这项研究得到了SANPORC和ALSA基金会赠款（Martin Marsala）的支持。国家可持续发展计划，项目编号LO1609（捷克教育，青年和体育部）;和RVO:67985904（Stefan Juhas和Jana Juhasova）。

这些研究是根据加利福尼亚大学圣地亚哥分校动物保健和使用委员会批准的方案进行的，并符合动物实验动物保护评估指南。所有研究都以使组大小和动物痛苦最小化的方式进行。 一般动物和手术准备<ol><li> 开始外科手术之前，解冻病毒（AAV9-UBI-GFP;5μL等分试样） 8 。通过将葡聚糖粉末在蒸馏水中混合，制备5％葡聚糖（10,000 MW）溶液。将病毒溶液与1％的5％葡聚糖溶液混合至2.5％的最终葡聚糖浓度。 <ol><li>将病毒溶液储存在冰上（4℃）。 </li></ol></li><li>使用成年C57BL / 6J小鼠（雄性和雌性，20-30克）。麻醉小鼠使用5％异氟烷（O 2，1 L / min），并保持在2-3％吸入异氟烷（O 2，1 L / min）由手术中的鼻锥，取决于呼吸频率和爪夹紧响应。 </li><li>用剃须刀剃刮动物的背部，并用2％氯己定清洁皮肤。 </li><li>在慢性恢复研究中遵循严格的无菌技术。 </li><li> 如果要进行腰椎间盘注射，用手术刀切开覆盖Th8-L1椎骨的皮肤，并用剪刀从Th10-12脊椎椎骨上分离椎旁肌。 <ol><li>使用鼠标脊柱夹将动物装入标准立体定位框架。 </li><li>使用牙钻（钻头:0.9mm，速度:20,000rpm）刮掉Th10-12椎骨椎板的两侧，直到出现裂缝。 </li><li>用镊子去除破裂的骨碎片，暴露脊髓背脊表面。 </li><li>使用30 G不锈钢针和镊子切开硬脑膜约1厘米。 </li></ol></li><li><strong&gt;如果要进行子宫颈腋下注射，使用剪刀将背部颈部皮肤切开1.5-2厘米，并暴露C1-C2段。 <ol><li>使用23G不锈钢针头和镊子取出大the the to膜。 </li><li>使用棉签清洁任何组织和骨碎片的切口部位。 </li><li>使用30 G不锈钢针和镊子将硬膜切开约2-3毫米。 </li></ol></li></ol> 2.打开瓣膜并插入AAV9交付的腋下针<ol><li>使用玻璃毛细管支架将34 G穿刺针插入XYZ操纵器的Z形臂（ 图1A和B ）。 注意:为了制造穿透针，使用具有金刚石研磨板的玻璃毛细管斜面（粗糙（5.0μm至50μm尖端尺寸））和34G针尖的原始斜角尖端磨削角度15 - 20°。然后将针的尖端（1mm长度，从尖端测量）轻轻地弯曲到约90°（ 图1B ，左插入物）。 </li><li> 使用外科手术解剖范围，设置为8-10 X放大倍数，使用X型手臂将穿刺针穿透皮瓣约1 mm（ 图1C ）。 <ol><li>将穿刺针的角度保持在5-10°的组织表面。 </li></ol></li><li>皮瓣打开后，使用X型手臂将ia穿刺针从水平空间（ 图 1D）水平取出。 