CN111050850A - 用于用表达lekti的重组微生物治疗内瑟顿综合征的组合物和方法 - Google Patents
用于用表达lekti的重组微生物治疗内瑟顿综合征的组合物和方法 Download PDFInfo
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- CN111050850A CN111050850A CN201880052930.8A CN201880052930A CN111050850A CN 111050850 A CN111050850 A CN 111050850A CN 201880052930 A CN201880052930 A CN 201880052930A CN 111050850 A CN111050850 A CN 111050850A
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Abstract
本公开内容尤其提供表达重组LEKTI结构域的经工程改造的微生物,所述重组LEKTI结构域有效治疗或改善内瑟顿综合征的症状。在某些实施方案中,提供包含表达LEKTI结构域的微生物的组合物、方法和试剂盒。
Description
相关申请
本申请要求于2017年6月16日提交的美国临时申请62/521,050的优先权,所述临时申请的全部内容通过引用以其整体结合到本文中。
公开内容的领域
本公开内容涉及用于使用经遗传修饰以在受试者的皮肤上表达一个或多个治疗性LEKTI结构域的一种或多种重组微生物治疗内瑟顿综合征(Netherton Syndrome)或改善其影响的方法、试剂盒和组合物。
发明背景
表皮是皮肤的鳞状复层上皮,由多个亚层组成,是身体抵抗外界的最重要屏障之一。角质层是表皮的最外层,由于角质形成细胞从有核表皮层中的细胞分化的最终无核步骤而形成。尽管认为角质层是最重要的物理屏障,但有核表皮层在屏障功能上也重要(Proksch,Brandner等2008)。总之,皮肤屏障保护免于一个方向上的大量水分流失(内外屏障)和免于来自环境中的有害物质入侵(外内屏障)(Proksch,Brandner等,2008)。屏障的维持对于基底层的平衡增殖和钙离子梯度的保持以及因此适当的表皮分化也重要(Lee, Jeong等2006)。
在皮肤治疗中存在许多当前的局限性。许多治疗,例如局部皮质类固醇或生物制剂,不治疗表皮中缺乏固有蛋白质或皮肤中微生物多样性失衡的根本问题。尽管重组蛋白在皮肤疾病的治疗中代表着有希望的一组治疗剂,但其在皮肤情况下的使用伴随着一些问题。
传统方法纯化和浓缩从细菌系统中提取的重组蛋白,然后将这样的制备物掺入递送系统中。重组蛋白的纯化常常是非常昂贵的获得蛋白的方法。此外,与这些传统方法关联许多问题,包括蛋白水解降解、无效率的递送和需要超时重复施用以达到治疗效果。
将会受益于改善的治疗方式的一种皮肤疾病为内瑟顿综合征(NS)。NS是一种罕见的常染色体皮肤疾病,表现为严重的皮肤炎症和脱屑、毛干缺陷、持续的过敏症状和免疫系统问题。患有NS的新生儿经常有红色和鳞屑状的皮肤,其可能渗漏液体,这造成皮肤或整个身体脱水和感染的风险。患病儿童也可能无法以正常速度生长。患有NS的较大儿童和成年人的健康通常改善,但那些个体常常体重不足且身材矮小。大多数具有NS的人也有免疫系统问题,例如食物过敏、枯草热、哮喘或湿疹。
NS由基因SPINK5 (5型kazal丝氨酸蛋白酶抑制剂)的功能丧失缺陷引起,该基因编码5型淋巴上皮kazal型相关抑制剂(LEKTI)蛋白。LEKTI为多结构域丝氨酸蛋白酶抑制剂,正常地在所有分层的上皮细胞和胸腺的Hassal小体中表达。编码LEKTI的SPINK5基因位于5号染色体上其它SPINK基因(例如SPINK6和SPINK9)的簇中,包含33个外显子,其编码由接头区域分隔的15个抑制结构域。SPINK5由于其编码的大量抑制结构域而在其它SPINK基因中脱颖而出。此外,SPINK5基因被转录成三种不同的转录物,导致C端区域不同的三种不同的LEKTI蛋白;即具有抑制结构域D1-D15的145 kDa全长蛋白、具有抑制结构域D1-D12的125 kDa (短)蛋白和具有扩展的接头区域13的148 kDa (长)蛋白。
LEKTI蛋白为Kazal型相关抑制剂。Kazal基序通过存在6个半胱氨酸残基定义,这些残基以特定的距离定位以允许以1-5、2-4和3-6模式形成三个二硫键。LEKTI的两个结构域(D2和D5)形成了这6个半胱氨酸基序,而其它结构域共享四个半胱氨酸残基,其产生一个刚性抑制环,认为该环模拟靶蛋白酶的底物并使靶蛋白酶催化位点失活。
LEKTI蛋白需要蛋白水解切割以激活其对许多蛋白酶的抑制功能。具体而言,全长蛋白质被切割成结构域D1-D5和D6-D15。D6-D15结构域然后进一步在多个步骤中被切割成D6-D9和D10-D15、→ D6和D7-D9→ D7和D8-D9 → D8。该过程导致包含1-6个抑制结构域的LEKTI蛋白,每种蛋白具有不同的抑制功能。例如,各种LEKTI抑制片段可以抑制各种激肽释放酶相关肽酶(KLK),例如KLK5、KLK7和KLK14。
缺陷的LEKTI蛋白可由于SPINK5基因的取代、插入或缺失突变而引起,所述突变常常导致无义或移码突变,导致提前终止密码子。剪接位点碱基的其它突变可导致转录的SPINK5基因的异常剪接事件。因此,许多SPINK5突变导致完全没有LEKTI结构域合成。LEKTI缺乏或有缺陷的LEKTI可导致蛋白酶活性失调,通过表皮分化和脂质代谢受损导致皮肤脱皮和表皮渗透,这导致有缺陷的皮肤屏障。此外,一些KLK蛋白的不受调节的活性导致桥粒切割和角质层脱离。
内瑟顿综合征是一种孤儿疾病,没有可用的特定治疗。鉴于前述,需要用于治疗NS的新型治疗剂。本申请旨在满足这些和其它需求。
发明概述
根据一个方面,本公开内容提供用于治疗皮肤疾病的组合物,所述组合物包含经遗传修饰以在哺乳动物的皮肤上表达和提供一个或多个LEKTI蛋白结构域的微生物,其中所述LEKTI蛋白结构域有效渗透哺乳动物皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的丝氨酸蛋白酶活性。
根据一些实施方案,所述微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间以提供LEKTI蛋白结构域的连续供应。根据一些实施方案,所述LEKTI蛋白结构域有效改善内瑟顿综合征的症状。在一个实施方案中,所述LEKTI结构域为结构域6。
根据一些实施方案,所述微生物通过用编码所述LEKTI蛋白结构域的重组DNA质粒转染/转化而遗传修饰。在一些实施方案中,LEKTI结构域与有效增强从微生物分泌和/或渗透哺乳动物的皮肤的一个或多个重组蛋白结构域可操作地连接。根据一些实施方案,至少一个LEKTI结构域与SecA结构域可操作地连接。根据一些实施方案,至少一个LEKTI结构域与RMR结构域可操作地连接。
根据一些实施方案,至少一个LEKTI结构域包含根据SEQ ID NO: 1的氨基酸序列。
根据一些实施方案,所述微生物适应于在哺乳动物的皮肤上繁殖。
根据一些实施方案,至少一个LEKTI结构域的表达受操纵子控制并且提供给哺乳动物的皮肤的LEKTI的量与外在因素的可用性成比例。在一些实施方案中,至少一个LEKTI结构域的表达受组成型活性的启动子控制。
根据一些实施方案,所述微生物已经通过用编码所述LEKTI蛋白结构域和一个或多个抗生素抗性基因的重组DNA质粒转染/转化而遗传修饰。
根据一些实施方案,所述微生物选自双歧杆菌属(Bifidobacterium)、短杆菌属(Brevibacterium)、丙酸杆菌属(Propionibacterium)、乳球菌属(Lactococcus)、链球菌属(Streptococcus)、葡萄球菌属(Staphylococcus)、乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)、片球菌属(Pediococcus)、明串珠菌属(Leuconostoc)或酒球菌属(Oenococcus)及其混合物。
根据一个方面,本公开内容提供治疗有需要的哺乳动物的皮肤疾病或改善有需要的哺乳动物的皮肤疾病的影响的方法,所述方法包括向哺乳动物的皮肤的表面上提供经遗传修饰以表达一个或多个LEKTI蛋白结构域的微生物,其中所述LEKTI蛋白结构域有效渗透哺乳动物皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的活性。
根据一些实施方案,所述微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间以提供LEKTI蛋白结构域的连续供应。
根据另一个方面,本公开内容提供用于治疗有需要的哺乳动物的皮肤疾病或改善有需要的哺乳动物的皮肤疾病的影响的试剂盒,其包含(1) 包含经遗传修饰以表达一个或多个LEKTI蛋白结构域的微生物的组合物,其中所述LEKTI蛋白结构域有效渗透哺乳动物皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的丝氨酸蛋白酶活性,和(2) 用于将所述组合物施用到哺乳动物的皮肤的试剂。
根据一些实施方案,所述微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间以提供LEKTI蛋白结构域的连续供应。
根据一个方面,本公开内容提供用于治疗皮肤疾病的组合物,其包含含有pJB38-LEKTI-完整质粒构建体的微生物。
根据一些实施方案,所述微生物选自双歧杆菌属、短杆菌属、丙酸杆菌属、乳球菌属、链球菌属、葡萄球菌属、乳杆菌属、肠球菌属、片球菌属、明串珠菌属或酒球菌属及其混合物。
根据一个方面,本公开内容提供包含pJB38-LEKTI-完整质粒构建体的组合物。在一些实施方案中,pJB38-LEKTI-完整质粒构建体在选自双歧杆菌属、短杆菌属、丙酸杆菌属、乳球菌属、链球菌属、葡萄球菌属、乳杆菌属、肠球菌属、片球菌属、明串珠菌属或酒球菌属及其混合物的微生物中表达。
附图简述
图1显示包含本发明的治疗性LEKTI结构域的载体构建体。质粒的蛋白编码区域包括SecA、6xHis标签、LEKTI D8-11和RMR标签,它们彼此可操作地连接并且处于CmR启动子的控制下。
图2显示根据本发明的一些实施方案的pJB38质粒的载体构建体。
图3为显示全长LEKTI多肽的结构域的示意图。
图4显示SDS-PAGE结果,其证明LEKTId6在大肠杆菌(E.coli) BL21 (De3)中高度可溶。
图5显示SDS-PAGE结果,其证明成功亲和纯化H6-LEKTId6 (8.8 kDa)。
图6显示SDS-PAGE结果,其证明LEKTId6-H6 (8.8 kDa)为潜在地N-端截短的。
图7A和图7B显示重组生产的LEKTI结构域6在体外抑制胰蛋白酶。图7A为所进行的实验的示意图。图7B为显示胰蛋白酶活性的图。
图8A和图8B显示与LEKTI结构域10-15相比重组生产的LEKTI结构域6 (ct His6标签)在体外抑制胰蛋白酶。图8A为所进行的实验的示意图。图8B为显示胰蛋白酶活性的图。
图9A和图9B显示重组生产的LEKTI结构域6在体外抑制KLK7,与LEKTI结构域10-15对KLK7的抑制相似。图9A为所进行的实验的示意图。图9B为显示KLK7活性的图。
图10A和图10B显示重组生产的LEKTI结构域6在体外以纳摩尔浓度抑制KLK5。图10A为所进行的实验的示意图。图10B为显示KLK5活性的图。
发明详述
