Name: evaluation of exon inclusion induced by splice switching antisense oligonucleotides in sma patient fibroblasts
Uploaded: 2018-05-11T14:30:00
Description: Watch this Scientific Journal Video about Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts at JoVE.com

作者感谢妮可 McRorie 的帮助与实验。这项工作得到了阿尔伯塔大学医学院和牙科学院的支持, Slipchuk SMA 研究基金会研究基金, 加拿大卫生研究院 (卫生研究院), 加勒特的朋友卡明研究基金会, Toupin 神经学科研经费、加拿大肌肉萎缩症、加拿大创新基金会、艾伯塔省企业和高级教育以及妇女儿童健康研究所 (WCHRI)。

1. 细胞培养
<ol><li>培养 SMA 患者成纤维细胞生长培养基 (Dulbecco 的改良鹰中等 1:1 F-12 (火腿) 与10% 胎牛血清 (血清) 和0.5% 青霉素/链霉素) 在 CO2孵化器在37°c。</li>
	<li>使用自动细胞计数器和稀释细胞到生长培养基中的 1 x 105细胞/mL, 然后在适当的板块上播种, 确定细胞浓度。使用12井板 (每井1毫升) 用于 rt-pcr 或 qPCR。</li>
	<li>在 CO2孵化器中, 在37摄氏度孵育24小时的板材。</li>
<...

<ol>
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这里描述的体外评估方法是快速、容易和敏感的, 足以测试各种 AONs。该协议广泛适用于外显子的跳跃和包含其他基因和各种细胞类型。此外, 大多数的怡人化学 (PMOs 除外) 可以用这种方法转染。AONs 可以调节内源和人工导入基因的前 mRNA 剪接, 并可与携带靶向基因的质粒载体进行联合转染。
该方法的一个关键步骤是, 当 AONs 转染成纤维细胞时, 细胞融合应约 65-80%。对于?...

脊髓肌萎缩 (SMA) 是一种遗传的致命神经肌肉疾病遗传性的常染色体隐性模式。它的特点是运动神经元的退化和渐进躯干和肢体肌肉麻痹1,2。大多数 SMA 的发生是由于在存活的马达神经元 1 (SMN1) 基因3的纯合突变。运动神经元 2 (SMN2) 基因的存活是SMN1的反向复制, 其几乎相同的序列与仅五个基4、5不同。位于外显子7中的SMN2中的 C 到 T 转换使该基因几乎无法正常进行, 因为该突变导致了在几乎90% 的SMN2记录 (图 1a) 中排除的必需的外显子7。SMN2基因缺少的外显子7无法补偿SMN1函数, 因为其蛋白质产品不稳定且快速降解。
反义疗法最近成为治疗 SMA6的非常有前途的策略。美国食品和药物管理局 (FDA) 最近批准了 nusinersen, 使它成为治疗 SMA7的第一种药物。Nusinersen 是一个18的反义寡核苷酸 (怡人) 与-2′-乙修改 (教育部) 和硫代核苷酸骨干。该药物的目标是内含子剪接消声器 N1 (ISS-N1) 位于 SMN2 基因的内含子 7. nusinersen 对 ISS-N1 的约束力通过诱导外显子7包含 (图 1)8,9,10, 促进从内源SMN2基因中恢复功能性全长 SMN 蛋白表达..目前, 对 SMA 治疗的许多研究都集中在研究新的可比 nusinersen 更有效和更少毒性的新型怡得化学药物。已经证明, 与其他化学成分的 AONs 也有效地诱导外显子包含在SMN2显子7两个在体内外和在体内11,12,13。
锁定核酸 (LNAs) 是化学修饰的 RNA 类似物, 包含一个亚甲桥连接 4′--碳与 furanose 结构内的-2′--氧(图 1b)14,15。与 dna 或 rna 相比, LNAs 具有更强的亲和力, 可以结合到互补 RNA 序列中, 并且对内源性核酸具有高度抵抗力。低噪声放大化学已被应用作为探针荧光原位杂交 (鱼) 和在 qPCR16,17。此外, 它被用来调节基因表达的在体外和在体内。GapmeR AONs 是单链 DNA 分子在5′和3′端两侧由几个 LNAs 的组合。它们通过对目标基因进行绑定互补来降低基因表达, 从而使它们被激活的 RNase H18所降解。低噪声 AONs/dna mixmers 是由 LNAs 之间集成的 dna 核苷酸组成的。在这个方向上, 它们可以绑定 miRNA 以抑制其功能 (低噪声 antimiR)19。一些低噪声 antimiRs 已达到临床发展。举例来说, miravirsen (AntimiR-122) 是一个低噪声的 AntimiR, 抑制 miR-122 治疗丙型肝炎感染, 而第二阶段临床试验目前正在进行19,20。最近, 已经证明, 低噪声/DNA mixmers 也可以调制 RNA 拼接21。它可以绑定一个特定序列的 mrna 和诱导外显子跳过肌萎缩蛋白 mrna 和显子包含在SMN2 mRNA在体内13,22.
在本文中, 我们概述了一种方法, 以诱导外显子夹杂使用 AONs 和评价的功效在 RNA 和蛋白质水平。为了举例说明这种方法, 我们在SMN2的内含子7中使用了低噪声/DNA mixmers 靶向 ISS-N1。AONs 转染 SMA 患者细胞的 lipotransfection 诱导外显子7包含在最后的SMN2转录和上调 SMN 蛋白生产。利用低噪声/DNA mixmers 诱导外显子夹杂的优点之一是, 转染的有效浓度明显低于其他化学试剂13。该方法除 phosphorodiamidate 吗啉代寡聚物 (PMOs) 外, 还可用于其他许多 AONs, 由于其中性电荷, 需要通过内-波特共转染试剂诱导的吞进入细胞。