注意:记住由地标（例如血管）穿透的部位。 </li><li>使用与PE-10或PE-20管连接到注射针的50μL微量注射器，装入带有AAV9-UBI-GFP病毒的钝性36G注射针头。 </li><li>将针安装到第二XYZ操纵器的Z形臂上（ 图1A </strong&gt;和B ）使用玻璃毛细管支架（ 图1B ，右插入）。 注意:为了制造亚型AAV9注射针，使用具有金刚石研磨板的玻璃毛细管斜面（粗糙（5.0μm至50μm尖端尺寸））抛光36 G针头的钝头，以去除尖锐边缘。然后将针的尖端（2-3mm长度，从尖端测量）轻轻弯曲至约90°。将穿刺和腋下注射针插入1-2厘米长的20G从属不锈钢管（距针头10毫米）并用环氧胶粘。需要使用20 G（0.91 mm直径）的管道，以确保与玻璃毛细管支架的连接。 </li><li>通过操纵第二操纵器的X，Y和Z臂，将AAV9注射针的尖端定位到穿刺部位，然后通过先前的膜片开口将其推进到约2-3mm的唾液空间中，使用X臂（ 图1E和F ）。 注:1）AAV9-UBI-GFP是根据先前报道的协议9，10中制备，并最终滴度每mL（GC /毫升）调节至1.2×10 13个基因组拷贝。 2）由于容易识别，所以不需要标记小开口的位置（ 图1D ）。 </li><li>使用50μL微量注射器将AAV9-UBI-GFP（1.5,3或5μL）注入涎腺空间（参见实验组的表1 ）。 </li><li>在完成AAV9-UBI-GFP注射后，从唾液空间中取出注射针。 </li><li>使用4.0单丝缝合线和手术夹将肌肉和皮肤关闭。 注意:没有必要密封开放的椎骨。 </li><li>让动物在加热垫上恢复。 </li><li>对于疼痛控制，每12小时注射丁丙诺啡0.05 mg / kg / sc术后2-3天</li></ol> 3.灌注固定，组织低温保护和免疫荧光染色<ol><li>在AAV9次注射后的预定时间点，用安乐死溶液深度麻醉小鼠（参见材料表 ，0.3mL），并用20mL肝素化盐水随后用20mL的4％多聚甲醛PBS溶液灌注。 </li><li>使用骨骨折解剖脊髓和脑，并在4℃下将其固定在4％甲醛的PBS中过夜。 </li><li>用30％蔗糖在PBS中冷冻保护脊髓和大脑至少5-7天。 </li><li>在低温恒温器上切割冠状，横向或纵向/横向冷冻切片（30μm厚），并将其储存在4°C的PBS中。 </li></ol> 4.脊髓和脑切片的免疫荧光染色（参见材料表） <ol><li>孵化自由浮动部分在初级y抗体过夜。 </li><li>与初级抗体孵育后，在PBS中洗涤切片三次，并与荧光结合的驴抗兔，驴抗鸡和驴抗山羊二抗孵育。 </li><li>将显微镜载玻片放置在室温下干燥，并用抗褪色培养基覆盖。 </li><li>使用荧光荧光显微镜（目标:10X，NA-0.3; 20X，NA-0.8;和63X，NA-1.4）捕获图像。 </li></ol>

