CN110172085A - 促转导素B（Protransduzin B）－基因转移的增强剂 - Google Patents
促转导素B（Protransduzin B）－基因转移的增强剂 Download PDFInfo
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Abstract
一种N端被保护的肽，具有如下序列：X‑Glu‑Cys‑Lys‑Ile‑Lys‑Gln‑Ile‑Ile‑Asn‑Met‑Trp‑Gln(SEQ ID NO:1)，其中，X为保护肽N端的基团。
Description
本申请是申请日为2014年4月30日、申请号为201480023747.7、发明名称为“促转导素B(Protransduzin B)－基因转移的增强剂”的发明专利申请的分案申请。
本发明涉及一种N末端被保护的肽，涉及含有所述肽的药物，涉及所述肽在基因治疗中的用途，涉及一种增强基因工程化病毒载体对细胞感染的方法，以及涉及所述肽在扩大转染或转导中的用途。
近年来，基因工程的重要性日益增加，因为在应用方法中所取得的重大进步，还因为不仅可以预测蛋白/肽类活性物质的生产，并且可以预测作为实验工具的、携带稳定基因的细胞转染，以及在细胞中引入基因作为基因缺陷的治疗手段最终将与许多疾病的治疗高度相关。
自从产生第一个人类基因的克隆和重组，由于基因转移的方法经历了持续不断的进步并且伴随着效率的增加，引入遗传材料以改变特定的细胞功能已经成为生物－医疗的基础性和应用性研究中必不可少的研究工具。基因引入的许多方法已经被优化。相应的经验已经积累了很多年的历史，这些经验的积累在最初是很慢的。
甚至在1953年F.克里克与J.沃森对脱氧核糖核酸(DNA)的功能进行阐述之前，F.格里菲斯(F.Griffith)就已经在20世纪20年代末成功完成了将非致病性肺炎球菌菌株转化为病原菌的实验。该转化是在一个幸运的情况下发生的，由于肺炎球菌具有罕见的DNA摄取的自然能力。将特定的DNA引入到原核生物中是由J.莱德伯格、M.德尔布吕克和S.Luria等人完成的，其中，借助于噬菌体，即所谓的转导。随着细胞培养体系的建立，在体外条件下培养真核细胞，已经开发了许多种用于转染的物理和化学方法。物理方法更为经常使用，然而需要更昂贵的设备，包括电穿孔和显微注射，它们与更简单适用的化学方法相竞争，如磷酸钙沉淀法(在20世纪80年代经常使用并至今仍使用)或基于阳离子脂质体或阳离子聚合物的方法(该方法在20世纪90年代早期被广泛使用)。然而，这些方法的使用总是依赖于细胞或DNA。并且，被引入细胞中的DNA通常在染色体外(瞬时转染)，因此它并没有传给子细胞。然而，噬菌体(如λ噬菌体)已知能够将它们的DNA整合到宿主基因组(前噬菌体、溶原性感染途径)。至此，它只是通往“建立逆转录酶病毒作为基因载体”(由Doehmer等人和Tabin等人)的一小步(1981/1982)。病毒是种类特异性和器官/组织特异性的，这就是为什么所有的病毒不能感染所有的(真核)细胞的原因。改变病毒包膜(糖蛋白交换，所谓的假型病毒)和添加主要为阳离子型的肽都被认为可提高转导效率。
在HIV的研究中，摄取病毒颗粒的第一代增强剂吸引了注意力。在对特定细胞的体外感染试验的分析过程中，观察到了血液的滤液肽对HIV融合的抑制作用(Munch等人，VIRIP)。
已经表明，出人意料的是，这些蛋白质片段是天然存在的，它们形成了纤维状结构，作为人类精子的增强剂，“衍生自精液的病毒感染增强剂”(“Semen derived Enhancerof Virus Infection"(SEVI))，其特征为淀粉样原纤维。这些纳米纤维提高了病毒与其靶细胞的对接，按10的数次方增加了病毒感染的比率。
这已用于改进逆转录病毒的基因转移，并用于基础研究以及可能的未来治疗应用。因此，能够表明，当SEVI蛋白存在下，用于基因治疗的慢病毒和γ-逆转录病毒，对不同的细胞类型(如人类T细胞、宫颈癌细胞、白血病细胞、造血干细胞、和胚胎干细胞)，均展现出高出许多倍的基因转移率(Wurm等人，J.Gene Med.2010，12，137-46；Wurm等人，Biol.Chem.2011,392,887-95)。