本公开内容的一个方面提供皮肤定殖细菌,其经遗传改变以表达重组蛋白以治疗或改善内瑟顿综合征。遗传改变的蛋白生产细菌能够通过表达和任选地分泌治疗NS或其症状的潜在原因的治疗性蛋白治疗所述疾病。根据一些实施方案,治疗性蛋白包含有效抑制哺乳动物的皮肤内或皮肤上的丝氨酸蛋白酶的一个或多个LEKTI结构域。根据一些实施方案,重组LEKTI结构域补偿哺乳动物中皮肤天然产生的有缺陷的内源性LEKTI蛋白。根据一些实施方案,遗传改变的细菌能够自我复制同时保留产生重组蛋白的能力,从而提供连续的治疗剂供应。
根据一些实施方案,本公开内容提供用于治疗皮肤疾病的组合物,其包含经遗传修饰以在哺乳动物的皮肤上表达和提供一个或多个LEKTI蛋白结构域的微生物,其中所述LEKTI蛋白结构域有效渗透哺乳动物皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的丝氨酸蛋白酶活性。
如本文所使用的术语“皮肤疾病”和其语法上的变化意指与人皮肤的正常或基线状况相比一般不期需或有害的皮肤状态或状况。异常的皮肤状况的实例包括,但不限于,内瑟顿综合征、银屑病、痤疮、特应性皮炎、过敏性接触性皮炎、表皮松解性角化过度、脂溢性皮炎、湿疹、皮肤干燥、过敏、疹、紫外线刺激皮肤、去污剂刺激的皮肤(包括由洗涤去污剂中使用的酶和分子以及月桂基硫酸钠引起的刺激)、皮肤变薄(例如老年人和儿童的皮肤)、大疱性类天疱疮、寻常型天疱疮、脓疱病、白癜风、脱发和多毛症。
如本文所使用的,术语“遗传修饰的”和其语法变化用于描述通过引入在微生物外制备的DNA遗传修饰或工程改造的微生物有机体(例如细菌)。例如,向细菌中引入包含新基因的质粒DNA将会允许细菌表达那些基因。或者,可将包含新基因的DNA引入细菌,然后整合入细菌的基因组,其中细菌将会表达那些基因。
如本文所使用的,术语“治疗”("treat"、"treating"、"treatment")和其语法上的变化意指提供给受试者方案、养生法、方法或疗法,其中期需在该受试者例如患者中获得生理反应或结果。特别地,本发明的方法和组合物可用于减缓疾病症状的发展或推迟疾病或病况的发病,或停止疾病发展的进展。然而,由于每一治疗的受试者可能不响应特定的治疗方案、养生法、方法或疗法,治疗不需要在每个和所有受试者或受试者群体例如患者群体中取得期需的生理反应或结果。相应地,给定的受试者或受试者群体例如患者群体可能不能响应或不适当地响应治疗。
在本发明中,受试者可为哺乳动物。如本文所使用的,“哺乳动物”和其语法上的变化意指任何哺乳动物类别。在本发明中,哺乳动物包括,例如,人、农场动物、家养动物、实验动物等。农场动物的一些实例包括奶牛、猪、马、山羊等。家养动物的一些实例包括狗、猫等。实验动物的一些实例包括灵长类、大鼠、小鼠、兔、豚鼠等。优选地,哺乳动物为人。
如本文所使用的,术语本文所公开的化合物或组合物的“有效量”或“治疗有效量”为当给予受试者时足以招致如本文所描述的有益的或期需的结果的这种化合物或组合物的量。有效的剂型、给予模式和剂量可依据经验确定,做出这种确定在本领域的技能内。本领域技术人员理解,剂量将会随给予途径、排泄率、治疗持续时间、给予的其它药物的身份、哺乳动物例如人患者的年龄、尺寸和物种以及医学和兽医学领域所熟知的类似因素不同而不同。一般而言,本发明的组合物的合适剂量会为作为有效产生期需效果的最低剂量的组合物量。本发明的组合物的有效剂量可作为2、3、4、5、6或更多个亚剂量给予,在全天内以适当的间隔分开给予。
微生物组合物:根据一些实施方案,本公开内容提供包含一种或多种适于在哺乳动物的皮肤上使用的宽范围的细菌的微生物组合物。实例包括,但不限于,非病原性和共生细菌。适于在本发明中使用的细菌包括,但不限于,双歧杆菌属、短杆菌属、丙酸杆菌属、乳球菌属、链球菌属、葡萄球菌属(例如,表皮葡萄球菌(S. epidermidis)和/或人葡萄球菌(S. hominis))、乳杆菌属(例如,嗜酸乳杆菌(L. acidophilus))、片球菌属、明串珠菌属或酒球菌属。根据一些实施方案,微生物组合物包含瓦氏葡萄球菌(Staphylococcuswarneri)、酿脓链球菌(Streptococcus pyogenes)、缓症链球菌(Streptococcus mitis)、痤疮丙酸杆菌(Propionibacterium acnes)、棒杆菌属(Corynebacterium)物种、约氏不动杆菌(Acinetobacter johnsonii)、铜绿假单胞菌(Pseudomonas aeruginosa)中的一种或多种。根据一些实施方案,使用皮肤上存在的其它相关或相似物种。
某些实施方案涉及细菌表皮葡萄球菌的用途。根据一些实施方案,所使用的表皮葡萄球菌的菌株不能产生生物膜。其实例为表皮葡萄球菌菌株ATCC 12228或NRRL B-4268。
根据一些实施方案,重组微生物适应于在哺乳动物的皮肤的表面上无限期地存活或存活受控的持续时间以提供LEKTI蛋白结构域的连续供应。在一些实施方案中,重组微生物与哺乳动物的皮肤上天然存在的共生微生物一起存活。在一些实施方案中,重组微生物排斥哺乳动物的皮肤上天然存在的共生微生物而存活。根据一些实施方案,重组微生物适应于在哺乳动物的皮肤上繁殖。在其它实施方案中,重组微生物不再活着,但包含有效量的治疗多肽例如LEKTI或其治疗有效的结构域。这样的细胞可为完整的或不依赖于向靶位点递送治疗肽(或其结构域)的细节。
如本文所使用的,术语“重组”和其语法上的变化意指与通过或使用包含来自不同来源或来自同一来源的不同部分的DNA片段的重组DNA形成的有机体、蛋白或遗传材料有关,或表示通过或使用包含来自不同来源或来自同一来源的不同部分的DNA片段的重组DNA形成的有机体、蛋白或遗传材料。例如,术语“重组DNA”意指通过重组方法剪接来自不同来源或来自同一来源的不同部分的DNA片段形成的DNA分子。在一些实施方案中,将两个或更多个不同来源的DNA使用限制酶切割并使用连接酶连接到一起。作为另一个实例,术语“重组蛋白”或“重组结构域”和其语法上的变化意指源于来自不同来源或来自同一来源的不同部分的剪接的DNA片段的通过重组方法形成的蛋白分子。作为另一个实例,术语“重组微生物”或“重组细菌”和其语法上的变化意指包含一个或多个重组DNA/蛋白分子的微生物/细菌。
根据一些实施方案,微生物选自双歧杆菌属、短杆菌属、丙酸杆菌属、乳球菌属、链球菌属、葡萄球菌属(例如,表皮葡萄球菌(S. epidermidis)和/或人葡萄球菌(S. hominis))、乳杆菌属(例如,嗜酸乳杆菌(L. acidophilus))、肠球菌属、片球菌属、明串珠菌属或酒球菌属和其混合物。
LEKTI基因:根据一些实施方案,重组微生物经工程改造以表达编码LEKTI蛋白的哺乳动物基因。LEKTI基因可从任何哺乳动物例如小鼠、大鼠、兔、山羊、绵羊、马、奶牛、狗、灵长类或人基因序列获得。根据一些实施方案,LEKTI基因序列为人基因序列。根据一些实施方案,重组微生物经工程改造以包含LEKTI基因的片段。
根据一些实施方案,经工程改造的微生物所表达的重组蛋白包含根据SEQ ID NO:1的肽序列(LEKTI D8-11)。根据一些实施方案,经工程改造的微生物所表达的重组蛋白包含根据SEQ ID NO: 2的肽序列。根据一些实施方案,根据SEQ ID NO: 2的肽序列的一个或多个片段由经工程改造的微生物表达。在一个实施方案中,所述片段包含一个或多个LEKTI结构域。在具体的实施方案中,LEKTI结构域为结构域6。
根据一些实施方案,重组微生物包含如本文所公开的序列,其与本文所列出的SEQID NO中的任何一个或多个具有至少约75%同一性或80%同一性或85%同一性或90%同一性或95%同一性。如本文所使用的,术语“同一性”和其语法上的版本意指在比对中两个核苷酸或氨基酸序列在相同位置具有相同残基的程度。百分比(%)同一性通过序列比对中匹配的数目乘以100并除以比对区域的长度(包括内部的缺口)计算。
根据一些实施方案,经工程改造的微生物所表达的重组蛋白包含LEKTI蛋白的一个或多个蛋白酶抑制结构域。一些非限制性的实例包括结构域D1、D2、D3、D4、D5、D6、D7、D8、D9、D10、D11、D12、D13、D14和D15中的一个或多个。根据一些实施方案,经工程改造的微生物所表达的重组蛋白包含LEKTI抑制结构域6或结构域D8-D11。
根据一些实施方案,LEKTI蛋白结构域有效改善内瑟顿综合征的症状。如本文所使用的,术语“改善”("ameliorate"、"ameliorating")和其语法上的变化意指降低受试者中疾病症状的严重性。在一些实施方案中,LEKTI蛋白结构域充当哺乳动物皮肤上或皮肤中存在的一种或多种蛋白酶的竞争性或非竞争性抑制剂。在一些实施方案中,LEKTI蛋白结构域充当丝氨酸蛋白酶抑制剂。如本文所使用的,术语“蛋白酶”和“蛋白水解酶”可互换使用,两个术语均指进行蛋白水解的酶。
根据一些实施方案,微生物通过用编码LEKTI蛋白结构域的重组DNA质粒转染/转化而遗传修饰。其它用于向微生物中引入DNA的常规或待发现的方法也可用在本发明中。根据一些实施方案,重组DNA质粒包含编码LEKTI蛋白结构域和一种或多种分泌肽和/或细胞渗透肽的序列。根据一些实施方案,LEKTI结构域与有效增强从微生物分泌和/或渗透哺乳动物的皮肤的一个或多个重组蛋白结构域可操作地连接。
术语“可操作地连接”指单个核酸片段上核酸序列的关联,以致于一个序列的功能受另一个序列调节或不受另一个序列阻碍。例如,当其能够调节编码序列的表达时(即编码序列在启动子的转录控制下),启动子与该编码序列可操作地连接。编码序列可以以有义或反义方向与调节序列可操作地连接。在另一个实例中,两个蛋白可可操作地连接,以致于任一蛋白的功能不受损。一般而言,可操作地连接意指连接的核酸序列是连续的,并且当需要以连接两个蛋白编码区时是连续的并且在同一读框中。
如本文所使用的术语“分泌肽”或“分泌序列”或“分泌标签”或“信号肽”或“输出信号”和其语法上的变化意指能够将合成的蛋白靶向细胞的分泌途径的任何肽序列。在一些实施方案中,分泌肽可位于重组蛋白的N端末端,并且可共翻译或翻译后地将标记的蛋白靶向分泌。根据一些实施方案,至少一个LEKTI结构域与SecA结构域(SEQ ID NO: 3)可操作地连接。
分泌肽:根据一些实施方案,治疗性LEKTI结构域与标记蛋白用于转运通过分泌途径的一个或多个分泌信号或输出信号可操作地连接。帮助LEKTI蛋白离开细菌细胞的任何分泌信号均可用作信号肽。分泌肽信号的非限制实例在下表1中列出:
表1
氨基酸序列 | SEQ ID NO: |
MKKLAFAITAASGAAAVLSHHDAEA | 9 |
WLDNRAFSKKFVPVVMATSVALFFLNLAFA | 10 |
MAKKFNYKLPSMVALTLFGTAFTAHQANA | 11 |
MKKRFLSICTMTIAALATTTMVNTSYA | 12 |
NLKKQSKLILIFICIFTFFIMIIQSQFLMG | 13 |
MKIFKLTSLTLAALTLAFPFSHVAQA | 14 |
MKKTVIASTLAVSLGIAGYGLSGHEAH | 15 |
MKKNKFLVYLLSTALITPTFATQTAFA | 16 |
MKTRQNKYSIRKFSVGASSILIAALLFMGGGSAQA | 17 |
MKNNNETRRFSIRKYTVGVVSIITGITIFVSGQHAQA | 18 |
MKKKLSYMITIMLAFTLSLALGLFFNSAHA | 19 |
根据一些实施方案,治疗性LEKTI结构域与衍生自表皮葡萄球菌的内源性蛋白的一个或多个信号序列可操作地连接。衍生自表皮葡萄球菌的内源性蛋白的分泌信号肽的非限制实例在下表2中列出:
表2 - 表皮葡萄球菌
