CN103370413B - Dna表达构建体 - Google Patents
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- CN103370413B CN103370413B CN201180067798.6A CN201180067798A CN103370413B CN 103370413 B CN103370413 B CN 103370413B CN 201180067798 A CN201180067798 A CN 201180067798A CN 103370413 B CN103370413 B CN 103370413B
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Abstract
本发明涉及最小化的基因表达构建体,其转移至细胞以及其在分子医药应用中用于基因表达的用途。根据本公开,提供了用于基因表达的DNA构建体,其中所述构建体是线性的和开链的DNA双链,其包含启动子序列、编码序列和终止信号,其中所述构建体包含至少一个L-DNA核苷酸。
Description
技术领域
本发明涉及最小化的(minimalistic)基因表达构建体,其至细胞的转移及其在分子医药应用中的基因表达的用途。
背景技术
可转移入目标细胞的基因表达构建体通常用于DNA疫苗接种、肿瘤治疗和肿瘤预防。用于这些目的的载体构建体必须保证成功的转染以及对患者最大的安全性。物理地或化学地转染入细胞的所谓基于质粒的环状闭合DNA双链构建体相对安全。然而,基于目标细胞类型,转染效率通常不是令人满意的。因此,质粒不适用于临床方案。此外,它们通常包括共转入细胞的若干原核蛋白(例如抗生素抗性基因)。原核启动子在真核细胞中并不是完全沉默的,这可能导致不期望的免疫作用。宿主免疫系统还可消除转染的细胞。此外,质粒载体中的非甲基化CG基序可以是免疫刺激性的,并负责在基因治疗方案中观察到的至少一些免疫毒性。
复制缺陷逆转录病毒或腺病毒载体由于它们较质粒高得多的转染效率而通常用于临床方案中。然而,它们具有与野生型病毒重组或激活癌基因的重大风险。此外,必须使用高病毒滴度可能引起炎症。
因此,有必要开发改善的基因表达构建体来克服质粒和病毒载体的不期望的副作用。EP0941318公开了一种小的、线性的、共价闭合的、哑铃型的新载体类型,用于最小化的免疫学定义的基因表达(MIDGE)。MIDGE是具最小大小的基因转移单元;它仅包含最小的必要序列:表达盒,包括启动子、基因和RNA-稳定化序列,两侧为两个短发夹寡聚核苷酸序列。此外,MIDGE的设计提供用于有效转运至目标细胞的手段。
MIDGE分子比传统使用的质粒或病毒载体小得多,因此更容易转入细胞并允许高表达率。它们在两端包括短发夹以保护表达盒免遭核酸酶的降解。
发明内容
相关于现有技术状态,本发明目的是提供用于在真核细胞中基因表达的有效的DNA构建体,其受保护免遭核酸酶的降解。
根据本公开,提供了用于基因表达的DNA构建体,其中所述构建体是线性的和开链的DNA双链,其包含启动子序列、编码序列和终止序列,其中所述构建体包含至少一个L-DNA核苷酸。包含至少一个L-DNA核苷酸的线性部分双链哑铃型DNA构建体也在本公开的范围之内。
还提供了一种DNA构建体,其中至少一个L-DNA核苷酸位于5’末端和/或3’末端。单链环可位于5’末端和/或3’末端,也与至少一个L-DNA核苷酸组合,所述至少一个L-DNA核苷酸位于5’末端和/或3’末端的最后2-5个核苷酸内。
根据本公开的构建体可包含部分或完全双链的DNA链。另外预期至少一个L-DNA或D-DNA核苷酸用选自包含羧基、胺基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基、硫醇基和醛基的组的官能团修饰。
核苷酸可连接至选自包含肽、蛋白质、碳水化合物、脂质、囊泡、抗体、合成分子、聚合物、微弹、金属颗粒、纳米颗粒或固相的组的化合物。