针对开发其他增强剂(比如，SEVI和精胶蛋白(seminogelin))的研究，产生了一种假设：来自病毒包膜蛋白的肽也可适于作为转染的增强剂，这令人惊奇地是一个出乎意料的巨大成功(MARAL Yolamanova，自然纳米技术)。因此表明，例如，与不同浓度(1-100微克/ml)的促转导素A(同义词:EF-C)进行预孵育的HIV，表现出对报道细胞的感染率有10的数次方的增加。至于作用机理，通常认为EF-C形成了纤丝结构，其能够结合和浓缩病毒，并相应地增加了进入细胞的病毒数量。除了用病毒颗粒进行感染，EF-C还高效地增强了慢病毒和逆转录病毒颗粒在应用于基因治疗的广泛多样的人细胞类型中(如T细胞、神经胶质细胞、成纤维细胞、造血干细胞)的转导(Jan Münch等，自然纳米技术，Vol.8,No.2,页码：130-136)。专利申请EP 2452947 A1也涉及了促转导素(protransduzin)A。
如上所述，由于基因技术的重要性日益增加，更有效的基因转移增强剂是所期望的。本发明的目的是提供一种改进的基因转移的增强剂。
出人意料地，已经发现，具有如下所示的序列的N端被保护的肽，实现了本发明的目的：
X-Glu-Cys-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln(SEQ ID NO:1)，
其中，X为保护肽的N端的基团。具体地，X代表1个或2个烷基，如甲基、乙基、丙基或丁基、酰基(如乙酰基或丙酰基)、或X-Glu基团为氨基酸焦谷氨酸：
出人意料地，已经发现，具体地，正是在体外用焦谷氨酸对N端进行的修饰(无细胞影响，尤其在酶的存在下)，导致了促转导素(protransduzin)在水溶液中的稳定性的极大提高。该结果可以从图1中清楚地看到。
在图1左栏(HPLC色谱图)中的在-20℃下储存0-13天和4℃(13天)的促转导素A的结果，与同等条件下的促转导素B的结果进行了比较。很明显，在4℃下储存13天后，促转导素A几乎降解至一半，而促转导素B在同样的储存条件下几乎完全没有降解(峰的高度对应于样本中所含的组分的浓度)。
在《生物化学期刊》(Journal of Biological Chemistry,Vol.286,No.45,页码：38825-38832)，S.Jawhar等人报道了关于淀粉样蛋白，尤其是焦谷氨酸修饰的淀粉样多肽的科学状态。这种淀粉样多肽含有大量的氨基酸，并且基本上不能与短链肽相比，而本发明所述的淀粉样蛋白也属于短链肽。顺便提及，该微评论涉及发生在体内条件下且酶存在下的细胞事件，这种体内条件与本发明中促转导素的稳定性得到提高的条件(即体外条件)是绝对没有可比性的。
本发明所述的肽还可以被用作药剂。
本发明还涉及本发明所述肽的用途，它被用于基因治疗，以治疗能够用基因治疗进行治疗的疾病。
本发明还涉及一种增强病毒对细胞的感染的方法，包括步骤：
提供权利要求1所述的肽，溶解于有机溶剂中；
将所述肽加入到水溶液中，以形成所述肽的不溶性聚集物；
混合上一步骤的溶液；和
培养细胞。
本发明还涉及本发明所述肽的用途，用于增强病毒对细胞的感染。
最后，含有本发明所述肽的试剂盒同样要求保护。
本发明所述的肽(促转导素B)可以，例如，根据Merrifield的方法，用Fmoc保护的氨基酸进行制备。
该方法可使用Fmoc保护的衍生物，即，用9-芴甲氧羰基保护的氨基酸，根据Merrifield原理进行逐步的固相合成，尤其在预负载了Fmoc-L-谷氨酰胺的、作为ABI-433合成仪上的固相载体的Wang树脂(0.59mmol/g，100-200目)上进行。
Fmoc－L－氨基酸的激活(典型地，Fmoc－L－氨基酸是10倍摩尔过量的)，是与[(2-(1H-苯并三唑-1-基)-1,1,3,3-四甲基脲六氟磷酸盐(HBTU，100mmol/l)在室温下进行的，并加入0.5M 1-羟基苯并三唑(HOBt)和2M二异丙基乙胺(DIEA)(溶于N-甲基-2-吡咯烷酮(NMP))。
各酰化反应进行45分钟，然后用20％的哌啶脱去Fmoc保护进行15分钟。
合成中还用到下述氨基酸衍生物以及相关的正交酸可裂解的侧链保护基团：
Fmoc-L-Asn(Trt)、Fmoc-L-Cys(Trt)、L-pGlu、Fmoc-L-Gln(Trt)、Fmoc-L-Ile、Fmoc-L-Lys(Boc)、Fmoc-L-Met和Fmoc-L-Trp(Boc)。
用94％三氟乙酸(TFA)、3％乙二硫醇(EDT)和3％的软化水从肽基树脂中裂解开树脂载体后，粗肽在冷的叔丁基甲基醚中沉淀，粗肽经离心获得，为沉淀物，而上清液被弃去。
随后的粗肽的色谱纯化，是通过梯度洗脱以制备型方式实现的。
专利申请EP2452947A1所述的促转导素A和促转导素B的区别，在于如下事实：促转导素B中，合成的L－焦谷氨酸(pGlu)被插入到N末端，以替换合成的L－谷氨酰胺(Gln)。原始的谷氨酰胺通过闭环被修饰成内酰胺。