<table border="0" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
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		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">SMA Fibroblasts</td>
			<td style="width:153px;">Coriell NIGMS human genetic cell repository</td>
			<td style="width:119px;">GM03813</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">11320033</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">F1051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15140122</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA (0.05%), phenol red</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">25300062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Serum-deprived media</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">31985070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Transfection reagent</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15338100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">guanidinium thiocyanate-phenol-chloroform (TRIzol)</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">15596-018</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RT-PCR Primers</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>Sequence 5&#39; to 3&#39;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SMN2 forward:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>CTGCCTCCATTTCCTTCTG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SMN2 reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>TGGTGTCATTTAGTGCTGCTC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH forward:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>TCCCTGAGCTGAACGGGAAG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>GGAGGAGTTTGGTCGCTGT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">qPCR Primers</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">full-length SMN2 forward:</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>GCTATCATACTGGCTATTATATGGGTTTT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">full-length SMN2 reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>CTCTATGCCAGCATTTCTCCTTAAT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#8710;7&#160;SMN2 forward:</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>TCTGGACCACCAATAATTCCCC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">&#8710;7&#160;SMN2 reverse:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>ATGCCAGCATTTCCATATAATAGCC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH forward:&#160;</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>GCAAATTCCATGGCACCGT</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GAPDH reverse:</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>AGGGATCTCGCTCCTGGAA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:153px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3803</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen, RNA grade</td>
			<td style="width:153px;">Thermo Fisher</td>
			<td style="width:119px;">R0551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ImageJ Software</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Protease cocktail inhibitor&#160;</td>
			<td style="width:153px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cathode Buffer</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>0.025 M Tris base + 40&#160;mM 6-aminocaproic acid + 20% Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Anode Buffer</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>0.03 M Tris Base + 20% Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Concentred Anode Buffer</td>
			<td style="width:153px;"></td>
			<td style="width:119px;"></td>
			<td>0.3 M Tris base + 20% Methanol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Beta Mercaptoethanol&#160;</td>
			<td style="width:153px;">Millipore</td>
			<td style="width:119px;">ES-007-E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PVDF membrane&#160;</td>
			<td style="width:153px;">GE</td>
			<td style="width:119px;">10600021</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Loading/sample buffer for Western blotting</td>
			<td style="width:153px;">NuPage Invitrogen</td>
			<td style="width:119px;">NP007</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">One-Step RT-PCR kit&#160;</td>
			<td style="width:153px;">Qiagen&#160;</td>
			<td style="width:119px;">210210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dNTPs</td>
			<td style="width:153px;">Clontech</td>
			<td style="width:119px;">3040</td>
			<td></td>
		</tr>
	</tbody>
</table>

evaluation of exon inclusion induced by splice switching antisense oligonucleotides in sma patient fibroblasts