预期根据本公开的构建体包含启动子,其选自包含启动子序列的组,其在人类、动物或真核细胞中是可操作的。该构建体可编码蛋白质、肽、抗体、激素、细胞因子或其他生物学活性物质,例如抑制性、调节性或刺激性RNA或免疫调节剂。
本公开的另一个目的是上述DNA构建体用于稳定或瞬时转染人、动物或真核细胞以及用于基因治疗或DNA疫苗接种的用途。术语“基因治疗”包括体内或离体,以及自体或异体(allogeneic)方法。
本公开的另一个目的是上述DNA构建体用于针对传染病或寄生虫的预防性或治疗性疫苗接种的用途。
根据本公开的DNA构建体可用于治疗癌症或自体免疫疾病,任选地与非编码免疫调节DNA构建体联合。
本发明的另一个目的是提供包含上述DNA构建体的药物组合物。这样的药物可进一步包含化疗剂。可选地,它可含有细菌、真菌、寄生虫或病毒来源的额外抗原。
具体实施方式
本公开含义范围内,线性开链DNA序列被指定为DNA构建体。所述DNA序列可以是单链的或完全或部分双链的。术语DNA构建体、DNA分子和表达构建体同义使用,不代表对相应DNA序列长度的限制。DNA构建体的单体成分是核苷酸。
DNA构建体可合成制备,或部分或完全为生物来源,其中生物来源包括DNA序列制备的基于遗传学的方法。
L-DNA或L-构象的核苷酸涉及包含L-脱氧核糖而不是天然存在的D-脱氧核糖作为糖残基的核苷酸。L-脱氧核糖是D-脱氧核糖的对映体(镜像)。部分或完全由L-构象的核苷酸组成的DNA构建体可部分或完全单链或双链;然而,L-构象的核苷酸不能与D-构象的核苷酸杂交(Hauseretal.,NucleicAcidRes.200634:5101-11)。L-DNA与D-DNA是同等可溶和选择性的。L-DNA还对天然存在的酶的酶促降解有抗性,因此L-DNA还被保护免遭生物降解(Urataetal.,NucleicAcidsRes.199220:3325-32)。因此,L-DNA可非常广泛地应用。
根据本公开,“茎(stem)”应理解为由同一DNA分子(那么其部分自互补)内或不同DNA分子(其部分或完全互补)内的碱基配对形成的DNA双链。分子内碱基配对指的是为同一DNA分子内的碱基配对,不同DNA分子之间的碱基配对称为分子间碱基配对。
本公开含义范围内,“环”应理解为茎结构内或末端处的非配对、单链区域。“发夹”是茎和环的独特结合,其当同一寡核苷酸的两个自互补区域杂交以形成在一端具有未配对环的茎时发生。
哑铃型描述在茎区域的两端具有发夹的线性DNA构建体。因此,在本公开内容中,“线性DNA构建体”描述了包含单链或双链DNA的线性开链DNA构建体或在双链DNA茎的两端包含单链环的线性哑铃型DNA构建体。
术语“DNA末端”,无论意思是DNA单链的5’末端或3’末端,涉及不仅末端核苷酸,而且包含末端三个核苷酸或者甚至关于各个DNA末端的最后五个核苷酸。DNA末端的修饰与各个核苷酸的至少一个有关。
核苷酸共价或非共价连接的“固相”涉及但不限于柱、基质、珠、玻璃(包括改性或官能化的玻璃)、二氧化硅或基于二氧化硅的材料(包括硅和改性硅)、塑料(包含聚丙烯、聚乙烯、聚氨酯等)、尼龙或硝化纤维素、树脂、多糖、碳以及无机玻璃和塑料。因此,微量滴定板也在根据本公开的固相范围之内。
根据本公开的免疫调节指免疫刺激和免疫抑制。免疫刺激优先意为免疫系统的效应细胞被刺激以增殖、迁移、分化或以任何其他的形式活化。例如可在无共刺激信号的情况下通过免疫刺激DNA分子诱导B细胞增殖,B细胞增殖通常要求来自辅助T细胞的共刺激信号。
另一方面,免疫抑制应理解为降低免疫系统的活化和效力。免疫抑制通常被有意诱导以防止例如移植器官的排斥,治疗骨髓移植后的移植物-抗-宿主疾病,或用于治疗自体免疫疾病例如类风湿关节炎或克罗恩氏病。
在此背景下,免疫调节还可指对免疫反应的性质或特征的影响,或者通过影响发育中或成熟中的免疫反应,或者通过调节已建立的免疫反应的特征。
在本公开中使用的术语“疫苗接种”指施用抗原性材料(疫苗)来产生对疾病的免疫力。疫苗可防止或减缓许多病原体(体例如病毒、真菌、寄生性原虫病、细菌)感染的作用,而且可防止或减缓变应性疾病和哮喘以及肿瘤的作用。疫苗一般含有一或多种佐剂,例如免疫刺激性核酸,用于增加强免疫应答。疫苗接种通常认为是防止传染病和其他疾病的最有效和低成本的方法。