纯化：
制备型分离：用购自于Gilson公司的HPLC进行纯化。UV/VIS探测器购自Kronwald公司，并且在波长230nm下检测分离。流速为40ml/min。
柱是Waters Prep-Pak C18柱(47x 300mm)。
洗脱液A：0.1％TFA(溶于软化水)，洗脱液B：0.1％TFA(溶于80％的乙腈和20％的软化水)。
促转导素B的梯度为35％-55％的洗脱液B，洗脱40分钟。即，每分钟0.5％的洗脱液B。
促转导素B在40％的洗脱液B中洗脱，在0.5-1分钟收集数个洗脱组分。经分析的纯的洗脱组分被合并且冻干。
应用方法：
冻干法：
购自Christ公司的、技术参数设置如下的单元Epsilon 1/45，被用于冷冻干燥：搁板面积3.78m2；冰容量约60kg；冰凝汽器性能最大值45kg/24h，真空泵的终分压为1×10-3/10-4毫巴(在有/无气镇的情况下)；冻干数据(用气镇手动操作单元)；终分压为1×10-2毫巴；冰冷凝器温度－50℃；搁板温度+15℃；搁板加热的工作点0.5毫巴；冷冻干燥时间长达3天。
用促转导素B进行细胞转导
溶解0.5mg的促转导素B于50μl DMSO。然后向该溶液中加入450μl的PBS，3分钟内形成细纤维状物质。将上述原液(1mg/ml)加入到载体中，获得浓度为25μg/ml的促转导素B。涡旋溶液1分钟，然后5000g离心5分钟。弃去上清液，将沉淀悬浮于少量PBS，并添加至细胞中。细胞在保温箱内孵育2天。
促转导素B显著增强了转导率。借助促转导素B，高达96％的细胞被转导。
序列表
<110> 法瑞斯生物技术有限公司
<120> 转导素B（Protransduzin B）－基因转移的增强剂
<130> 140997wo
<150> EP13166266.0
<151> 2013-05-02
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 12
<212> PRT
<213> 人工序列(artificial sequence)
<400> 1
Glu Cys Lys Ile Lys Gln Ile Ile Asn Met Trp Gln
1 5 10
Claims (6)
1.一种N端被保护的肽，具有如下序列：
X-Glu-Cys-Lys-Ile-Lys-Gln-Ile-Ile-Asn-Met-Trp-Gln，
其中，X为保护肽的N端的基团，X为1个或2个烷基，例如甲基、乙基、丙基、或丁基、酰基，例如乙酰基或丙酰基。
2.一种药剂，其特征在于，所述药剂含有权利要求1所述的肽。
3.如权利要求1所述的肽在基因治疗方面的用途，其特征在于，用于治疗可以用基因治疗进行治疗的疾病。
4.一种增强病毒对细胞的感染的方法，包括步骤：
提供权利要求1所述的肽，溶解于有机溶剂中；
将所述肽加入到水溶液中，以形成所述肽的不溶性聚集物；
混合上一步骤的溶液；和
培养细胞。
5.如权利要求1所述肽的用途，其特征在于，用于增强病毒对细胞的感染。
6.一种试剂盒，其特征在于，含有权利要求1所述的肽。
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| EP13166266.0 | 2013-05-02 | ||
| EP13166266 | 2013-05-02 | ||
| CN201480023747.7A CN105209480B (zh) | 2013-05-02 | 2014-04-30 | 促转导素B(Protransduzin B)－基因转移的增强剂 |
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| CN201910487826.8A Pending CN110172085A (zh) | 2013-05-02 | 2014-04-30 | 促转导素B（Protransduzin B）－基因转移的增强剂 |
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| US20210154327A1 (en) | 2017-08-29 | 2021-05-27 | Pharis Biotec Gmbh | Protransduzin-d - improved enhancer of gene transfer |
| EP4067375A1 (en) | 2021-03-30 | 2022-10-05 | Universität Ulm | Fibril peptides |