脊髓肌萎缩 (SMA), 一个致命的神经系统疾病造成的损失的SMN1, 提出了一个独特的情况下, 反义寡核苷酸 (怡) 介导治疗领域。虽然SMN1突变负责该疾病, 但已显示在SMN2(包括 FDA 批准的 nusinersen) 中的 AONs 目标内含子拼接消声器 (ISS) 站点已被证明可以恢复 SMN 表达式并改善症状。目前, 对 SMA 治疗的许多研究都侧重于调查以SMN2为目标的新型怡得化学药物, 其效果可能比 nusinersen 更有效、毒性更小。在这里, 我们描述了一个体外的协议, 利用 AONs 的 lipotransfection 进行反向转录聚合酶链反应 (rt-pcr)、定量聚合酶链反应 (qPCR) 和西方印迹的研究, 对显子夹杂物进行评价。这种方法可用于各种类型的安怡化学。使用这种方法, 我们证明由交替锁定核酸 (LNAs) 和 dna 核苷酸 (低噪声/dna mixmers) 组成的 AONs, 导致有效的SMN2外显子包含和恢复 SMN 蛋白在极低浓度, 因此, 放大器/基于 DNA mixmer 的反义寡核苷酸可能是治疗遗传疾病引起的剪接缺损的一种很有吸引力的治疗策略。这里描述的体外评估方法是快速、容易和敏感的, 足以测试各种新颖的 AONs。

利用 SMA 患者成纤维细胞, 我们瞄准了SMN2基因内含子7中的 ISS-N1 消声器区域, 八种不同的反义放大器/DNA mixmers 被转染为 5 nM 浓度 (图 3)。每一个 mixmers 包含一个修改过的 phosphorothioated 骨干, 使他们能够抵御核酸酶退化。为了评估 mixmers 的有效性, 通过 rt-pcr 和 qPCR, 使用SMN2的引物, 量化了SMN2外显子7包含率。外显子7的包含效率从 78-98%...

Watch this Scientific Journal Video about Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts at JoVE.com

Evaluation of Exon Inclusion Induced by Splice Switching Antisense Oligonucleotides in SMA Patient Fibroblasts

剪接开关反义寡核苷酸诱导的 SMA 患者成纤维细胞外显子夹杂的评价

各种反义寡核苷酸 (AONs) 被证明可以诱导外显子夹杂 (剪接调制) 和抢救 SMN 表达的脊髓肌萎缩 (SMA)。在这里, 我们描述了一个协议, 以 lipotransfection 诱导外显子包含在SMN2基因和评价方法, 以确定的有效性, SMA 患者成纤维细胞。

Spinal muscular atrophy (SMA), a lethal neurological disease caused by the loss of SMN1, presents a unique case in the field of antisense oligonucleotide ...

Research

JoVE Journal

Medicine

Vascular Occlusion Training for Inclusion Body Myositis: A Novel Therapeutic Approach

血管闭塞包涵体肌炎培训：一种新的治疗方法

Inclusion body myositis (IBM) is a rare idiopathic inflammatory myopathy. It is known to produces remarkable muscle weakness and to greatly compromise ...

科研

医学

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

Mouse Model of Intraluminal MCAO: Cerebral Infarct Evaluation by Cresyl Violet Staining

小鼠模型的腔内MCAO脑梗塞评价甲酚紫染色

Stroke is the third  cause of mortality and the leading cause of disability in the World. Ischemic  stroke accounts for approximately 80% of all strokes. ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

多巴酚丁胺诱导的心脏压力测试和监控通过在小鼠心脏导管插入术

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

评价肺转移的小鼠乳腺肿瘤模型定量实时荧光定量PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy

多外显子跳跃在犬X连锁肌营养不良用鸡尾酒反义寡核苷酸

Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon ...

A Model of Glaucoma Induced by Circumlimbal Suture in Rats and Mice

Circumlimbal 缝合法诱导大鼠和小鼠青光眼模型的建立

The circumlimbal suture is a technique for inducing experimental glaucoma in rodents by chronically elevating intraocular pressure (IOP), a well-known ...

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

Quantitative [18F]-Naf-PET-MRI Analysis for the Evaluation of Dynamic Bone Turnover in a Patient with Facetogenic Low Back Pain

定量 =18F_-Naf-PET-MRI 分析,用于评估面部腰痛患者的动态骨质周转率

Imaging techniques that reflect dynamic bone turnover may aid in characterizing a wide range of bone pathologies. Bone is a dynamic tissue undergoing ...

Invasive Hemodynamic Assessment for the Right Ventricular System and Hypoxia-Induced Pulmonary Arterial Hypertension in Mice

小鼠右心室系统及缺氧诱发肺动脉高血压的侵入性血液动力学评估

Pulmonary arterial hypertension (PAH) is a chronic and severe cardiopulmonary disorder. Mice are a popular animal model used to mimic this disease. ...

Characterizing Exon Skipping Efficiency in DMD Patient Samples in Clinical Trials of Antisense Oligonucleotides

反义寡核苷酸临床试验中DMD患者样本的Exon跳过效率特征

Duchenne muscular dystrophy (DMD) is a degenerative muscle disease that causes progressive loss of muscle mass, leading to premature death. The mutations ...