摘要
携带外源性治疗基因的靶向性载体是基因治疗的一个中心环节,靶向导入治疗的外源基因能够极大地减少不利的副作用。本课题组在反义寡核苷酸抑制乙型肝炎病毒(hepatitis B virus, HBV)的表达、HDV核酶体外和细胞内切割HBV活性等方面作了大量工作,研究证实了HDV核酶作为HBV反义抑制剂的可行性。目前存在的问题是如何构建高效、安全且具有靶向性的转移载体携带HDV核酶进入肝细胞内发挥作用,使其更接近于临床治疗途径。
自从80年代中期逆转录病毒(retrovirus, RV)载体面世以来,其结构功能较为清楚、感染效率高、无须辅助病毒等优点使遗传病的基因治疗获得了一次飞跃性进展。典型的逆转录病毒基因组可分为3个独立的转录单位,gag、pol和env。gag区即所谓组特异抗原,编码病毒核心蛋白;pol区编码蛋白酶、逆转录酶和整合酶;env区编码病毒表面蛋白或称包膜蛋白。逆转录病毒的宿主范围主要由病毒包膜蛋白组成,它决定了病毒与宿主细胞结合的能力;另一个重要因素是启动子和增强子,它们影响病毒在特定细胞中的表达能力。
逆转录病毒的包膜蛋白由两部分组成:表面蛋白(surface protein, SU protein)包含受体结合区域;跨膜蛋白(transmembrane protein, TM protein)包含融合肽。SU与细胞表面受体结合后诱导TM构象发生变化,暴露出融合肽,激活了病毒包膜与宿主细胞膜的融合作用,最终使得病毒核心进入细胞。在通过修饰包膜蛋白构建靶向性逆转录病毒载体的研究中,国内外学者做了大量的工作。HBV preS2基因编码产物由55个氨基酸组成,含有聚合人血白蛋白受体(plymerized human serum albumin receptor, pHSA-R)。血液中的pHSA可与肝细胞膜上的相应受体以及HBV包膜上的特异性受体相结合,这样,HBV颗粒通过pHSA就可以与肝细胞膜结合在一起,从而介导HBV进入肝细胞中。本课题将主要利用preS2基因的编码产物进行肝细胞靶向性重组逆转录病毒载体的构建及验证。
有关HBV侵入肝细胞的机制尚没有一致的结论。除了聚合人血清白蛋白受体激活机制外,HBV也可以以PreS1肽的某一区域附着于细胞,而且这种结合属于非肝细胞特异的。然而,这两种机制的主从关系尚无探讨。本研究额外构建了两种PreS1肽相关的重组质粒并进行病毒包装,构建肝细胞靶向性病毒载体的同时间接探讨在HBV入侵肝细胞过程中PreS1肽与PreS2肽发挥的作用。
本室前期的pcDNA3.1(-)-env-preS2融合表达载体是将来源于PT67包装细胞的Env蛋白与HBV preS2基因编码产物相融合的基础上构建的。PT67包装细胞的Env蛋白是嵌合蛋白,它来源于10A1 Amphotropic MLV、4070A AmphotropicMLV和MoMLV,可与天然靶细胞表面的pit1和pit2受体结合且相互影响,因此,为了比较重组逆转录病毒载体的靶向性,必须阻断其经此途径感染肝细胞以及其他靶细胞。研究表明,MoMLV Env蛋白的第60位Tyr和第61位Val氨基酸残基对pit1和pit2受体的识别起着决定性的作用,相关基因序列突变后的逆转录病毒感染原靶细胞效率很低甚至不发生感染。
研究目的:
探讨以pHSA介导的,重组逆转录病毒导入肝源细胞的靶向性。
研究方法:
1.生产重组逆转录病毒相关表达载体的构建:利用DNA重组技术将本课题组前期构建的表达载体pcDNA3.1(-)-env-preS2进行定点突变,封闭掉MLV的天然受体结合位点,同时构建PreS1相关的新的表达载体及对照性载体。
2.重组逆转录病毒的生产:将构建好的上述四种载体及pcDNA3.1(-)-env-preS2分别与质粒pL–EGFP、pcDNA4/HisMaxA-gag-pol共转染293T包装细胞系中,收获逆转录病毒颗粒。
3.重组逆转录病毒载体感染性的测定:包装的五种重组逆转录病毒分别感染肝源细胞系HepG2215及非肝源细胞NIH 3T3、HEK,荧光定量PCR测定各逆转录病毒的感染效率。
研究结果:
1.经酶切鉴定和PCR鉴定,构建了四种新的表达载体,分别命名为pcDNA3.1(-)-envm-preS2、pcDNA3.1(-)-envm、pcDNA3.1(-)-envm-preS1、pcDNA3.1(-)-envm-preS1+S2。
2.利用磷酸钙沉淀法将env相关质粒与质粒pL–EGFP、pcDNA4/HisMaxA-gag-pol共转染到293T细胞中,收集含有短暂产生病毒颗粒的培养上清,-70℃贮存备用。
3.将获得的病毒上清分别感染肝源细胞系HepG2215及非肝源细胞NIH 3T3、HEK,利用荧光定量PCR方法证明以质粒pcDNA3.1(-)-envm-preS2为基础构建的重组逆转录病毒对肝细胞有较好的靶向性。
4.在pHSA介导下,preS1和preS2相关的重组逆转录病毒都可以感染肝细胞,但聚合人血清白蛋白受体激活机制占主导地位。
5. pHSA可增强HBV PreS1相关重组病毒感染肝细胞的作用。
6. pHSA存在或缺失的情况下,PreS2相关的重组逆转录病毒也可以感染非肝源细胞且病毒感染力相当,但是感染力比较低,推测可能是由于HBV PreS2肽与非肝源细胞表面某种或者某类受体相互作用引起的。
结论:
本实验构建了具有肝细胞靶向性的基因治疗载体系统,在细胞水平上验证具有可行性,为其进入临床治疗提供实验依据;证明了HBV主要通过聚合人血清白蛋白受体激活机制感染肝细胞,为HBV分子生物学理论研究、相关的基因治疗及研发新型治疗载体建立基础。
