, subtle differences in size, structure, and elasticity of tissues surrounding the intrathecal space require accommodations in the approach. This article describes a method for performing lumbar puncture in juvenile rats to deliver an adeno-associated serotype 9 vector. Here, 25-35 &#181;L of vector were injected into the lumbar cistern, and a green fluorescent protein (GFP) reporter was used to evaluate the transduction profile resulting from each injection. The benefits and challenges of this approach are discussed.

著者スポットライト:若年ラットにおける髄腔内遺伝子治療の有効性の評価

A surgical procedure is described to perform injections into the lumbar cistern of the juvenile rat. This approach has been used for the intrathecal delivery of gene therapy vectors, but it is anticipated that this approach can be used for a variety of therapeutics, including cells and drugs.

腰椎貯水槽注射による幼若ラットの髄腔内ベクター送達

Gene therapy is a powerful technology to deliver new genes to a patient for the treatment of disease, be it to introduce a functional gene, inactivate a ...