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| US20120122177A1 (en) * | 2010-11-16 | 2012-05-17 | Frank Kirchhoff | Viral Infection Enhancing Peptide |
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| CN1293204A (zh) * | 1999-10-18 | 2001-05-02 | 上海博容基因开发有限公司 | 一种新的多肽－人溴基功能域蛋白72和编码这种多肽的多核苷酸 |
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| WO2009032702A2 (en) | 2007-08-28 | 2009-03-12 | Uab Research Foundation | Synthetic apolipoprotein e mimicking polypeptides and methods of use |
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| US9944675B2 (en) * | 2011-09-01 | 2018-04-17 | University Of Southern California | Methods for preparing high throughput peptidomimetics, orally bioavailable drugs and compositions containing same |
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| Publication number | Publication date |
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| HRP20181138T1 (hr) | 2018-09-21 |
| EP3412681B1 (de) | 2024-04-17 |
| CA2909744A1 (en) | 2014-11-06 |
| US20190100557A1 (en) | 2019-04-04 |
| AU2014261381A1 (en) | 2015-11-12 |
| AU2018202618A1 (en) | 2018-05-10 |
| CN105209480A (zh) | 2015-12-30 |
| WO2014177635A1 (de) | 2014-11-06 |
| JP6667432B2 (ja) | 2020-03-18 |
| AU2014261381B2 (en) | 2018-01-18 |
| EP3412681A1 (de) | 2018-12-12 |
| LT2992004T (lt) | 2018-08-10 |
| CY1120511T1 (el) | 2019-07-10 |
| US20160185820A1 (en) | 2016-06-30 |
| CA2909744C (en) | 2021-06-15 |
| TR201809579T4 (tr) | 2018-07-23 |
| PT2992004T (pt) | 2018-10-19 |
| HUE040476T2 (hu) | 2019-03-28 |
| RS57506B1 (sr) | 2018-10-31 |
| ES2681424T3 (es) | 2018-09-13 |
| EP2992004B1 (de) | 2018-06-06 |
| US10125169B2 (en) | 2018-11-13 |
| DK2992004T3 (da) | 2018-09-17 |
| PL2992004T3 (pl) | 2018-10-31 |
| JP2016524594A (ja) | 2016-08-18 |
| CN105209480B (zh) | 2019-08-16 |
| SI2992004T1 (en) | 2018-08-31 |
| EP2992004A1 (de) | 2016-03-09 |
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