施用的材料可以例如是病原体(例如真菌、细菌或病毒)的活的但是弱化形式,这些病原体的杀死或灭活形式,纯化的材料例如蛋白质、编码抗原的核酸或例如肿瘤细胞或树突细胞的细胞。特别地,最近已开发了DNA疫苗接种。DNA疫苗接种通过将编码抗原的DNA插入(和表达,触发免疫系统识别)至人或动物细胞而工作。能识别所表达蛋白的免疫系统的某些细胞将对这些蛋白和表达它们的细胞发动攻击。DNA疫苗的一个优点是它们非常容易生产和储存。此外,DNA疫苗具有优于传统疫苗的许多优点,包括引起更广范围免疫应答类型的能力。
疫苗接种可用作预防性方法,在已接种疫苗的健康个体暴露于抗原之后,引起其针对所述抗原的免疫力。可选地,治疗性免疫接种能通过引导个体的免疫系统至抗原而引起被接种疫苗的患病个体免疫系统的改善的应答。预防性和治疗性疫苗接种可应用于人以及动物。
本公开中所用术语“基因治疗”指对个体细胞和/或生物组织的瞬时或永久的基因修饰(例如基因的插入、改变或移除)以治疗疾病,例如肿瘤和自体免疫疾病。最常见形式的基因治疗包括将功能基因插入非特定的基因组位置以取代突变基因,然而其他形式包括直接纠正突变或修饰使得病毒能够感染的正常基因,或甚至将基因或基因片段转入细胞进行其转录。
“自体基因治疗”指使用相同个体的组织或细胞。分离的细胞或组织将通过基因治疗修饰,并重新引入供体中。相对而言,“异体基因治疗”指将来自不是受体个体的个体的细胞用于基因治疗。遗传修饰之后,所述异体细胞被引入受体。
术语“离体基因治疗”指这样的治疗方法,其中来自个体的细胞,例如造血干细胞或造血祖细胞,离体遗传修饰,并随后引入待治疗的个体。术语“体内基因治疗”指这样的治疗方法,其中来自个体的细胞,例如造血干细胞或造血祖细胞,用例如病毒载体或其他表达构建体在体内遗传修饰。
基因治疗还可分为“生殖系细胞基因治疗”和“体细胞基因治疗”。在“生殖细胞基因治疗”的情况下,生殖细胞,即精子或卵细胞,被遗传修饰。遗传改变通常整合到它们的基因组中。因此,由于治疗产生的改变可以遗传,并将传递至后代。该方法可用于治疗遗传紊乱和遗传病。在“体细胞基因治疗”的情况下,治疗性基因被转移入个体的体细胞。任何修饰和效应将仅被限制至该个体,将不会由个体的子孙或后代遗传。
术语“癌症”包含治疗或预防的癌性疾病或肿瘤,其选自于包含乳腺癌、黑色素瘤、皮肤癌、胃肠肿瘤(包括结肠癌、胃癌、胰腺癌、直肠癌、小肠癌)、卵巢癌、宫颈癌、肺癌、前列腺癌、肾细胞癌和/或肝转移的组。
根据本公开的自体免疫疾病包含类风湿关节炎、克罗恩氏病、全身性红斑狼疮(SLE)、自身免疫性甲状腺炎、桥本氏甲状腺炎、多发性硬化、格氏病(Graves’disease)、重症肌无力、乳糜泻和爱迪生氏病(Addison’sdisease)。
根据本发明的传染病包括细菌、病毒、真菌或真核寄生虫感染,例如HIV、肝炎、流感、利什曼病、细菌性肺炎、肺结核、麻疹、百日咳、破伤风、脑膜炎、梅毒、疟疾和霍乱,以及动物特异性疾病,例如猫免疫缺陷病毒感染(FIV)、猫白血病病毒感染(FeLV)、牛疱疹病毒感染(BHV)和牛病毒性腹泻。
由包含最小化的基因表达单元的包含L-DNA的脱氧核糖核酸构建体获得不会被核酸酶降解的基因表达构建体。实验数据证实如此保护的基因表达构建体适合目标细胞中有效的基因转移和表达。
根据本发明的构建体转染可实现的基因表达率高于可由如EP0941318中公开的共价闭合分子(MIDGE载体)实现的比率。MIDGE载体已经表明达到比可比的质粒载体更高的转染效率以及更高的表达率(Schakowskiet.al.,invivo,21:17-24,2007)。通过避免EP0941318的单链环,本发明的分子在大小上进一步降低,所述大小便于基因转移。由于L-DNA修饰,提供了稳定性。事实上,在生产过程中使用DNA降解酶去除未修饰的DNA强调了该稳定性。应当指出,提供包含L-DNA的具有安置在双链一端的至少一个发夹的DNA构建体也在本发明范围之内。