<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>

Martin Marsala是Neurgain Technologies，Inc.（美国圣地亚哥）的联合创始人。

目前的研究描述了一种在成年小鼠中的辅助载体（AAV9）递送技术。如所附视频所示，该方法和技术可以有效地被使用，只要根据既定和测试的规格，正确地制造所需的仪器和穿刺针和腋下注射针。 在小鼠中进行一致和安全的唾液注射的关键技术变量: 如所证明的，在背侧颈椎或腰椎板切除术后，可以在成年麻醉的小鼠中容易地进行支气管注射。有几个关键?...

使用AAV载体治疗各种脊髓和CNS神经退行性疾病正在成为一个被广泛接受的平台，可有效地上调或沉默感兴趣的基因的表达。更有效地利用该技术治疗CNS /脊髓障碍的关键限制之一是将AAV载体递送到成年哺乳动物的深部脑或脊髓实质的能力有限。 据证实，例如，AAV9的成年啮齿动物，猫，或非人灵长类动物的全身性递送是在脑和脊髓1，2，3个神经元诱导转基因表达仅适度有效。 AAV9载体更有效的鞘内输送也已显示导致在解剖学定义的神经元池中只有有限的转基因表达。更具体地说，它已经是恶魔认为在非人灵长类动物，猪或啮齿动物中的顺式或骶 - 囊性鞘内AAV9传递导致脊髓α运动神经元和节段背根神经节神经元中高水平的转基因表达。然而，在脊柱的interneurons或升序或在白质下行轴突最小的或没有表达看到4个 ，5，6，7。总的来说，这些数据表明，存在高度有效的生物解剖屏障，其防止鞘内递送的AAV扩散进入更深的脊髓实质。 在以前使用成年大鼠和猪的研究中，开发了一种新型的唾液腺载体递送技术8 。使用这种方法，在单次推荐的subpial AAV9递送后证明了高效和多节段的转基因表达。一贯看到强烈的GFP表达在神经元，神经胶质细胞和下降/上升的轴突通过注射的脊髓段。这项研究首次表明，该皮肤表现出限制从鞘内空间扩散到脊髓实质的有效AAV9的主要障碍。虽然这种先前开发的技术和亚型注射装置相对容易在大型啮齿动物（如大鼠）或成年猪中使用，但该系统不适合用于小型动物，例如成年小鼠。由于各种神经变性疾病的可用转基因小鼠模型的数量很多，所以显然需要在小鼠中发展有效的脊髓实质载体递送技术。这种技术的可用性将允许研究特异性基因沉默（ 例如，使用shRNA）或使用细胞非特异性（ 例如巨细胞病毒-CNV或泛素）或细胞特异性（ 例如，突触素或胶质细胞）的上调的作用纤维酸性蛋白质（GFAP））启动子。 因此，在本研究中，我们开发和验证了一种可以有效地用于成年小鼠的微型载体递送系统。类似地，如在以前的大鼠和猪研究中，这项工作在小鼠中单次推注AAV9单次递送后，证明在整个脊髓实质中有力的转基因表达。这种方法的简单性，注射的小鼠对于亚临床AAV9递送的非常好的耐受性以及脊髓实质中转基因表达的高效力表明该技术可以在任何实验室设置中有效地实施并用于靶向脊髓基因表达的实验中。