根据一些实施方案,治疗性LEKTI结构域与衍生自其它细菌的内源性蛋白的一个或多个分泌信号序列可操作地连接。衍生自各种细菌的内源性蛋白的分泌信号肽的非限制性实例在附录A中列出。
根据一些实施方案,重组LEKTI结构域与增强LEKTI结构域穿过细胞膜的能力的细胞渗透肽序列可操作地连接。如用于描述细胞渗透肽/LEKTI的术语“增强”意指相对于缺乏细胞渗透序列的重组LEKTI结构域,细胞渗透序列改善重组LEKTI结构域通过细胞膜。
细胞渗透肽:根据一些实施方案,使用一种或多种细胞渗透肽介导在体内递送治疗蛋白而不使用细胞表面受体并且不引起显著的膜损伤。根据一些实施方案,一种或多种细胞渗透肽与治疗蛋白可操作地连接以帮助进入皮肤细胞(例如角质形成细胞)。非限制性实例在下表3中列出:
表3
<u>细胞渗透序列</u> | <u>SEQ ID NO</u> |
GRKKRRQRRRPPQ | 77 |
GWTLNS AGYLLGKINLKALAALAKKIL | 78 |
KLALKLALKALKAALKLA | 79 |
WEAKLAKALAKALAKHLAKALAKALKACEA | 80 |
KETWWETWWTEWSQPKKKRKV | 81 |
RRRRRRRRR | 82 |
LGTYTQDFNKFHTFPQTAIGVGAP | 83 |
RQIKWFQNRRMKWKK | 84 |
YGRKKRRQRRR | 85 |
RGGRLSYSRRRFSTSTGR | 86 |
RRLSYSRRRF | 87 |
PIRRRKKLRRLK | 88 |
RRQRRTSKLMKR | 89 |
RRRRNRTRRNRRRVR | 90 |
KMTRAQRRAAARRNRWTAR | 91 |
TRRQRTRRARRNR | 92 |
GRKKRRQRRRPPQ | 93 |
GRRRRRRRRRPPQ | 94 |
GWTLNSAGYLLGKINLKALAALAKKIL | 95 |
KLALKLALKLALALKLA | 96 |
MGLGLHLLVLAAALQGAWSQPKKKRKV | 97 |
GALFLGWLGAAGSTMGAWSQPKKKRKV | 98 |
GALFLGFLGAAGSTMGAWSQPKKKRKV | 99 |
GALFLGFLGAAGSTMGAWSQPKSKRKV | 100 |
KETWWETWWTEWSQPKKKRKV | 101 |
KETWFETWFTEWSQPKKKRKV | 102 |
根据一些实施方案,细胞渗透肽包含周期性氨基酸序列。周期性细胞渗透序列的非限制性实例包括:聚精氨酸,R x n (其中4<n<17);聚赖氨酸,K x n (其中4<n<17);用6-氨基己酸残基间隔的精氨酸重复(RAca),其中存在2-6个精氨酸重复;用4-氨基丁酸间隔的精氨酸重复(RAbu),其中存在2-6个精氨酸重复;用甲硫氨酸间隔的精氨酸重复,其中存在2-6个精氨酸重复;用苏氨酸间隔的精氨酸重复,其中存在2-6个精氨酸重复;用丝氨酸间隔的精氨酸重复,其中存在2-6个精氨酸重复;和用丙氨酸间隔的精氨酸重复,其中存在2-6个精氨酸重复。
根据一些实施方案,LEKTI结构域与RMR结构域(SEQ ID NO: 4)可操作地连接。
根据一些实施方案,LEKTI结构域的表达受操纵子控制,并且提供给哺乳动物的皮肤的LEKTI的量与外在因素的可用性成比例。例如,在一些实施方案中重组LEKTI基因可处于木糖诱导型启动子(例如木糖阻遏物(xylR)、木糖操纵基因(sylO)、包含顺式作用分解代谢物效应元件(CRE)的木糖异构酶基因(xylA))的控制下,并且哺乳动物皮肤可用的重组LEKTI蛋白的量受重组微生物可用的外源木糖的量控制。根据一些实施方案,LEKTI结构域的表达受组成型活性的启动子控制。根据一些实施方案,LEKTI结构域的表达受根据SEQ IDNO: 8的CmR启动子控制。
根据一些实施方案,微生物通过用编码LEKTI蛋白结构域和一个或多个抗生素抗性基因的重组DNA质粒转染/转化而遗传修饰。例如,重组DNA质粒的一些实施方案包含卡那霉素抗性基因和/或甲氧苄啶抗性基因例如dfrA (SEQ ID NO: 5)。根据一些实施方案,用抗生素(重组微生物对其耐受)处理哺乳动物的皮肤可用于使共生微生物群体向更大比例的产LEKTI微生物偏斜。重组DNA质粒中可能存在的其它元件包括,但不限于,复制蛋白基因,例如复制蛋白Rep超家族的成员。例如,在一些实施方案中重组DNA质粒包含repF基因(SEQ ID NO: 6)。
根据一些实施方案,重组DNA质粒包含pJB38载体的一个或多个序列。在一些实施方案中,重组LEKTI与pJB38载体中的诱导型启动子、核糖体结合位点、输出信号和/或细胞渗透肽可操作地连接。如本文所使用的,术语“pJB38-LEKTI-完整”意指包含pJB38载体和一个或多个LEKTI结构域的重组DNA质粒构建体。根据一些实施方案,重组DNA质粒包含根据SEQ ID NO: 1542的pJB38载体。根据一些实施方案,根据SEQ ID NO: 1的LEKTI结构域与根据SEQ ID NO: 1542的pJB38载体可操作地连接。
根据一些实施方案,重组DNA质粒包含根据SEQ ID NO: 7的pKK30-LEKTI-完整序列(附录B)。根据一些实施方案,本公开内容提供用于治疗皮肤疾病的组合物,其包含含有pKK30-LEKTI-完整质粒构建体的微生物。根据一些这样的实施方案,微生物选自双歧杆菌属、短杆菌属、丙酸杆菌属、乳球菌属、链球菌属、葡萄球菌属(例如,表皮葡萄球菌和/或人葡萄球菌)、乳杆菌属(例如,嗜酸乳杆菌)、肠球菌属、片球菌属、明串珠菌属或酒球菌属和其混合物。
根据一些实施方案,治疗性LEKTI蛋白的可用性的量或持续时间受含有LEKTI的载体在微生物中的稳定性控制。例如,重组载体的持续性可受质粒的一个或多个元件控制,所述元件包括提供宿主有益基因、质粒稳定性机制和质粒共适应的那些。例如,一些质粒可提供稳定的复制、活动分区机制和确保质粒可靠遗传至数代内的子细胞的机制。(参见,例如,J.C. Baxter, B.E. Funnell, Plasmid partition mechanisms, Microbiol. Spectr.,2 (2014) PLAS-0023-2014和Nils Hülter等, An evolutionary perspective onplasmid lifestyle modes, Current Opinion in Microbiology, 第38卷,2017年8月,第74-80页,其各自通过引用以其整体结合本文中)。根据一些实施方案,本发明包括使用本领域技术人员已知的所有常规选择和稳定方法。
根据一个方面,本公开内容提供治疗有需要的哺乳动物的皮肤疾病或改善所述皮肤疾病的影响的方法,其包括在哺乳动物的皮肤表面上提供经遗传修饰以表达一个或多个LEKTI蛋白结构域的微生物,其中LEKTI蛋白结构域有效渗透哺乳动物的皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的活性。根据一些实施方案,微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间和提供连续的LEKTI蛋白结构域供应。
根据另一个方面,本公开内容提供用于治疗有需要的哺乳动物的皮肤疾病或改善所述皮肤疾病的影响的试剂盒,其包含:(1) 包含经遗传修饰以表达一个或多个LEKTI蛋白结构域的微生物的组合物,其中LEKTI蛋白结构域有效渗透哺乳动物的皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的丝氨酸蛋白酶活性;和(2) 用于将组合物施用到哺乳动物的皮肤上的试剂。根据一些实施方案,微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间和提供连续的LEKTI蛋白结构域供应。
除了以上组分之外,主题试剂盒将进一步包含使用所述组分和/或实践主题方法的说明书。这些说明书可以以各种形式存在于主题试剂盒中,所述形式的一种或多种可存在于试剂盒中。这些说明书可能存在的一种形式为作为在合适的介质或基底上的印刷信息,例如其上印刷有信息的一片或多片纸,在试剂盒的包装中或在包装插页中。又另一个手段会为计算机可读介质,例如磁盘或CD,其上记录信息。进一步地,说明书可藉其存在的另一个手段为经由互联网使用以在远离的位点访问信息的网站地址。任何方便的手段均可存在于试剂盒中。
试剂盒的组分可以以水性介质或以冻干的形式包装。试剂盒一般将会包装为包含至少一个小瓶、试管、烧瓶、瓶子、注射器或其它容器手段,所述试剂可放置在其中,并且优选地在其中适当等分。当提供额外的组分时,试剂盒一般将会还包含第二个、第三个或其它额外的容器,这样的组分可放置于其中。
本公开内容的试剂盒通常还将会包含用于密闭容纳试剂容器用于商业出售的手段。这样的容器可包括注射或吹塑的塑料容器,期需的小瓶保持在其中。
制剂
根据一些实施方案根据本发明使用的制剂可包含任何药学有效量的重组细菌以产生治疗有效量的期需多肽或其治疗有效的结构域,例如,至少约0.01%、约0.05%、约0.1%、约0.2%、约0.3%、约0.4%、约0.5%、约0.6%、约0.7%、约0.8%、约0.9%、约1.0%、约1.5%、约2.0%、约3.0%、约4.0%、约5.0%、约6.0%、约7.0%、约8.0%、约9.0%、约10.0%、约11.0%、约12.0%、约13.0%、约14.0%、约15.0%、约16.0%、约17.0%、约18.0%、约19.0%、约20.0%、约25.0%、约30.0%、约35.0%、约40.0%、约45.0%、约50.0%或更多重量的重组细菌,其上限为约90.0%重量的重组细菌。
根据一些实施方案,根据本发明使用的制剂可包含,例如,至少约0.01%-约30%、约0.01%-约20%、约0.01%-约5%、约0.1 %-约30%、约0.1%-约20%、约0.1%-约15%、约0.1 %-约10%、约0.1%-约5%、约0.2%-约5%、约0.3%-约5%、约0.4%-约5%、约0.5%-约5%、约1%-约5%或更多重量的重组细菌。
根据一些实施方案,局部制剂可呈适于施用到身体表面的任何形式,例如乳膏、洗剂、喷雾剂、溶液、凝胶、软膏、糊剂、膏药、涂剂、生物粘附剂、混悬剂、乳剂等,和/或可以制备成含有脂质体、胶束和/或微球。这样的制剂可与闭合的覆盖层组合使用以便在施用到身体表面时和其后将从身体表面蒸发的水分维持在制剂内。根据一些实施方案,制剂可包含活细胞培养物组合物并且可包含至少一种经工程改造的产生治疗有效的重组多肽或其治疗有效的结构域的细菌菌株。该经工程改造的活细胞培养物组合物可将多肽直接递送到皮肤用于治疗或预防异常的皮肤状况。
局部制剂包括其中将任何其它活性成分溶解或分散在本领域已知的皮肤病学溶媒中的那些(例如水性或非水性凝胶、软膏、油包水或水包油乳剂)。这种溶媒的组分可包括水、水性缓冲溶液、非水性溶剂(例如乙醇、异丙醇、苯甲醇、2-(2-乙氧基乙氧基)乙醇、丙二醇、丙二醇单月桂酸酯、糖原质或甘油)、油(例如矿物油例如液体石蜡油、天然或合成的甘油三酯类例如Miglyol™,或硅油例如二甲硅油)。尤其,根据制剂的性质以及其意图的用途和施用位点,使用的皮肤病学溶媒可包含选自以下列表的一种或多种组分(例如,当制剂为水性凝胶时,除了水之外的组分):增溶剂或溶剂(例如β-环糊精,例如羟丙基β-环糊精(bydroxypropyl β- cyclodextrin),或醇或多元醇例如乙醇、丙二醇或甘油)、增稠剂(例如羟乙基纤维素、羟丙基纤维素、羧甲基纤维素或卡波姆)、胶凝剂(例如聚氧乙烯-聚氧丙烯共聚物)、防腐剂(例如苯甲醇、苯扎氯铵、氯己定、氯丁醇、苯甲酸酯、山梨酸钾或EDTA或其盐)和pH缓冲剂(例如磷酸二氧盐和磷酸氢盐的混合物,或柠檬酸和磷酸氢盐的混合物)。