根据本公开的DNA构建体可用于所编码基因在目标细胞中的人工基因表达。在这方面,DNA疫苗接种、肿瘤治疗和肿瘤预防需要安全和有效地能用于临床方案中的表达构建体。标准表达构建体是例如基于质粒的载体。然而,这些载体具有两个缺陷。首先,它们的效率相当低,其次,基于质粒的载体包含数个基因和对于期望多肽的表达来说不必要的遗传元件,由此带有不可预知的免疫副作用的危险。在较久和重复应用中,很可能期望的免疫响应由于可出现严重并发症而被这些副作用掩蔽。
现有技术的其他表达构建包括复制缺陷逆转录病毒或腺病毒载体。尽管由于它们可用于转导多种细胞类型的甚至非分裂的细胞而非常有效,它们具有与野生型病毒重组,由此产生新的病原体病毒以及激活癌基因的风险。此外,病毒蛋白,尤其是在高滴度时,非常有可能引起免疫副作用。
根据本公开的表达构建体的最小化的表达单元可编码但不限于MHC-I或MHC-II呈递肽、细胞因子或细胞周期调节组分、或调节性RNA分子和反义RNA、核酶或编辑mRNA的RNA分子。此外,根据本公开的DNA构建体允许它们吸附或共价结合或离子性结合至例如肽、蛋白质、碳水化合物、脂质或糖肽配体,以及微弹,所述微弹允许将构建体通过弹道转移转移进细胞,尤其转移进皮肤、肌肉组织、胰腺和肝脏。
附图说明
本公开通过实例和图将进一步解释,并不限于公开的实施方式。其显示:
图1根据本公开的DNA构建体的示意图。
图2酶消化后DNA构建体的琼脂糖凝胶电泳。
图3使用L-DNA构建体2L-M的电穿孔的转染效率。
图4使用L-DNA构建体2L-M的脂质转染的转染效率。
图5比较L-DNA和D-DNA表达构建体的荧光强度。
图6使用L-DNA构建体DL-M和LD-M和MIDGE载体的电穿孔的转染效率的比较。
图7小鼠用编码乙肝表面抗原小蛋白的MIDGE或L-MIDGE的免疫。
附图详细说明
图1显示根据本公开的DNA构建体(2L-M)的示意图。如表1中所示,末端寡核苷酸包含L构象的核苷酸,其在图1中在双链构建体的末端由灰色框指示。由此,整个DNA构建体被保护免遭核酸外切酶的降解。图1中描述的构建体在一末端或两末端不具有或不需要保护免遭降解的发夹环。
图2显示经过来自T7噬菌体的T7聚合酶消化的DNA构建体的琼脂糖凝胶。各DNA构建体6μg与10单位的T7聚合酶孵育,总反应体积为20μl。0、1、5、30、60和1500分钟后,将3μl孵育混合物的等份从样品移除,并用5μl含甲酰胺的Sanger染料稀释。将所有等份上样至3%琼脂糖凝胶,其在100伏下运行100分钟。
泳带1显示根据EP0941318的MIDGE载体。泳带2显示用于制备根据EP0941318的MIDGE载体的未保护表达盒。泳带3显示根据本发明的DNA构建体,两端都被L-DNA保护(以下称“2L-M”)。泳带4和5显示根据本发明的可选择的DNA构建体,一端被L-DNA保护,另一端被发夹保护。泳带4中的DNA构建体包含在启动子5’末端的L-DNA保护端,以及3’末端的发夹,然而泳带5的构建体分别由启动子3’末端的L-DNA和其5’末端的发夹保护。这些构建体称为“DL-M”和“LD-M”(详情参见实施例部分)。
图3显示与EP0941318中公开的MIDGE(M)相比较,使用本公开的含L-DNA的2L-M表达构建体(2L-M)的电穿孔的转染效率,二者都编码eGFP。使用鲑鱼精作为阴性对照的试验结果显示在右侧。左侧黑色柱代表活细胞的数目,灰色中间柱表明转染细胞的数目,以及白色右侧柱显示死亡细胞的数目。黑色线表明电穿孔后细胞的总数目。
新的2L-M含L-DNA构建体的转染效率高于使用哑铃型MIDGE构建体约三分之一。如上所述,MIDGE载体已显示具有高于可比的质粒构建体的转染效率(Schakowskiet.al.,invivo,21:17-24,2007)。
结果不仅显示根据本公开的表达构建体适于基因表达,而且令人惊讶地表明所述构建体与其他表达构建体相比具有意想不到的高效率。所公开的表达构建体显然足够稳定以引起相当大量的基因表达,另一方面,当使用电穿孔以及使用脂质转染时,构建体至细胞中的吸收或转移看起来都运作非常良好。