<table><tbody><tr><td>C57BL/6J Mice</td><td>Jackson Labs</td><td>664</td><td></td></tr><tr><td>Lab Standard Stereotaxic for Mice</td><td>Harvard Apparatus</td><td>72-9568</td><td></td></tr><tr><td>Mouse Spinal Adaptor</td><td>Harvard Apparatus</td><td>72-4811</td><td></td></tr><tr><td>XYZ Manipulator</td><td>Stoelting</td><td>51604</td><td></td></tr><tr><td>Manual Infusion Pump</td><td>Stoelting</td><td>51218</td><td></td></tr><tr><td>34G Beveled Nanofill Needle</td><td>World Precision Instruments</td><td>NF34BV-2</td><td></td></tr><tr><td>36G Blunt Nanofill needle</td><td>World Precision Instruments</td><td>NF-36BL-2</td><td></td></tr><tr><td>Fluriso, Isoflurane</td><td>MWI Veterinary Supply</td><td>502017</td><td></td></tr><tr><td>Chlorhexidine Solution</td><td>MWI Veterinary Supply</td><td>501027</td><td></td></tr><tr><td>20G Stainless Steel Needle</td><td>Becton-Dickinson</td><td>305175</td><td></td></tr><tr><td>23G Stainless Steel Needle</td><td>Becton-Dickinson</td><td>305145</td><td></td></tr><tr><td>30G Stainless Steel Needle</td><td>Becton-Dickinson</td><td>305128</td><td></td></tr><tr><td>Cotton Tipped Applicator</td><td>MWI Veterinary Supply</td><td>27426</td><td></td></tr><tr><td>Glass Capillary Beveller&amp;nbsp;</td><td>Narishige International</td><td>SM-25B</td><td></td></tr><tr><td>Slide Microscope Superfrost</td><td>Leica Microsystems</td><td>M80</td><td></td></tr><tr><td>50 &amp;mu;L Microsyringe&amp;nbsp;</td><td>Hamilton</td><td>81242</td><td></td></tr><tr><td>BD Intramedic PE-20 Tubing</td><td>Becton, Dickinson</td><td>427406</td><td></td></tr><tr><td>BD Intramedic PE-10 Tubing</td><td>Becton, Dickinson</td><td>427401</td><td></td></tr><tr><td>4-0 monofilament suture</td><td>VetOne</td><td>V1D397</td><td></td></tr><tr><td>Glass Capillary Beveller&amp;nbsp;</td><td>Narishige</td><td>Pipet Micro Grinder EG-40&amp;nbsp;</td><td></td></tr><tr><td>5 min Epoxy (Epoxy Clear)</td><td>Devcon</td><td>14310</td><td></td></tr><tr><td>Euthanasia Solution</td><td>MWI Veterinary Supply</td><td>11168</td><td></td></tr><tr><td>Heparin Inj 1,000 U/mL</td><td>MWI Veterinary Supply</td><td>54254</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Sucrose</td><td>Sigma-Aldrich</td><td>S0389</td><td></td></tr><tr><td>Anti NeuN Antibody</td><td>EMD-Millipore</td><td>ABN78</td><td>Primary Rabbit Polyclonal Antibody, 1:1000</td></tr><tr><td>Anti-Choline Acetyltransferase (CHAT) Antibody</td><td>EMD-Millipore</td><td>AB144P</td><td>Primary Goat Polyclonal Antibody, 1:100</td></tr><tr><td>Anti GFP Antibody</td><td>Aves Labs</td><td>GFP-1020</td><td>Primary Chicken Polyclonal Antibody, 1:1000</td></tr><tr><td>Donkey anti-Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594</td><td>ThermoFisher Scientific</td><td>A21207</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>&amp;nbsp;Donkey anti-Rabbit IgG Secondary Antibody, Alexa Fluor 680</td><td>ThermoFisher Scientific</td><td>A10043</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>Donkey anti-Chicken IgY Secondary Antibody, Alexa Fluor 488</td><td>Jackson Immunoresearch Labs</td><td>703-545-155</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>Donkey Anti-Goat IgG H&amp;amp;L (Alexa Fluor 647</td><td>Abcam</td><td>ab150131</td><td>Secondary Antibody, 1:1000</td></tr><tr><td>Slide Microscope Superfrost</td><td>Fisher Scientific</td><td>12-550-143</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant</td><td>Fisher Scientific</td><td>P36930</td><td></td></tr><tr><td>Epifluorescence Microscope</td><td>Zeiss</td><td>Zeiss AxioImager M2</td><td></td></tr><tr><td>Fluorescence Confocal Microscope</td><td>Olympus</td><td>Olympus FV1000</td><td></td></tr><tr><td>Dextran</td><td>Polysciences, Inc</td><td>19411</td><td></td></tr><tr><td>AAV9-UBC-GFP</td><td>UCSD Viral Vector Core Laboratory</td><td></td><td></td></tr></tbody></table>

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

已经报道了在成年大鼠和猪中成功开发涎腺腺相关病毒9（AAV9）载体递送技术。已经证明使用低位放置的聚乙烯导管（PE-10或PE-5）用于AAV9递送，通过脊髓实质（白色和灰色物质）在低水平注射的脊髓节段中的有效转基因表达。由于神经变性疾病的转基因小鼠模型的广泛范围，强烈希望在成年小鼠中发展有效的中枢神经系统（CNS） - 靶向载体递送技术。因此，本研究描述了在成年C57BL / 6J小鼠中开发脊髓辅助载体递送装置和技术以允许安全和有效的脊髓AAV9递送。在脊髓固定和麻醉的小鼠中，使用XYZ操纵器用尖锐的34G针切开皮肤（颈部1和腰部1-2脊柱节段水平）。第二个XYZ马然后使用咬合器将平坦的36G针推进到腰部和/或颈部的腋下空间中。然后，首先注射编码绿色荧光蛋白（GFP）的AAV9载体（3-5μL; 1.2×10 13个基因组拷贝（gc））。注射后，定期评估神经功能（运动和感觉），动物在用4％多聚甲醛递送AAV9后14天灌注固定。水平或横向脊髓切片的分析显示在整个脊髓中的转基因表达，包括灰色和白色物质。此外，在运动皮层，细胞核和网状细胞的下降的运动轴突和神经元中观察到强烈的逆行介导的GFP表达。任何动物均未观察到神经功能障碍。这些数据表明，唾液腺向量传递技术可以成功地用于成年小鼠，而不会引起与程序相关的脊髓损伤，并且与高效转基因表达有关整个脊髓神经轴突。