药学上可接受的载体也可掺入本发明的制剂中并且可为本领域常规使用的任何载体。其实例包括水、低级醇、高级醇、多元醇、单糖、二糖、多糖、烃油、脂肪和油、蜡、脂肪酸、硅油、非离子型表面活性剂、离子型表面活性剂、硅酮表面活性剂以及这些载体的水基混合物和乳液基混合物。术语“药学上可接受的”或“药学上可接受的载体”在本文中用于指可掺入药学制剂中而不引起不期需的生物学作用或不想要的与制剂其它组分的相互作用的化合物或组合物。如本文所使用的“载体”或“溶媒”指适于掺入局部施用的组合物中的载体材料。如本文所使用的载体和溶媒包括本领域已知的任何这样的材料,其为无毒的并且不与包含其的制剂的其它组分以有害的方式相互作用。术语“水性”指包含水或在施用到皮肤或粘膜组织之后变得包含水的制剂。
当其干燥时,成膜剂在施用的位点上形成保护性的膜。膜抑制移除活性成分和保持其与所治疗的位点接触。适于在本发明中使用的成膜剂的实例为Flexible Collodion,US P。如Remington: The Science and Practice of Pharmacy,第19版(Easton, PA:Mack Publishing Co., 1995)在第1530页中所描述的,火棉胶为包含火棉(硝酸纤维素)的乙醚/乙醇溶液,其蒸发留下火棉膜。成膜剂可另外充当载体。干燥以形成膜的溶液有时称为涂剂。乳膏,如药学制剂领域所熟知的,为粘稠液体或半固体的水包油或油包水乳剂。
乳膏基质为可水洗的,包含油相、乳化剂和水相。油相也称为“内部”相,一般包含矿脂和脂肪醇例如十六烷基或十八烷基醇。尽管不一定,水相通常在体积上超过油相,一般包含湿润剂。乳膏制剂中的乳化剂一般为非离子型的、阴离子型的、阳离子型的或两性表面活性剂。
洗剂为施用到皮肤表面而无摩擦的制备物,其通常为液体或半流体的制备物,其中颗粒(包括活性剂)存在于水或醇基质中。洗剂一般为固体悬浮液,优选地包含水包油型的液体油性乳剂。洗剂为本文中用于治疗大身体区域的优选制剂,这是因为容易施用更流体的组合物。一般需要洗剂中的不溶物质为细碎的。
洗剂通常将会包含助悬剂以产生更好的分散体以及可用于将活性剂定位和维持在与皮肤接触的化合物例如甲基纤维素、乙氧基甲基纤维素钠等。
溶液为通过将一种或多种化学物质(溶质)溶解在液体中以致于溶解的物质的分子分散在溶剂的那些之中制备的均质混合物。溶液可包含其它药学上或化妆品可接受的化学品以缓冲、稳定或保藏溶质。制备溶液中使用的溶剂的常见实例为乙醇、水、丙二醇或任何其它可接受的溶媒。如当然是众所周知的,凝胶为半固体、悬浮类型的系统。单相凝胶包含大体上均一分布在整个载体液体(通常为水性的,但优选地也包含醇,和任选地油)中的有机大分子。优选的“有机大分子”,即胶凝剂,为交联的丙烯酸聚合物例如聚合物的“卡波姆”家族,例如可以在Carbopol商标下市售获得的羧基聚亚烷基。同样优选的为亲水聚合物例如聚氧乙烯、聚氧乙烯-聚氧丙烯共聚物和聚乙烯醇;纤维素聚合物例如羟基丙基纤维素、羟基乙基纤维素、羟基丙基甲基纤维素、羟基丙基甲基纤维素邻苯二甲酸酯和甲基纤维素;树胶例如黄芪胶和黄原胶;海藻酸钠和明胶。为了制备均一的凝胶,可加入分散剂例如醇或甘油,或胶凝剂可通过研磨、机械混合或搅拌或其组合分散。如本领域同样熟知的,软膏为通常基于矿脂或其它石油衍生物的半固体制备物。如本领域技术人员将会理解的,待使用的具体的软膏基质为将会提供许多期需特性例如润肤等的软膏基质。正如其它载体或溶媒,软膏基质应为惰性的、稳定的、无刺激性的和不敏感的。如Remington: The Scienceand Practice of Pharmacy,第19版(Easton, PA: Mack Publishing Co., 1995)在第1399-1404页中所解释的,软膏基质可分组为4类:油质基质、可乳化基质、乳剂基质和水溶性基质。油质软膏基质包括,例如,植物油、获自动物的脂肪和获自石油的半固体烃类。
可乳化的软膏基质也称为可吸收的软膏基质,包含很少的水或不包含水,包含例如硫酸羟基三硬脂酸甘油酯、无水羊毛脂和亲水矿脂。
乳剂软膏基质为油包水(W/O)乳剂或水包油(O/W)乳剂,并包括例如乙酰基醇、单硬脂酸甘油脂、羊毛脂和硬脂酸。优选的水溶性软膏基质从不同分子量的聚乙二醇制备;进一步的信息参见Remington: The Science and Practice of Pharmacy。
糊剂为半固体剂型,其中活性剂悬浮在合适的基质中。根据基质的性质,糊剂分为脂肪糊剂或从单相水性凝胶制成的那些。脂肪糊剂中的基质一般为矿脂或亲水矿脂等。从单相水性凝胶制成的糊剂一般掺入羧甲基纤维素等作为基质。
增强剂为那些亲脂性共增强剂,通常称为“塑化”增强剂,即具有分子量在约150-1000范围内、水溶性小于约1 wt.%、优选小于约0.5 wt.%并且最优选小于约0.2 wt.%的增强剂。塑化增强剂的Hildebrand溶解度参数δ在约2.5-约10的范围内,优选地在约5-约10的范围内。优选的亲脂性增强剂为脂肪酯、脂肪醇和脂肪醚。具体的和最优选的脂肪酸酯的实例包括月桂酸甲酯、油酸乙酯、丙二醇单月桂酸酯、丙烯甘油二月桂酸酯(propyleneglycerol dilaurate)、甘油单月桂酸酯、甘油单油酸酯、正癸酸异丙酯和肉豆蔻酸辛基十二烷基酯。脂肪醇包括例如硬脂醇和油醇,而脂肪醚包括其中二醇或三醇优选C2-C4烷二醇或三醇用一个或两个脂肪醚取代基取代的化合物。
额外的渗透增强剂为局部药物递送领域的普通技术人员所熟知,和/或在相关文本和文献中描述。参见,例如,Percutaneous penetration Enhancers, 编辑 Smith等(CRC Press, 1995)(通过引用结合到本文中)。
除上文鉴定的那些之外,本发明的组合物可包含各种其它添加剂。这些包括,但不限于,抗氧化剂、收敛剂、香料、防腐剂、润肤剂、色素、染料、湿润剂、推进剂和遮光剂,以及其存在可为药学上或以其他方式期需的其它种类的材料。任选的用于包含在本发明的制剂中的添加剂的典型实例如下:防腐剂例如山梨酸酯;溶剂例如异丙醇和丙二醇;收敛剂例如甲醇和乙醇;润肤剂例如聚亚烷基甲基葡糖苷;湿润剂例如甘油;乳化剂例如甘油硬脂酸酯、PEG-100硬脂酸酯、聚甘油基-3羟基月桂基醚和聚山梨酯60;山梨醇和其它多羟基醇例如聚乙二醇;遮光剂例如辛基甲氧基肉桂酸酯(作为Parsol MCX市售可得)和丁基甲氧基苯甲酰甲烷(在商品名Parsol 1789下可得);抗氧化剂例如抗坏血酸(维生素C)、α生育酚(维生素E)、β-生育酚、γ-生育酚、δ-生育酚、ε-生育酚、ζι -生育酚、ΖΛ-生育酚、η-生育酚和视黄醇(维生素A);精油、神经酰胺、必需脂肪酸、矿物油、植物油(例如,豆油、棕榈油、乳木果油的液体部分、葵花籽油)、动物油(例如全氢鲨烯)、合成油、硅油或蜡(例如环甲硅油和二甲硅油)、氟化油(一般地全氟聚醚)、脂肪醇(例如鲸蜡醇)和蜡(例如蜂蜡、巴西棕榈蜡和石蜡);皮肤感觉修饰剂;和增稠剂和结构化剂例如膨胀粘土和可在Carbopol商标下市售获得的交联羧基聚亚烷基。其它添加剂包括有益剂例如调理皮肤(特别地,角质层中皮肤的上层)和通过阻止降低其含水量保持其柔软和/或保护皮肤的那些材料。这种调理剂和保湿剂包括,例如,吡咯烷羧酸和氨基酸;有机抗微生物剂例如2,4,4'-三氯-2-羟基二苯醚(三氯生)和苯甲酸;抗炎剂例如乙酰基水杨酸和甘草次酸;抗皮脂溢剂例如视黄酸;血管扩张剂例如烟酸;黑色素生成抑制剂例如曲酸;及其混合物。进一步的其它活性剂包括,例如,α羟基酸、α酮酸、聚合羟基酸、保湿剂、胶原、海洋提取物和抗氧化剂例如抗坏血酸(维生素C)、α-生育酚(维生素E)、β-生育酚、γ-生育酚、6-生育酚、ε-生育酚、ζι -生育酚、ζ2-生育酚、η-生育酚和视黄醇(维生素A)和/或其药学上可接受的盐、酯、酰胺或其它衍生物。优选的生育酚化合物为α-生育酚。额外的试剂包括能够改善皮肤组织中的氧供应的那些,如例如在均转让给Lancaster Group AG的Gross等WO 94/00098和Gross等WO 94/00109 (通过引用结合到本文中)中所描述的。遮光剂和UV吸收化合物也可包括在内。这样的遮光剂和UV吸收化合物的非限制性实例包括氨基苯甲酸(PABA)、阿伏苯宗、西诺沙酯、二羟苯宗、胡莫柳酯、邻氨基苯甲酸甲酯、氰双苯丙烯酸辛酯(oxtocrylene)、辛基甲氧基肉桂酸酯、水杨酸辛酯、氧苯酮、帕地马酯(padirnate) O、苯基苯并咪唑磺酸、舒利苯酮、二氧化钛、水杨酸三乙醇胺、氧化锌、恩索利唑、meradiraate、桂皮酸盐、辛水杨酯和氰双苯丙烯酸辛酯。参见标题21第1章子章节D第352部分"Sunscreen drug products for over-the-counter humanuse",其以其整体结合到本文中。
其它实施方案可包含各种帮助用本发明的制剂治疗的非致癌的、无刺激性的愈合材料。这种愈合材料可包括营养素、矿物质、维生素、电解质、酶、草药、植物提取物、腺或动物提取物或可加入制剂中以帮助愈合皮肤病症的安全的治疗剂。
这些各种添加剂的量为化妆品领域常规使用的那些,和范围为例如局部制剂的总重量的约0.01%-约20%。
本发明的制剂还可包含常规添加剂例如不透明剂、芳香剂、着色剂、稳定剂、表面活性剂等。在某些实施方案中,还可加入其它试剂,例如抗微生物剂,以防止储存时变质,即抑制微生物例如酵母和霉菌生长。
合适的抗微生物剂通常选自对羟基苯甲酸的甲基和丙基酯(即甲基和丙基对羟基苯甲酸酯)、苯甲酸钠、山梨酸、咪脲和其组合。在其它实施方案中,也可加入其它试剂,例如阻遏物和诱导物,即,抑制(即葡萄糖)或诱导(即木糖)目的多肽的生产。只要其与制剂的功能相容并且不干扰制剂的功能,这些添加剂就可使用。
制剂还可包含刺激减轻添加剂以最小化或消除所给予的化学实体或组合物的其它组分引起皮肤刺激或皮肤损伤的可能性。
合适的刺激减轻添加剂包括,例如:α-生育酚;单胺氧化酶抑制剂特别是苯基醇例如2-苯基-1-乙醇;甘油;水杨酸盐;抗坏血酸;离子载体例如莫能菌素;两亲胺;氯化铵;N-乙酰半胱氨酸;辣椒素;和氯喹。若存在,刺激减轻添加剂可以以有效减轻刺激或皮肤损伤的浓度掺入组合物中,通常表示不超过制剂的约20 wt.%,更通常不超过制剂的约5 wt.%。
在某些实施方案中可掺入本制剂中并因此与活性剂一起局部施用的进一步合适的药理学活性剂包括但不限于以下:改善或根除色素沉着的或非色素沉着的老人斑;角化病和皱纹的剂;抗微生物剂;抗细菌剂;止痒和抗干燥剂;抗炎剂;局部麻醉剂和镇痛剂;皮质类固醇;类维生素A;维生素;激素;和抗代谢药。
局部药理学活性剂的一些实例包括阿昔洛韦、两性霉素、氯己定、克霉唑、酮康唑、益康唑、咪康唑、甲硝唑、米诺环素、制霉菌素、新霉素、卡那霉素、苯妥英、对氨基苯甲酸酯、甲氧基肉桂酸辛酯、水杨酸辛酯、氧苯酮、二氧苯酮、生育酚、生育酚乙酸酯、硫化硒、吡硫锌、苯海拉明、普莫卡因、利多卡因、普鲁卡因、红霉素、四环素、克林霉素、克罗米通、对苯二酚及其单甲基和苄基醚、萘普生、布洛芬、色甘酸、视黄醇、视黄醇棕榈酸酯、视黄醇乙酸酯、煤焦油、灰黄霉素、雌二醇、氢化可的松、氢化可的松21-乙酸酯、氢化可的松17-戊酸酯、氢化可的松17-丁酸酯、孕酮、戊酸倍他米松酯、二丙酸倍他米松、曲安奈德、氟轻松醋酸酯、丙酸氯倍他索、米诺地尔、双嘧达莫、苯妥英、过氧苯甲酰、和5-氟尿嘧啶。
可将乳膏、洗剂、凝胶、软膏、糊剂等铺展在受影响的表面上并轻轻揉搓。溶液可以以相同的方式施用,但更通常将会用滴管、拭子等施用并小心施用到受影响的区域。
施用方案将取决于可容易地测定的许多因素,例如状况的严重性和其对初始治疗的响应性,但正常地将会涉及在持续进行的基础上每天一次或多次施用。普通技术人员可容易地确定所给予的制剂的最佳量、给予方法学和重复率。一般而言,考虑本发明的制剂将会在每周一次或两次至直每天一次或两次的范围内施用。