为了检验图3中所示结果是否与电穿孔相关,将等摩尔量的如EP0941318中公开的MIDGE和含L-DNA的DNA构建体2L-M利用脂质转染转移入细胞(图4)。使用鲑鱼精作为阴性对照。每个白色柱显示活细胞的数目,灰色柱转染细胞的数目,以及黑色柱表明死亡细胞的数目。
使用1.5μgDNA脂质转染对于根据本公开的2L-ML-DNA构建体来说获得较多数目的转染细胞。尽管当使用L-DNA构建体时,使用0.5μgDNA的转染细胞的数目较低,但是使用L-DNA构建体时荧光强度更高。
图5显示用于转染的DNA量(质量)与在个体细胞中引起的平均荧光强度之间的关系。实线表示根据本发明的2L-M构建体,虚线显示使用如EP0941318中所公开的MIDGE的结果。
为了获得同等的光强度,与所公开的2L-M构建体相比,必须应用多约30%的哑铃型DNA构建体的DNA(质量)。因此,根据本公开的DNA构建体的效率出人意料地高于使用哑铃型构建体。
图6比较了获自CHO-K1细胞的荧光,所述细胞通过使用MIDGE载体和构建体LD-M和DL-M以及鲑鱼精作为对照的电穿孔,用eGFP进行修饰。出人意料地,在所使用的所有浓度下,与MIDGE载体相比,DL-M和LD-M构建体都引起来自细胞的较高的荧光信号。因此,与MIDGE载体相比,含一个L-DNA修饰末端和一个D-DNA发夹的L-DNA构建体出人意料地显示提高的转染效率。明显地,这些构建体被细胞非常良好地摄入,并允许稳定和有效的表达,获得所述载体改善的表现。
为了测试所公开的表达构建体在动物模型中的效率,使用编码乙肝病毒表面抗原(HBsAg)小蛋白的DNA序列。图7中描绘了显示用MIDGE和L-MIDGE免疫的小鼠组之间显著差异的线性表示(在IgG2a情况下,p=0.033)。与使用MIDGE相比,使用L-MIDGE作为表达构建体产生更高的抗体浓度。
本公开提供用于基因表达的线性构建体,其被保护免遭核酸酶的降解,并适合于转染至细胞后的有效基因表达。证实获自本公开表达构建体转染的基因产物正诱导特异性抗体是可能的。因此,已证实所公开的表达构建体适合用于真核细胞中有效的基因表达。
实施例
DNA构建体的制备
根据本公开的DNA构建体的制备类似于EP0941318。然而,发夹寡核苷酸被所谓的“L-接头”取代,如表1中所总结。两种接头都分别由单独的嵌合DNA分子SEQIDNo.1和SEQIDNo.2(L-接头1)或SEQIDNo.3和SEQIDNo.4(L-接头2)组成(表1)。接头由等摩尔浓度的根据表1的单链DNA分子在40mMTris-HCl、10mMMgCl2、10mMDTT、0.5mMATP(25℃下pH7.8)中在逐渐从95℃降低至25℃的温度下以0.28mg/ml杂交40min产生。在将两种接头连接到如EP0941318中所合成的表达盒之后,进行随后的由T7聚合酶去除线性D-DNA和产品的最终HPLC纯化。该构建体称为“2L-M”。
可选地,其中一个L-接头被相应的如EP0941318中描述的MIDGE载体中所使用的发夹取代。这获得在启动子一侧具有L-DNA保护,在另一侧具有发夹的DNA构建体。这些构建体分别称为“DL-M”(L-接头1和发夹)和“LD-M”(L-接头2和发夹)。
表1:用于产生DNA构建体的DNA分子。除指定的核苷酸外,所有核苷酸为D-构象。
SEQ ID | 名称 | 序列(5’-3’) | L-构象的核苷酸 |
SEQ ID No.1 | CKm364 | AGGGGTCCAGTTTTTT | 14,15 |
SEQ ID No.2 | CKm365 | AAAAACTGGAC | 1,2 |
SEQ ID No.3 | CKm362 | TTTTTCTAAGCTT | 1,2 |
SEQ ID No.4 | CKm363 | GGGAAAGCTTAGAAAAAT | 16,17 |
L-接头1 | SEQ ID No.1/SEQ ID No.2 | 见上 | |