<html> 亚型AAV9注射片段中强有力的转基因表达: 在AAV9递送14天后脊髓切片中转基因（GFP）表达的分析显示整个脊髓实质中具有AAV9-剂量依赖性GFP表达。首先，注射到上腰椎间质空间中的两次双侧3μL注射AAV9-UBI-GFP与全部腰脊髓中白色和灰质的近完全感染相关，延伸至上胸段（ 图2A和2B ，左列和中间列）。两只双侧1.5μL注射AA...

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

本研究的目的是通过在成年小鼠中使用新的唾液基因传递技术来开发和验证脊髓腺相关病毒9（AAV9）介导的基因递送的效力和安全性。

在成年小鼠中的亚型腺相关病毒9（AAV9）载体递送

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

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

17.4K 次观看.  University of California, San Diego. 本研究的目的是通过在成年小鼠中使用新的唾液基因传递技术来开发和验证脊髓腺相关病毒9（AAV9）介导的基因递送的效力和安全性。 

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

Preparation of rAAV9 to Overexpress or Knockdown Genes in Mouse Hearts

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation of gene functions. Their therapeutic potential in the heart can also be determined. There are limited approaches for in vivo molecular intervention in the mouse heart. Recombinant adeno-associated virus (rAAV)-based genome engineering has been utilized as an essential tool for in vivo cardiac gene manipulation. The specific advantages of this technology include high efficiency, high specificity, low genomic integration rate, minimal immunogenicity, and minimal pathogenicity. Here, a detailed procedure to construct, package, and purify the rAAV9 vectors is described. Subcutaneous injection of rAAV9 into neonatal pups results in robust expression or efficient knockdown of the gene(s) of interest in the mouse heart, but not in the liver and other tissues. Using the cardiac-specific TnnT2 promoter, high expression of GFP gene in the heart was obtained. Additionally, target mRNA was inhibited in the heart when a rAAV9-U6-shRNA was utilized. Working knowledge of rAAV9 technology may be useful for cardiovascular investigations.

In this manuscript, a method to prepare recombinant adeno-associated virus 9 (rAAV9) vectors to manipulate gene expression in the mouse heart is described.

在小鼠心脏rAAV9的制备过表达或敲除基因

Controlling the expression or activity of specific genes through the myocardial delivery of genetic materials in murine models permits the investigation ...

科研

遗传学

Systemic Delivery of MicroRNA Using Recombinant Adeno-associated Virus Serotype 9 to Treat Neuromuscular Diseases in Rodents

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.

Here we describe the delivery of microRNA using a recombinant adeno-associated virus serotype 9 in a mouse model of a neuromuscular disease. A single peripheral administration in mice resulted in sustained miRNA overexpression in muscle and motor neurons, providing an opportunity to study miRNA function and therapeutic potential in vivo.

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. ...

使用球后注射技术对小鼠进行有效的全身递送

Systemic Treatment for Postnatal, Juvenile, and Runted Adult Mice by Retrobulbar Sinus Injection

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for systemic delivery with fewer limitations. First, tail vein injections (TVIs) are limited to adult mice where the size of the tail vein is suitable for access. Being restricted to treating adult mice can be problematic when dealing with mouse models that do not survive to adulthood. Second, TVIs are not feasible for mouse models with growth retardation phenotypes where the mice never achieve the size of adult wildtype mice. Therefore, retrobulbar injections can be successfully used to treat both young and small adult mice. Lastly, retrobulbar injections are performed under anesthesia, which is less stressful on the mice than TVIs that are commonly performed without anesthesia. This article presents a protocol and detailed instructions for retrobulbar injections that can be used for systemic delivery to small and young mice.