Targeting retroviral entry is a central theme in the development of vectors for gene therapy. The attractiveness of the approach is the selective delivery of a therapeutic gene, which would immensely reduce unfavorable side effects and ease the clinical application of gene therapy. Recently, the study on the relation between HDV ribozyme structure and activity have been done at home and abroad, and our laboratory have proved that HDV ribozyme is a new potent of the antisense regent for HBV gene therapy. But how to carry HDV ribozyme gene into defined target cells or tissues has been a tackling problem.
The ability of viruses to introduce foreign DNA into target cells is being exploited in many gene therapy strategies aimed at treating genetic diseases, including cancer. Of the various viral vectors developed for this purpose, those based on retroviruses are best understood and the most widely used. These vectors integrate their genomes stably into host cell DNA allowing long term expression of inserted therapeutic genes. The processes of viral entry and genome integration do not require viral protein synthesis. In order to produce vector particles,the viral proteins such as the core protein, the reverse transcriptase and the envelope protein are provided in trans in the packaging cell lines. These cell lines release vector genomes packaged into infectious particles that are free from contaminating helper virus and replication-competent recombinant virus.
The host range of the retroviral vector is dependent upon its envelope glycoprotein (Env), which recognizes and binds to a specific cell surface receptor protein. After binding, the envelope protein undergoes conformational changes allowing induction of membrane fusion. The murine leukemia virus (MLV) Env protein, like all retroviral Envs, consists of two subunits: SU, which contains the receptor binding domain, and TM which contains the fusion peptide. Binding of SU to the receptor is thought to induce a conformational change in TM, resulting in the exposure of the fusion peptide and activation of the process of fusion of the viral and cellular membranes, and eventual delivery of the viral core into the cell. So the host range of a virus that does not infect human cells may be extended to a predetermined human cell type. The ecotropic MLV envelope protein has most frequently been used for this approach. Since ecotropic MLV recognizes receptors only on rodent cells and not on human cells, its host range is restricted to rodent cells. This targeting approach requires the inclusion of a novel attachment site and the induction of fusion via a novel receptor interaction.