L-接头2 | SEQ ID No.3/SEQ ID No.4 | 见上 |
转染
为了将DNA构建体转染到细胞中,可使用不同的转染方法,例如脂质转染或电穿孔。如下进行脂质转染:将6x104CHO-K1细胞接种在3.8cm2组织培养基质上,24h后用指定数量的表达eGFP的DNA通过1:4混合FugeneHD(罗氏)并如由制造商建议处理进行转染。转染后一天,通过胰蛋白酶消化收获细胞,并通过流式细胞术分析荧光(计数10.000事件)。
电穿孔
如下进行电穿孔:将2x106CHO-K1细胞悬浮于500μl生长培养基中,与指定数量的表达eGFP的DNA混合,加上11μg鲑鱼精,以270V在1650μF脉冲处理。随后,将细胞接种在9.5cm2细胞培养基质上并培养。转染后一天,通过胰蛋白酶消化收获细胞,并通过流式细胞术分析荧光(计数10,000事件)。
小鼠免疫
将HBsAg编码序列置于强病毒PCMV启动子的控制下。使用ODNCKm362-365(comp.表1)生产编码HBsAg的L-MIDGE载体,根据EP0941318生产编码HBsAg的MIDGE载体。用10μg/25μlL-MIDGE-HBsAg载体(8.14pmol)或10μg/25μlMIDGE-HBsAg载体(8.179pmol)两次(分开3周)皮内免疫Balb/c小鼠(每组6只动物)。第二次免疫后两周,获得血清,并使用包被HBsAg的ELISA板(DadeBehring;EnzygnostAnti-HBsII;cat.no.OQNE17或OQNE11)通过ELISA分析HBsAg特异的IgG1和IgG2。兔-抗-鼠IgG1和IgG2a(BD;货号559626和553391)作为二抗,鼠-抗-HBsAgIgG2a(AffinityBioReagents,货号MA1-19264)作为标准物,和鼠-抗-HBsAgIgG1(AffinityBioReagents,货号MA1-19263)作为标准物。
Claims (15)
1.用于基因表达的DNA构建体,其中所述构建体是线性的和开链的DNA双链,其包含启动子序列、编码序列和终止信号,其中所述构建体仅在5’-末端和/或3’-末端的最后五个核苷酸内包含至少一个L-DNA核苷酸。
2.权利要求1的构建体,其中所述DNA构建体的至少一端包含单链环。
3.权利要求1或2的构建体,其中所述构建体是部分或完全双链的。
4.权利要求1或2的构建体,其中至少一个L-DNA或D-DNA核苷酸用官能团修饰,所述官能团选自包含羧基、胺基、酰胺基、醛亚胺基、缩酮基、缩醛基、酯基、醚基、二硫基、硫醇基和醛基的组。
5.权利要求4的构建体,其中修饰的核苷酸与化合物连接或与固相连接,所述化合物选自包含肽、蛋白质、碳水化合物、合成分子、聚合物、微弹、金属颗粒、纳米颗粒、脂质的组。
6.权利要求1或2的构建体,其中所述启动子选自包含启动子序列的组,所述启动子序列在真核细胞、人类或动物中可操作。
7.权利要求1或2的构建体,其中所述构建体编码蛋白质、肽、激素或其他生物学活性物质。
8.权利要求7的构建体,其中所述生物学活性物质是免疫调节剂。
9.药物组合物,其包含权利要求1至8任一项的DNA构建体。
10.权利要求9的药物组合物,其还包含化疗剂。
11.权利要求1至8任一项的DNA构建体或权利要求9或10的药物组合物在制备用于稳定或瞬时转染人或动物细胞的药物中的用途。
12.权利要求1至8任一项的DNA构建体或权利要求9或10的药物组合物在制备用于稳定或瞬时转染真核细胞的药物中的用途。
13.权利要求1至8任一项的DNA构建体或权利要求9或10的药物组合物在制备用于离体基因治疗或DNA疫苗接种的药物中的用途。
14.权利要求1至8任一项的DNA构建体或权利要求9或10的药物组合物在制备用于治疗癌症或自体免疫疾病的药物中的用途。
15.权利要求13或14的用途,其中所述药物与非编码免疫调节DNA构建体组合。
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