本发明的药物组合物包含一种或多种活性成分例如治疗剂与一种或多种药学上可接受的稀释剂或载体和任选一种或多种其它化合物、药、成分和/或材料的混合物。无论所选择的给予途径为何,本发明的试剂/化合物通过本领域技术人员所已知的常规方法配制成药学上可接受的剂型。参见,例如,Remington, The Science and Practice ofPharmacy (第21版,Lippincott Williams和Wilkins, Philadelphia, Pa.)。
药学上可接受的稀释剂或载体为本领域所熟知(参见,例如,Remington, TheScience and Practice of Pharmacy (第21版,Lippincott Williams和Wilkins,Philadelphia, Pa.)和The National Formulary (American PharmaceuticalAssociation, Washington, D.C.))并且包括糖(例如,乳糖、蔗糖、甘露醇和山梨醇)、淀粉、纤维素制备物、钙磷酸盐(例如,磷酸二钙、磷酸三钙和磷酸氢钙)、柠檬酸钠、水、水溶液(例如,盐水、氯化钠注射液、林格注射液、葡萄糖注射液、葡萄糖和氯化钠注射液、乳酸林格注射液)、醇(例如,乙醇、丙醇和苯甲醇)、多元醇(例如,甘油、丙二醇和聚乙二醇)、有机酯(例如油酸乙酯和甘油三酯)、生物可降解聚合物(例如聚乳酸-聚乙交酯、聚(原酸酯)和聚(酸酐))、弹性基质、脂质体、微球体、油(例如玉米、胚芽、橄榄、蓖麻、芝麻、棉籽和落花生)、可可脂、蜡(例如,栓剂蜡)、石蜡、硅酮、滑石、硅酸盐(silicylate)等。本发明的药物组合物中使用的每一药学上可接受的稀释剂或载体必须是从与制剂的其它成分相容和不伤害受试者的意义上“可接受的”。对于选择的剂型和意图的给予途径合适的稀释剂或载体是本领域所熟知的,对于选择的剂型和给予方法可接受的稀释剂或载体可使用本领域普通技术确定。
本发明的药物组合物可任选包含药物组合物中通常使用的额外成分和/或材料。这些成分和材料为本领域所熟知,并且包括(1) 填充剂或增充剂,例如淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸;(2) 粘合剂,例如羧甲基纤维素、海藻酸盐、明胶、聚乙烯吡咯烷酮、羟丙基甲基纤维素、蔗糖和阿拉伯胶;(3) 湿润剂,例如甘油;(4) 崩解剂,例如琼脂、碳酸钙、土豆或木薯淀粉、海藻酸、某些硅酸盐、羟基乙酸淀粉钠、交联的羧甲基纤维素钠和碳酸钠;(5) 溶液阻滞剂,例如石蜡;(6) 吸收加速剂,例如季胺类化合物;(7)润湿剂,例如鲸蜡醇和甘油单硬脂酸酯;(8) 吸收剂,例如高岭土和膨润土;(9) 润滑剂,例如滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇和十二烷基硫酸钠;(10) 助悬剂,例如乙氧基化的异硬脂醇、聚氧乙烯山梨醇和失水山梨醇酯、微晶纤维素、偏氢氧化铝、膨润土、琼脂和黄芪胶;(11) 缓冲剂;(12) 赋形剂,例如乳糖、牛奶糖、聚乙二醇、动物和植物脂肪、油、蜡、石蜡、可可脂、淀粉、黄芪胶、纤维素衍生物、聚乙二醇、硅酮、膨润土、硅酸、滑石、水杨酸盐、氧化锌、氢氧化铝、硅酸钙和聚酰胺粉末;(13) 惰性稀释剂,例如水或其它溶剂;(14) 防腐剂;(15) 表面活性剂;(16) 分散剂;(17) 控制释放或吸收延迟剂,例如羟丙基甲基纤维素、其它聚合物基质、生物可降解的聚合物、脂质体、微球体、单硬脂酸铝、明胶和蜡;(18) 遮光剂;(19) 佐剂;(20) 润湿剂;(21) 乳化和助悬剂;(22)增溶剂和乳化剂,例如乙醇、异丙醇、碳酸乙酯、乙酸乙酯、苯甲醇、苯甲酸苄酯、丙二醇、1,3-丁二醇、油(具体地,棉籽油、落花生油、玉米油、胚芽油、橄榄油、蓖麻油和芝麻油)、甘油、四氢呋喃醇、聚乙二醇和失水山梨醇脂肪酸酯;(23) 推进剂,例如氯氟烃和易挥发的未取代烃类,例如丁烷和丙烷;(24) 抗氧化剂;(25) 致使制剂与预期受体的血液等渗的试剂,例如糖和氯化钠;(26) 增稠剂;(27) 包衣材料,例如卵磷脂;和(28) 甜味剂、调味剂、着色剂、香料和防腐剂。每一这种成分或材料必须是从与制剂的其它成分相容和不伤害受试者的意义上“可接受的”。对于选择的剂型和意图的给予途径合适的成分和材料是本领域所熟知的,对于选择的剂型和给予方法可接受的成分和材料可使用本领域普通技术确定。
用于局部或透皮给予的剂型包括粉剂、喷雾剂、软膏、糊剂、乳膏、洗剂、凝胶、溶液、贴剂、滴剂和吸入剂。活性剂/化合物可在无菌条件下与合适的药学上可接受的稀释剂或载体混合。软膏、糊剂、乳膏和凝胶可包含赋形剂。粉剂和喷雾剂可包含赋形剂和推进剂。
适于胃肠外给予的本发明的药物组合物可包含一种或多种试剂/化合物以及一种或多种药物上可接受的无菌等渗水性或非水溶液、分散体、悬浮液或乳液,或可在临用前重构在无菌的可注射溶液或分散体中的无菌粉剂,其可包含合适的抗氧化剂、缓冲剂、致使制剂与预期的受体的血液等渗的溶质或助悬或增稠剂。可例如通过使用包衣材料,在分散体的情况下通过维持需要的颗度和通过使用表面活性剂维持适当的流动性。这些药物组合物还可包含合适的佐剂,例如润湿剂、乳化剂和分散剂。其还可能期需包含等渗剂。另外,可注射的药物形式的延长的吸收可通过包含延迟吸收的试剂带来。
提供下列实施例以进一步说明本发明的方法。这些实施例仅为说明性的,不意在以任何方式限制本发明的范围。
实施例1
细菌
在一些实施方案中,金黄色葡萄球菌RN4220菌株的细菌可用在载体的制备中(Kreiswirth, BN等1983)。在一些这样的实施方案中,菌株的储液在50%甘油中在LB或TS肉汤中储存在-20 ℃。
根据一些实施方案,可使用表皮葡萄球菌菌株ATCC 12228或NRRL B-4268的细菌(Zhang, YQ., et ah 2003)。在一些这样的实施方案中,菌株的储液在50%甘油中在LB或TS肉汤中储存在-20 ℃。细菌在LB肉汤或TS肉汤中培养。孵育16小时后,将细菌通过离心收获并在LB肉汤或TS肉汤中以2 x l09个细菌/100 μL 10倍浓缩。细菌的储存制备物通过用表皮葡萄球菌接种5 mL肉汤和30 ℃生长过夜制备。然后,将3 mL完全生长的培养物加入1 mL60%甘油中并储存在-80 ℃。
表达载体
根据一些实施方案,质粒构建体pKK30-LEKTI-完整可包括具有LEKTI结构域插入物的pKK30载体。根据一些实施方案,LEKTI结构域可与SecA分泌信号、6xHis标签和/或RMR细胞渗透序列可操作地连接,在氯霉素抗性(CmR)启动子序列(来自pDB114E)的控制下表达。在一些实施方案中,pKK30载体包含二氢叶酸还原酶(dfrA)选择基因。
转化
根据一些实施方案,可将含有LEKTI序列的载体转化入表皮葡萄球菌菌株。可制备/转化含有LEKTI序列的载体,其包括以下步骤:制备感受态金黄色葡萄球菌细菌细胞,转化金黄色葡萄球菌,从金黄色葡萄球菌分离质粒DNA,制备感受态表皮葡萄球菌细菌细胞,转化表皮葡萄球菌,生长转化的表皮葡萄球菌细菌和储存转化的表皮葡萄球菌。
在一些实施方案中,在准备以转化入表皮葡萄球菌中备选的中间菌株也可用于转化和分离质粒DNA。这些菌株可包括但不限于大肠杆菌菌株等菌株,包括甲基化缺陷的那些。
根据一些实施方案,可将金黄色葡萄球菌RN4220细胞通过在LB或TS培养基中在37℃生长50 mL培养物过夜,然后用10 mL过夜培养物接种100 mL新鲜LB或TS培养基而制成电转化感受态。当OD600达到0.2-0.3,将细胞沉淀并用1×体积的4 ℃ 10%蔗糖重悬。该过程重复3次,然后将细胞用0.1×体积的4 ℃ 10%蔗糖重悬、沉淀和用1 mL 10%蔗糖重悬。
对于转化RN4220,可将200-500ug LEKTI质粒(例如pKK30-LEKTI-完整)与电转化感受态细胞混合并使用电穿孔在室温在2.5 kV下使用MicroPulser Electroporator(Bio-Rad, Hercules, CA)转化。将转化的细胞在28 ℃铺板在选择性LB或TB培养基上过夜,在选择性LB或TB培养基中生长过夜,然后用于分离DNA。
根据一些实施方案,电转化感受态表皮葡萄球菌ATCC 12228或NRRL B-4268使用以下方法制作。首先,将来自-80 ℃储液的ATCC 12228或NRRL B-4268的50 ml过夜培养物在37 ℃在B2培养基(1.0%胰蛋白胨, 2.5%酵母提取物, 0.5%葡萄糖, 2.5% NaCl, 0.1%K2PO4, pH至7.5)中生长。将10 mL过夜培养物稀释入新鲜预热的B2培养基中并振荡直至OD600达到0.5-0.6,然后在4 ℃沉淀10 min。接着,将细胞用1、1/2、1/20和1/50体积的冷10%甘油洗涤,洗涤之间在4 ℃沉淀。最终的沉淀重悬在700 μL冷10%甘油中。
根据一些实施方案,将电转化感受态ATCC 12228或NRRL B-4268使用电穿孔在2.5kV, 25 uF, 100Ω (使用Micropulser Electroporator (Bio-Rad, Hercules, CA)正常阅取为4.5-5毫秒)用从金黄色葡萄球菌分离的pKK30-LEKTI-完整转化。细胞然后在28 ℃下铺板在选择性LB或TB培养基上。在一些实施方案中,细菌转化也可经由备选的转化方法实施,所述方法包括但不限于备选的中间菌株、噬菌体转导和热激。
蛋白表达分析
根据一些实施方案,将转化细胞分级并通过SDS-PAGE电泳和蛋白质印迹分析。将表达重组LEKTI的细菌细胞和细菌对照细胞沉淀并用CelLytic B细胞裂解试剂(Sigma-Aldrich, St. Louis, MO)裂解。将来自诱导样品的上清液收集并浓缩。将样品重悬在还原型样品缓冲液中,然后,在4-15% Tris-丙烯酰亚胺凝胶上用Tris-HCl电泳缓冲液电泳。电泳之后,将凝胶转移至PVDF膜,继而用抗LEKTI结构域8-11或His标签的初级山羊单克隆抗体探查。然后缀合辣根过氧化物酶的驴抗山羊抗体(sc-2020)探查,第二抗体通过使用增强的化学发光底物(SuperSignal West Pico, Thermo Scientific)放射自显影(SyngeneGeneGnome Bio Imaging System)检测。
上清液和细胞裂解物的分析证明当用包含目的蛋白的质粒转化时成功表达和分泌治疗多肽。使用备选的方法检测蛋白表达和分泌也是可能的,当前的实施例不应解释为对本发明的限制。
人受试者的治疗
根据一些实施方案,可将1x109个集落形成单位(CFU)的包含重组LEKTI的表皮葡萄球菌添加至药学上可接受的载体。前述组合物可用于在有需要的受试者中治疗或预防内瑟顿综合征导致的异常皮肤状况。所述组合物可至少每天一次,至多例如每天约3-4次或按照需要或遵医嘱施用。在一些实施方案中,取得治疗效果仅需要单次施用。可需要多久就使用所述组合物多久以确保治疗所述状况或持续预防所述状况。治疗的持续时间可从约1天直至约10-14天或更长变化。在某些情况下,可给予长期或慢性治疗。
实施例2
检验重组LEKTI的丝氨酸蛋白酶抑制活性
根据一些实施方案,针对在与不同的分泌肽和细胞渗透肽可操作地连接时取得的不同,检验重组LEKTI的蛋白酶抑制活性。根据一些实施方案,分泌肽和细胞渗透肽的特定组合可对LEKTI结构域的蛋白酶抑制功能具有预料不到的效果,因此可经验性地确定。
在一些实施方案中,将与分泌标签、6xHis标签和细胞渗透标签可操作地连接的LEKTI结构域D8-D11克隆入昆虫表达载体用于大规模生产纯化的重组蛋白和评估对一种或多种蛋白酶(例如纤溶酶、组织蛋白酶G、弹性蛋白酶和胰蛋白酶)的抑制活性。