作者聚焦:通过在小鼠模型中进行球后注射克服有机酸血症基因治疗的挑战

This article provides a protocol and an accompanying video for the retrobulbar sinus injection of up to a total volume of 150 &#181;L for postnatal, juvenile, and runted adult mice. This procedure is particularly well suited for the injection of small mice (15 g) when tail vein injection is not feasible.

球后窦注射对出生后、幼年和发育成年小鼠的全身治疗

While tail vein injections are frequently used as a systemic route of delivery in adult mice, retrobulbar injections are an alternative method for ...

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.

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

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

神经科学

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.

重组腺相关病毒在成年小鼠中的直接注射

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.

与腺相关病毒的向量的生产、纯化和质量控制

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

Subconjunctival Administration of Adeno-associated Virus Vectors in Small Animal Models

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ease of accessibility via multiple administration routes. Subconjunctival (SCJ) injections offer advantages over other intraocular administration routes as they are simple, safe, and usually performed in an outpatient setting. SCJ injections in small animals usually require the assistance of an operating microscope due to the size of the eye. Previous work has demonstrated that SCJ injection of specific adeno-associated virus (AAV) serotypes is a valid gene delivery strategy for targeted transduction of the ocular surface, eye muscle, cornea, and optic nerve, providing a potential approach for the treatment of many ocular diseases.
Herein, a detailed protocol is presented for SCJ injections in a mouse model using an injection system consisting of a programmable infusion/withdrawal syringe pump (which allows for consistent and precise injection speed and pressure) and a gastight removable syringe coupled with microinjection needles. The injection system is also adaptable for other intraocular administration routes such as intrastromal, intracameral, intravitreal, and subretinal injections in small animals. Although the delivery of adeno-associated viral vectors for ocular gene therapy studies is described, the protocol herein can also be adapted for a variety of ophthalmic solutions in small animal models. The key practical steps in the administration route, setup for the injection platform, preparation of the injection, and tips from direct experience will be discussed in detail. In addition, common validation techniques for AAV delivery confirmation to the desired tissues will also be briefly discussed.

In this manuscript, subconjunctival injection is demonstrated as a valid vector delivery method for ocular tissues in mice using an injection system consisting of an infusion/withdrawal syringe pump and a gastight removable syringe coupled with microinjection needles. This injection system is also adaptable for other intraocular administration routes.

小动物模型中腺相关病毒载体的结膜下给药

Ocular diseases include a wide range of inherited genetic and acquired disorders that are appealing targets for local drug delivery due to their relative ...

医学

幼年大鼠腰椎穿刺 AAV9 递送用于 CNS 基因治疗

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.

作者聚焦：评估幼年大鼠的鞘内基因治疗疗效

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.

通过腰椎池注射在幼年大鼠中鞘内载体给药

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 ...

在成年小鼠中的亚型腺相关病毒9（AAV9）载体递送

摘要

探索更多视频

在小鼠心脏rAAV9的制备过表达或敲除基因

重组腺相关病毒9型 MicroRNA 系统分娩治疗啮齿动物神经肌肉疾病

球后窦注射对出生后、幼年和发育成年小鼠的全身治疗

球后窦注射对出生后、幼年和发育成年小鼠的全身治疗

球后窦注射对出生后、幼年和发育成年小鼠的全身治疗

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

重组腺相关病毒在成年小鼠中的直接注射

与腺相关病毒的向量的生产、纯化和质量控制

小动物模型中腺相关病毒载体的结膜下给药

通过腰椎池注射在幼年大鼠中鞘内载体给药