昆虫细胞和试剂
下列试剂可如所标明的市售获得:草地夜蛾细胞系草地贪夜蛾(Spodopterafrugiperda) (Sf9)、低熔点琼脂糖、cellFECTIN, pFASTBAC1、pCRII-TOPO、大肠杆菌感受态DH10BAC、粉纹夜蛾卵细胞系粉纹夜蛾(Trichoplusia ni) 5B1-4 (High Five)和最终的无血清昆虫培养基(来自Invitrogen (Carlsbad, CA) );限制性内切核酸酶(来自NewEngland Biolabs (Beverly, MA) );TALON Superflow(来自Clontech Laboratory (PaloAlto, CA));Insect-XPRESS培养基和胎牛血清(来自BioWhittaker (Walkersville,MD));YM10 Centriplus(来自Millipore Corp. (Bedford, MA));预制的SDS-PAGE凝胶、蛋白测定试剂盒、SEC-250尺寸柱和预染标记物(来自Bio-Rad (Hercules, CA));BSA(来自Kabi Pharmacia (Franklin, OH)); DTT和甘油(来自Boehringer MannheimBiochemicals (Indianapolis, IN));和五-His mAb和六-His标记的蛋白梯(来自QIAGENInc. (Valencia, CA))。
LEKTI D8-D11的克隆和表达
可将与分泌肽和细胞渗透肽的各种排列可操作地连接的6xHis标记的LEKTI结构域(例如SEQ ID NO: 1)按照制造商的说明书克隆入pFASTBAC1载体。重组LEKTI复合病毒然后如Gao, M.等, (1996) J. Biol. Chem. 271, 27782-27787 (通过引用以其整体结合到本文中)先前所描述的产生。为了检验重组LEKTI复合病毒的重组LEKTI表达,可将sf9细胞在不同的感染复数下用重组病毒感染,并每24-96 h收集细胞裂解物和培养基。组氨酸标记的蛋白的存在情况可通过蛋白质印迹分析使用针对六-组氨酸标签的五-His mAb按照制造商的推荐证实。可选择显示最高表达水平的LEKTI复合病毒用于进一步的实验和旋转瓶。
重组LEKTI蛋白可通过在含10%血清Insect-XPRESS培养基中以8噬斑形成单位(PFU)的感染复数感染sf9细胞的旋动培养物(16亿细胞)大规模生产。感染3天后,可将细胞沉淀收获并使用负荷Co2+的Sepharose亲和柱(TALON)接着SEC-250尺寸柱层析从细胞裂解物选择性纯化重组LEKTI,如Jayakumar, A.等, (1995) Proc. Natl. Acad. Sci. U.S.A.92, 8695-8699先前所描述的。可将包含均质的LEKTI的级分合并并通过超滤浓缩。蛋白可使用Bio-Rad Protein Assay Kit II定量。
蛋白酶抑制测定试剂和方案
以下酶、生色底物和试剂可如所标明的市售获得:人纤溶酶、人组织蛋白酶L、人组织蛋白酶S、人胰蛋白酶、人组织蛋白酶G、人糜蛋白酶和人嗜中性细胞弹性蛋白酶(HNE)(来自Athens Research & Technology, Inc. (Athens, GA));枯草杆菌蛋白酶A(来自Calbiochem-Novabiochem (San Diego, CA));木瓜蛋白酶(来自Roche MolecularBiochemicals (Indianapolis, IN));弗林蛋白酶(来自New England BioLabs);琥珀酰-Ala-Ala-Pro-Phe-对硝基酰苯胺(Succ-AAPF-pNA)、琥珀酰-Ala-Ala-Val-pNA (Succ-AAVpNA)和D-Val-Leu-Lys-pNA (VLK-pNA) (来自Sigma Chemical Co. (St. Louis,MO));H-Glu-Gly-Arg-pNA (EGRpNA)和苄氧羰基-Phe-Arg-pNA (Z-FR-pNA) (来自BachemBioscience, Inc. (King of Prussia, PA));和甲氧基-Succ-Arg-Pro-Tyr-pNA (MeO-Succ-RPY-pNA) (来自Chromogenix Instrumentation Laboratory SpA (Milan,Italy))。PBS反应缓冲液(137 mM NaCl, 27 mM KCl, 和10 mM磷酸盐缓冲液(pH 7.4))可与胰蛋白酶、纤溶酶、组织蛋白酶G、HNE和糜蛋白酶一起使用。组织蛋白酶反应缓冲液(0.1%CHAPS, 50 mM乙酸钠(pH 5.5), 1 mM EDTA)可与组织蛋白酶K、L和S和木瓜蛋白酶一起使用。独特的反应缓冲液可与枯草杆菌蛋白酶A一起使用(PBS和0.1%吐温20)。
蛋白酶抑制活性可通过重组LEKTI阻断切割小生色肽底物的能力检测,如通过Schick, C.等, (1998) Biochemistry 37, 5258-5266 (通过引用以其整体结合到本文中)先前所描述的光谱学技术所测定的。对蛋白酶的抑制可在用重组LEKTI在25 ℃在100 μL测定缓冲液中预孵育酶2 min之后评估。该混合物可加入到1 mL石英比色皿中的890或880μL测定缓冲液中。蛋白酶活性可通过加入10-20 μL适当的pNA底物开始。405 nm处的吸光度变化(A405 = 8.8 10-3 M-1 cm-1) 可使用分光光度计(Beckman Instruments, Inc.,Fullerton, CA)跟随长达10 min。受抑制的和对照反应的速率变化(ΔA405/min)可从速度绘图确定。
根据一些实施方案,分泌标签和细胞渗透标签的不同组合可对每一检验的蛋白酶(例如胰蛋白酶、纤溶酶、组织蛋白酶G、HNE、枯草杆菌蛋白酶A和糜蛋白酶)导致不同的LEKTI蛋白酶活性。此外,在个体蛋白酶中分泌标签和细胞渗透标签的离散组合可导致不同LEKTI蛋白酶活性。
实施例3
渗透肽介导的递送
根据一些实施方案,分泌标签和细胞渗透标签的不同组合可以以或多或少的程度影响重组LEKTI蛋白穿过细胞膜的能力。因此,可在细胞培养中测试不同的重组LEKTI产物以评估分泌标签和细胞渗透标签的不同组合的影响。
根据一些实施方案,贴壁成纤维细胞HS-68、NIH-3T3、293、Jurkat T或Cos-7细胞系可在补充有1% (vol/vol) 200 mM谷氨酰胺、1% (vol/vol)抗生素(链霉素,10,000 µg/ml;青霉素,10,000 IU/ml)和10% (wt/vol) FBS的Dulbecco改良的Eagle培养基(DMEM)中在37℃在包含5% CO2的潮湿大气中培养。对于肽介导的重组LEKTI蛋白递送,可将纯化的重组LEKTI产物(如上获得的)加载在DMEM或PBS (500 µL包含0.25 µg蛋白的DMEM)中并在37℃孵育30 min。然后将生长至75%汇合的细胞用这些重组LEKTI蛋白培养基覆盖。在37℃孵育30 min 后,将1 mL补充有10% FBS的新鲜DMEM加至细胞,不移除重组LEKTI蛋白的覆盖物,并将细胞返回培养箱另外30 min。然后将细胞用PBS广泛洗涤并检查重组LEKTI蛋白。可将细胞按照制造商的说明书经由首先用2%甲醛(Sigma)固定、透化然后用初级抗-6xHis标签抗体和第二抗体孵育通过免疫荧光观察。或者可获得细胞裂解物并通过蛋白质印迹观察His标记的重组LEKTI的存在情况,如上所述。
根据一些实施方案,分泌蛋白和渗透肽的某些组合对重组LEKTI蛋白穿过细胞膜的能力具有不同的影响。
实施例4
LEKTI蛋白需要蛋白水解切割以活化其对许多蛋白酶的抑制功能。将全长蛋白切割成结构域D1-D5和D6-D15。将D6-D15结构域然后在多步中进一步分切割成D6-D9和D10-D15、→D6和D7-D9 → D7和D8-D9 → D8。全长LEKTI多肽、结构域和天然切割产物的示意图在图3中示出。在选择具体的结构域表达时,考虑结构域的以下标准:(1) 对不同的激肽释放酶相关肽酶(KLK)例如KLK5和KLK7有活性;(2) 耐受蛋白酶;(3) 小(非代谢负荷);(4) 包含最小的二硫键含量。将结构域6选作表达的LEKTI片段。全长LEKTI蛋白的氨基酸序列如SEQ IDNO:103所列出。15个个体结构域也各自在下文以fasta格式列出:
LEKTI氨基酸序列残基1-1064 (SEQ ID NO:103):
MKIATVSVLLPLALCLIQDAASKNEDQEMCHEFQAFMKNGKLFCPQDKKFFQSLDGIMFINKCATCKMILEKEAKSQKRARHLARAPKATAPTELNCDDFKKGERDGDFICPDYYEAVCGTDGKTYDNRCALCAENAKTGSQIGVKSEGECKSSNPEQDVCSAFRPFVRDGRLGCTRENDPVLGPDGKTHGNKCAMCAELFLKEAENAKREGETRIRRNAEKDFCKEYEKQVRNGRLFCTRESDPVRGPDGRMHGNKCALCAEIFKQRFSEENSKTDQNLGKAEEKTKVKREIVKLCSQYQNQAKNGILFCTRENDPIRGPDGKMHGNLCSMCQAYFQAENEEKKKAEARARNKRESGKATSYAELCSEYRKLVRNGKLACTRENDPIQGPDGKVHGNTCSMCEVFFQAEEEEKKKKEGKSRNKRQSKSTASFEELCSEYRKSRKNGRLFCTRENDPIQGPDGKMHGNTCSMCEAFFQQEERARAKAKREAAKEICSEFRDQVRNGTLICTREHNPVRGPDGKMHGNKCAMCASVFKLEEEEKKNDKEEKGKVEAEKVKREAVQELCSEYRHYVRNGRLPCTRENDPIEGLDGKIHGNTCSMCEAFFQQEAKEKERAEPRAKVKREAEKETCDEFRRLLQNGKLFCTRENDPVRGPDGKTHGNKCAMCKAVFQKENEERKRKEEEDQRNAAGHGSSGGGGGNTQDECAEYREQMKNGRLSCTRESDPVRDADGKSYNNQCTMCKAKLEREAERKNEYSRSRSNGTGSESGKDTCDEFRSQMKNGKLICTRESDPVRGPDGKTHGNKCTMCKEKLEREAAEKKKKEDEDRSNTGERSNTGERSNDKEDLCREFRSMQRNGKLICTRENNPVRGPYGKMHINKCAMCQSIFDREANERKKKDEEKSSSKPSNNAKDECSEFRNYIRNNELICPRENDPVHGADGKFYTNKCYMCRAVFLTEALERAKLQEKPSHVRASQEEDSPDSFSSLDSEMCKDYRVLPRIGYLCPKDLKPVCGDDGQTYNNPCMLCHENLIRQTNTHIRSTGKCEESSTPGTTAASMPPSDE
LEKTI结构域在下文列出:
LEKTI结构域1 (残基23-77; SEQ ID NO:104)
KNEDQEMCHEFQAFMKNGKLFCPQDKKFFQSLDGIMFINKCATCKMILEKEAKSQ
LEKTI结构域2 (残基91-153; SEQ ID NO:105)
APTELNCDDFKKGERDGDFICPDYYEAVCGTDGKTYDNRCALCAENAKTGSQIGVKSEGECKS
LEKTI结构域3 (残基155-216; SEQ ID NO:106)
NPEQDVCSAFRPFVRDGRLGCTRENDPVLGPDGKTHGNKCAMCAELFLKEAENAKREGETRI
LEKTI结构域4 (残基219-285; SEQ ID NO:107)
NAEKDFCKEYEKQVRNGRLFCTRESDPVRGPDGRMHGNKCALCAEIFKQRFSEENSKTDQNLGKAEE
LEKTI_结构域5 (残基291-352; SEQ ID NO:108)
REIVKLCSQYQNQAKNGILFCTRENDPIRGPDGKMHGNLCSMCQAYFQAENEEKKKAEARAR
LEKTI_结构域6 (残基356-423; SEQ ID NO:109)
ESGKATSYAELCSEYRKLVRNGKLACTRENDPIQGPDGKVHGNTCSMCEVFFQAEEEEKKKKEGKSRN
LEKTI结构域7 (残基_431-489; SEQ ID NO:110)
ASFEELCSEYRKSRKNGRLFCTRENDPIQGPDGKMHGNTCSMCEAFFQQEERARAKAKR
LEKTI结构域8 (残基490-550; SEQ ID NO:111)
EAAKEICSEFRDQVRNGTLICTREHNPVRGPDGKMHGNKCAMCASVFKLEEEEKKNDKEEKG
LEKTI结构域9 (残基_561_622; SEQ ID NO:112)
EAVQELCSEYRHYVRNGRLPCTRENDPIEGLDGKIHGNTCSMCEAFFQQEAKEKERAEPRAK
LEKTI结构域10 (残基626-688; SEQ ID NO:113)
EAEKETCDEFRRLLQNGKLFCTRENDPVRGPDGKTHGNKCAMCKAVFQKENEERKRKEEEDQR
LEKTI结构域11 (残基701-757; SEQ ID NO:114)
GNTQDECAEYREQMKNGRLSCTRESDPVRDADGKSYNNQCTMCKAKLEREAERKNEY
LEKTI结构域12 (残基768-830; SEQ ID NO:115)
ESGKDTCDEFRSQMKNGKLICTRESDPVRGPDGKTHGNKCTMCKEKLEREAAEKKKKEDEDRS
LEKTI结构域13 (残基843-905; SEQ ID NO:116)
NDKEDLCREFRSMQRNGKLICTRENNPVRGPYGKMHINKCAMCQSIFDREANERKKKDEEKSS
LEKTI结构域14 (残基910-970; SEQ ID NO:117)
NNAKDECSEFRNYIRNNELICPRENDPVHGADGKFYTNKCYMCRAVFLTEALERAKLQEKPS
LEKTI结构域15 (残基987-1048; SEQ ID NO:118)
SLDSEMCKDYRVLPRIGYLCPKDLKPVCGDDGQTYNNPCMLCHENLIRQTNTHIRSTGKCEE
LEKTI核酸序列在下文如SEQ ID NO:119所列出。
LETKI全长核酸序列(SEQ ID NO:119)
ATGAAGATAGCCACAGTGTCAGTGCTTCTGCCCTTGGCTCTTTGCCTCATACAAGATGCTGCCAGTAAGAATGAAGATCAGGAAATGTGCCATGAATTTCAGGCATTTATGAAAAATGGAAAACTGTTCTGTCCCCAGGATAAGAAATTTTTTCAAAGTCTTGATGGAATAATGTTCATCAATAAATGTGCCACGTGCAAAATGATACTGGAAAAAGAAGCAAAATCACAGAAGAGGGCCAGGCATTTAGCAAGAGCTCCCAAGGCTACTGCCCCAACAGAGCTGAATTGTGATGATTTTAAAAAAGGAGAAAGAGATGGGGATTTTATCTGTCCTGATTATTATGAAGCTGTTTGTGGCACAGATGGGAAAACATATGACAACAGATGTGCACTGTGTGCTGAGAATGCGAAAACCGGGTCCCAAATTGGTGTAAAAAGTGAAGGGGAATGTAAGAGCAGTAATCCAGAGCAGGATGTATGCAGTGCTTTTCGGCCCTTTGTTAGAGATGGAAGACTTGGATGCACAAGGGAAAATGATCCTGTTCTTGGTCCTGATGGGAAGACGCATGGCAATAAGTGTGCAATGTGTGCTGAGCTGTTTTTAAAAGAAGCTGAAAATGCCAAGCGAGAGGGTGAAACTAGAATTCGACGAAATGCTGAAAAGGATTTTTGCAAGGAATATGAAAAACAAGTGAGAAATGGAAGGCTTTTTTGTACACGGGAGAGTGATCCAGTCCGTGGCCCTGACGGCAGGATGCATGGCAACAAATGTGCCCTGTGTGCTGAAATTTTCAAGCAGCGTTTTTCAGAGGAAAACAGTAAAACAGATCAAAATTTGGGAAAAGCTGAAGAAAAAACTAAAGTTAAAAGAGAAATTGTGAAACTCTGCAGTCAATATCAAAATCAGGCAAAGAATGGAATACTTTTCTGTACCAGAGAAAATGACCCTATTCGTGGTCCAGATGGGAAAATGCATGGCAACTTGTGTTCCATGTGTCAAGCCTACTTCCAAGCAGAAAATGAAGAAAAGAAAAAGGCTGAAGCACGAGCTAGAAACAAAAGAGAATCTGGAAAAGCAACCTCATATGCAGAGCTTTGCAGTGAATATCGAAAGCTTGTGAGGAACGGAAAACTTGCTTGCACCAGAGAGAACGATCCTATCCAGGGCCCAGATGGGAAAGTGCATGGCAACACCTGCTCCATGTGTGAGGTCTTCTTCCAAGCAGAAGAAGAAGAAAAGAAAAAGAAGGAAGGTAAATCAAGAAACAAAAGACAATCTAAGAGTACAGCTTCCTTTGAGGAGTTGTGTAGTGAATACCGCAAATCCAGGAAAAACGGACGGCTTTTTTGCACCAGAGAGAATGACCCCATCCAGGGCCCAGATGGAAAAATGCATGGCAACACCTGCTCCATGTGTGAGGCCTTCTTTCAACAAGAAGAAAGAGCAAGAGCAAAGGCTAAAAGAGAAGCTGCAAAGGAAATCTGCAGTGAATTTCGGGACCAAGTGAGGAATGGAACACTTATATGCACCAGGGAGCATAATCCTGTCCGTGGCCCAGATGGCAAAATGCATGGAAACAAGTGTGCCATGTGTGCCAGTGTGTTCAAACTTGAAGAAGAAGAGAAGAAAAATGATAAAGAAGAAAAAGGGAAAGTCGAGGCTGAAAAAGTTAAGAGAGAAGCAGTTCAGGAGCTGTGCAGTGAATATCGTCATTATGTGAGGAATGGACGACTCCCCTGTACCAGAGAGAATGATCCTATTGAGGGTCTAGATGGGAAAATCCACGGCAACACCTGCTCCATGTGTGAAGCCTTCTTCCAGCAAGAAGCAAAAGAAAAAGAAAGAGCTGAACCCAGAGCAAAAGTCAAAAGAGAAGCTGAAAAGGAGACATGCGATGAATTTCGGAGACTTTTGCAAAATGGAAAACTTTTCTGCACAAGAGAAAATGATCCTGTGCGTGGCCCAGATGGCAAGACCCATGGCAACAAGTGTGCCATGTGTAAGGCAGTCTTCCAGAAAGAAAATGAGGAAAGAAAGAGGAAAGAAGAGGAAGATCAGAGAAATGCTGCAGGACATGGTTCCAGTGGTGGTGGAGGAGGAAACACTCAGGACGAATGTGCTGAGTATCGGGAACAAATGAAAAATGGAAGACTCAGCTGTACTCGGGAGAGTGATCCTGTACGTGATGCTGATGGCAAATCGTACAACAATCAGTGTACCATGTGTAAAGCAAAATTGGAAAGAGAAGCAGAGAGAAAAAATGAGTATTCTCGCTCCAGATCAAATGGGACTGGATCAGAATCAGGGAAGGATACATGTGATGAGTTTAGAAGCCAAATGAAAAATGGAAAACTCATCTGCACTCGAGAAAGTGACCCTGTCCGGGGTCCAGATGGCAAGACACATGGCAATAAGTGTACTATGTGTAAGGAAAAACTGGAAAGGGAAGCAGCTGAAAAAAAAAAGAAAGAGGATGAAGACAGGAGCAATACAGGAGAAAGGAGCAATACAGGAGAAAGGAGCAATGACAAAGAGGATCTGTGTCGTGAATTTCGAAGCATGCAGAGAAATGGAAAGCTTATCTGCACCAGAGAAAATAACCCTGTTCGAGGCCCATATGGCAAGATGCACATCAATAAATGTGCTATGTGTCAGAGCATCTTTGATCGAGAAGCTAATGAAAGAAAAAAGAAAGATGAAGAGAAATCAAGTAGCAAGCCCTCAAATAATGCAAAGGATGAGTGCAGTGAATTTCGAAACTATATAAGGAACAATGAACTCATCTGCCCTAGAGAGAATGACCCAGTGCACGGTGCTGATGGAAAGTTCTATACAAACAAGTGCTACATGTGCAGAGCTGTCTTTCTAACAGAAGCTTTGGAAAGGGCAAAGCTTCAAGAAAAGCCATCCCATGTTAGAGCTTCTCAAGAGGAAGACAGCCCAGACTCTTTCAGTTCTCTGGATTCTGAGATGTGCAAAGACTACCGAGTATTGCCCAGGATAGGTTATCTTTGTCCAAAGGATTTAAAGCCTGTCTGTGGTGACGATGGCCAAACCTACAACAATCCTTGCATGCTCTGTCATGAAAACCTGATACGCCAAACAAATACACACATCCGCAGTACAGGGAAGTGTGAGGAGAGCAGCACCCCAGGAACCACCGCAGCCAGCATGCCCCCGTCTGACGAA
在大肠杆菌BL21 (De3)中的可溶性
原核生物产生可溶的和包涵体结合的蛋白质。可溶性受温度、蛋白电荷和蛋白结构及尺寸影响。不溶的包涵体结合蛋白常常是错误折叠的,通常是无活性的,并且以非常纯的不溶的包涵体分离。将包涵体结合蛋白在体外分离和重折叠,然后纯化。可溶蛋白呈折叠的结构,常常是有功能的,与其余蛋白质组一起存在于细胞质中。
进行了第一组实验以确定结构域6是否在大肠杆菌中可靠地产生。可溶性蛋白通过亲和纯化和缓冲液交换分离,然后纯化。可溶性测试测定用于测定包涵体蛋白和可溶性蛋白部分之间的分布。简言之,将结构域6蛋白表达细胞(大肠杆菌BL21 (De3))用水性缓冲液裂解。使用高速离心和包涵体纯化分离可溶部分和包涵体部分。分离的部分经受十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS–PAGE)。图4显示SDS-PAGE结果,证明LEKTId6 (8.8kDa)在大肠杆菌BL21 (De3)中高度可溶。大肠杆菌GFP (33.8 kDa)用作阳性对照,无载体用作阴性对照。实验在三种不同的诱导温度下进行:18、30和37℃。如图4中所示,在His6_LEKTId6实验组的可溶部分中检测到8.8 kDa处的带。箭头指示8.8 kDa处的带。
图5显示SDS-PAGE结果,证明对于H6-LEKTId6 (8.8 kDa)成功地进行了亲和纯化。箭头指示8.8 kDa处的带。图6显示LEKTId6-H6 (8.8 kDa)可能是N端截短的。在图5和图6二者中,对于实验组使用以下缩写:
SN = 澄清的细胞裂解物(上清液)
FT = 非Ni2+结合蛋白(流过)
W1-4 = 来自一系列洗涤(1-4)的洗脱液。注意在W4中一些来自邻近的梯的污染。
L = SDS-PAGE蛋白梯(SeeBlue Plus2, ThermoFisher Scientific)
E1-6 = 咪唑处理后来自柱的洗脱液(即所得的亲和纯化的蛋白)。随着将柱处理,收集不同的洗脱液部分(1-6)。
实施例5
对纯化的重组LEKTI结构域6 (LETKId6)片段在体外发挥丝氨酸蛋白酶抑制剂功能的能力进行评估。
首先,测定重组生产的LEKTId6在体外抑制胰蛋白酶的能力。酶活性使用BApNA (Nα-苯甲酰-l-精氨酸-对硝基酰苯胺)作为胰蛋白酶特异性底物测量。图7A显示测定的示意性概述。测定通过混合80 μL浓度(.25、2.5、25 uM)的LEKTId6与20 μL胰蛋白酶(35 ug/mL)和100 uL 2x 胰蛋白酶测定缓冲液(100 mM Tris-HCl, pH 8.0, 300 mM NaCl, 100 mMCaCl2, 0.02% Triton-X-100, 500 uM L-BAPNA)进行。在反应混合物中,组分终浓度为LEKTId6 (0.1、1、10 μM),胰蛋白酶(3.5 μg/mL),测定缓冲液(50 mM Tris-HCl, pH 8.0,150 mM NaCl, 50 mM CaCl2, 0.01% Triton-X-100)和L-BAPNA (250 μM)。让反应在37 ºC进行15 min。胰蛋白酶抑制剂亮抑酶肽用作阳性对照。产物的形成用微板读数器在405 nm测量。使用空白对照。胰蛋白酶活性定义为在建立的条件下每分钟405 nm处的吸光度(L-BAPNA切割的指示物)变化的速率。如图7中所示,LEKTI结构域6在体外抑制胰蛋白酶活性。
接着,测定LEKTI结构域6 (ct His6 tag)对胰蛋白酶抑制的影响并与LEKTI结构域10-15对胰蛋白酶抑制的影响比较。胰蛋白酶抑制测定如上所述进行,其中酶活性使用L-BAPNA (Nα-苯甲酰-l-精氨酸-对硝基酰苯胺)作为胰蛋白酶特异性底物测量。图8显示测定的示意性概述。将LETKId6 (10、30、100、1000 nm)或LEKTI结构域10-15 (10、30、100 nm)与L-BAPNA (终浓度250 uM)在25 ℃混合10 min。胰蛋白酶抑制剂亮抑酶肽用作阳性对照。测定重组生产的LEKTId6在体外抑制激肽释放酶7和5 (KRK7和KRK5)的能力。简言之,将蛋白酶KLK7和KLK5用增加的浓度的LEKTId6在25℃孵育5 min,然后加入其最佳的肽底物,最佳的肽底物对于KLK7为Suc-Arg-Pro-Tyr-对硝基-酰苯胺以及对于KLK5为D-Ile-Pro-Arg-对硝基-酰苯胺。产物的形成用微板读数器在405 nm测量。使用空白对照。KLK7测定和KLK5测定的示意性概述分别在图9A和10A中显示。对于KLK7,使用逐渐增加的浓度的LEKTId6 (10、30、100、300、1000 nm)和逐渐增加的浓度的LEKTId10-15 (10、30、100 nm)。胰蛋白酶抑制剂亮抑酶肽用作阴性对照。对于KLK5,使用逐渐增加的浓度的LEKTId6 (10、30、100、300、1000 nm)。如图9B中所示,重组生产的LEKTI结构域6在体外抑制KLK7,LEKTI结构域10-15差不多一样在体外抑制KLK7。如图10B中所示,重组生产的LEKTId6在体外以纳摩尔浓度抑制KLK5。同时,高浓度的LETKId6显示为刺激性的,不受理论束缚,这可能是因为测定的缓冲液组分所致,特别是亲和纯化后遗留在LEKTId6样品中的残余咪唑。
实施例6
治疗性LETKId6表皮葡萄球菌菌株的功效将在条件内瑟顿小鼠模型中评估。简言之,诱导Cre重组后1、2和4周我们将验证CRISPR建立的内瑟顿综合征小鼠(条件SPINK5 -/-)的皮肤中LEKTI的不存在。具有经验证的内瑟顿综合征表型的小鼠将用局部施用重组LEKTI治疗以消退Spink5条件突变体中的皮肤病况。首先使用纯化的LEKTI的原理为避免对构建表皮葡萄球菌菌株的依赖,以致于我们可迅速证明在体内局部施用的功效。第二,我们将评估纯化的表皮葡萄球菌或LEKTI的能力以证明益生菌定殖对持续修复的价值。作为对照,我们将局部定殖相同的小鼠pre-Cre-诱导的SPINK5条件突变。为了评估LETKId6在小鼠模型中的效果,我们将进行纵向测定(1x/周)(在可能的情况下)和终点测定(定殖3周后)以检验施用治疗的表皮葡萄球菌是否将(1) 在体外产生可测量的量的LEKTI,如通过皮肤的免疫组织化学分析测量的(终点),(2) 减少皮肤疾病严重性,如通过DASI测定的(纵向和终点),(3)改善TEWL (纵向)和渗透率分数(终点),(4) 改善皮肤形态学,如通过组织学分析测量的(终点),和(5) 导致蛋白水解活性变化,如使用靶向KLK5和KLK7的比色测定测量的(终点)。
通过引用结合
本文中提及的各个专利文献(包括专利申请文献)、科学文章、政府报告、网站和其它参考文献的全部公开内容通过引用以其整体结合到本文中用于所有目的。在术语冲突的情况下,以本说明书为准。本文公开的所有序列表或Seq. ID.号以其整体结合到本文中。
就其提供与本文阐述的那些补充的示例性的程序或其它细节而言,以下参考文献通过应用具体地结合到本文中。
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尽管已经在本文中描述本发明的说明性实施方案,应理解本发明不限于所描述的那些,可由本领域技术人员作出各种其它变化或修饰而不脱离本发明的范围或精神。
Claims (22)
1.用于治疗皮肤疾病的组合物,所述组合物包含:
经遗传修饰以在哺乳动物的皮肤上表达和提供一个或多个LEKTI蛋白结构域的微生物,其中所述LEKTI蛋白结构域有效渗透哺乳动物皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的丝氨酸蛋白酶活性。
2.权利要求1的组合物,其中所述微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间以提供LEKTI蛋白结构域的连续供应。
3.权利要求1的组合物,其中所述LEKTI蛋白结构域有效改善内瑟顿综合征的症状。
4.权利要求1的组合物,其中所述微生物通过用编码所述LEKTI蛋白结构域的重组DNA质粒转染/转化而遗传修饰。
5.权利要求1的组合物,其中所述LEKTI结构域与有效增强从微生物分泌和/或渗透哺乳动物的皮肤的一个或多个重组蛋白结构域可操作地连接。
6.权利要求1的组合物,其中至少一个LEKTI结构域与SecA结构域可操作地连接。
7.权利要求1的组合物,其中至少一个LEKTI结构域与RMR结构域可操作地连接。
8.权利要求1的组合物,其中至少一个LEKTI结构域包含根据SEQ ID NO: 1的氨基酸序列。
9.权利要求1的组合物,其中所述微生物适应于在哺乳动物的皮肤上繁殖。
10.权利要求1的组合物,其中至少一个LEKTI结构域的表达受操纵子控制并且提供给哺乳动物的皮肤的LEKTI的量与外在因素的可用性成比例。
11.权利要求1的组合物,其中至少一个LEKTI结构域的表达受组成型活性的启动子控制。
12.权利要求1的组合物,其中所述微生物已经通过用编码所述一个或多个LEKTI蛋白结构域和一个或多个抗生素抗性基因的重组DNA质粒转染/转化而遗传修饰。
13.权利要求1的组合物,其中所述微生物选自双歧杆菌属(Bifidobacterium)、短杆菌属(Brevibacterium)、丙酸杆菌属(Propionibacterium)、乳球菌属(Lactococcus)、链球菌属(Streptococcus)、葡萄球菌属(Staphylococcus)、乳杆菌属(Lactobacillus)、肠球菌属(Enterococcus)、片球菌属(Pediococcus)、明串珠菌属(Leuconostoc)或酒球菌属(Oenococcus)及其混合物。
14.治疗有需要的哺乳动物的皮肤疾病或改善有需要的哺乳动物的皮肤疾病的影响的方法,所述方法包括:
向哺乳动物的皮肤的表面上提供经遗传修饰以表达一个或多个LEKTI蛋白结构域的微生物,其中所述LEKTI蛋白结构域有效渗透哺乳动物皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的活性。
15.权利要求14的方法,其中所述微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间以提供LEKTI蛋白结构域的连续供应。
16.用于治疗有需要的哺乳动物的皮肤疾病或改善有需要的哺乳动物的皮肤疾病的影响的试剂盒,所述试剂盒包含:
(1) 包含经遗传修饰以表达一个或多个LEKTI蛋白结构域的微生物的组合物,其中所述LEKTI蛋白结构域有效渗透哺乳动物皮肤的一个或多个层并有效抑制哺乳动物的皮肤中或皮肤上的至少一种丝氨酸蛋白酶的丝氨酸蛋白酶活性;和
(2) 用于将所述组合物施用到哺乳动物的皮肤的试剂。
17.权利要求16的试剂盒,其中所述微生物适应于在哺乳动物的皮肤表面上存活受控的持续时间以提供LEKTI蛋白结构域的连续供应。
18.用于治疗皮肤疾病的组合物,所述组合物包含含有pJB38-LEKTI-完整质粒构建体的微生物。
19.权利要求18的组合物,其中所述微生物选自双歧杆菌属、短杆菌属、丙酸杆菌属、乳球菌属、链球菌属、葡萄球菌属、乳杆菌属、肠球菌属、片球菌属、明串珠菌属或酒球菌属及其混合物。
20.包含pJB38-LEKTI-完整质粒构建体的组合物。
21.权利要求20的组合物,其中pJB38-LEKTI-完整质粒构建体在选自双歧杆菌属、短杆菌属、丙酸杆菌属、乳球菌属、链球菌属、葡萄球菌属、乳杆菌属、肠球菌属、片球菌属、明串珠菌属或酒球菌属及其混合物的微生物中表达。
22.能够分泌多肽的重组微生物,其中所述重组微生物包含表达载体,所述表达载体包含含有能够表达所述多肽的基因的第一编码序列和含有能够表达细胞渗透肽的基因的第二编码序列的。
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EP3638369A4 (en) | 2021-03-17 |
WO2018232300A1 (en) | 2018-12-20 |
CA3067364A1 (en) | 2018-12-20 |
KR20200021991A (ko) | 2020-03-02 |
US20240218049A1 (en) | 2024-07-04 |
JP2023138994A (ja) | 2023-10-03 |
US20190040116A1 (en) | 2019-02-07 |
US11773154B2 (en) | 2023-10-03 |
JP2020527357A (ja) | 2020-09-10 |
AU2018285080A1 (en) | 2020-01-16 |
EP3638369A1 (en) | 2020-04-22 |
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