The mechanism for the entry of Hepatitis B Virus (HBV)-particles into target cells, in particular into hepatocytes is yet not understood. Several receptors on liver cells have been previously suggested which could bind either to wild-type HBV particles or to genetically engineered virus. The envelope of the HBV particle is composed of three related surface (S) proteins. The major or small S protein is 226 amino acids in length. The two other S proteins include the small S sequence at their carboxyl termini. In addition, the middle S protein contains an amino-terminal extension to the small S protein of 55 amino acids (PreS2). The large S protein contains a further amino-terminal extension to the middle S protein of 108 to 119 (depending on subtype ay or ad, respectively) amino acids (PreS1). It has been suggested that the large S protein performs the attachment function since a synthetic peptide containing a PreSl amino acid sequence and an antiserum raised against this peptide inhibited the attachment of cultured hepatoma (HepG2) cells to immobilized HBsAg particles. It also has been suggested that the middle S protein, which binds to polymerized human serum albumin (pHSA), may use pHSA to attach to hepatocytes, which express albumin receptors. In this study, we have investigated whether Moloney murine leukemin virus (MoMLV)-based retroviral vector that contains the HBV PreS2 peptide fused to aa +1 at the N-terminus of Env could be applied to target hepatocytes.
Amphotropic murine leukemin virus (A-MLV) and MoMLV use sodium-dependent phosphate symporters, Pit1 and Pit2 as receptors for infection. Studies of naturally occurring MLVs have identified the amino-terminal domain of the SU glycoprotein as responsible for receptor recognition and binding. Specially, residues Tyr-60 and Val-61 of MoMLV VRA are critical for receptor recognition. We use site-specific mutagenesis to investigate the new infectivity of recombinant retroviruses, mediated by HBV PreS1 or PreS2 peptide.
Purpose
To explore the possibility of targeting hepatocytes mediated by pHSA by recombinant retrovirus vector which carries pHSA-R.
Methods
1. The construction of recombinant retrovirus-associated expression vectors: The env gene of vector pcDNA3.1(-)-env-preS2 was mutated by PCR and additional preS1-associated expression vectors were constructed.
2. To obtain the supernatant of the packaging cell lines. The plasmids pL–EGFP and pcDNA4/HisMaxA-gag-pol were cotransfected into the packaging 293T cell lines in combined with env-associated expression vectors, respectively, by calcium phosphate precipitation method.
3. The detection of recombinant retroviral vectors infectivity was studied in HepG2215, NIH3T3 and HEK cells by real-time quantitative PCR.
Results
1. The design of expression vectors. Four eukaryote expression vectors pcDNA3.1(-)-envm-preS2, pcDNA3.1(-)-envm, pcDNA3.1(-)-envm-preS1 and pcDNA3.1(-)-envm-preS1+S2, were successfully constructed and identified by DNA sequencing and digestion with restriction enzymes.
2. Transient transfection was performed on 293T cells by calcium phosphate treatment. The high-titer retrovirus carrying eGFP gene were obtained from supernatant of the packaging cell line.
3. The packaged recombinant retrovirus based on pcDNA3.1(-)-envm-preS2 had a better hepatocellular tropism.
4. In the presence of polymerized human serum albumin, both preS1 and preS2- associated recombinant retrovirus were capable of infect hepatocytes. preS2-associated recombinant retrovirus had a higher infectivity.
5. pHSA could enhance the preS1-associated recombinant retrovirus infectivity to NIH 3T3 and HEK cells.
6. preS2-associated recombinant retrovirus could infect NIH 3T3 and HEK cells with or without pHSA with a similar titer, though the infectivity was lower. Our results indicated that the transduction of NIH 3T3 and HEK cells was caused by the interactions between HBV PreS2 peptide and some receptors rather than pHSA-R on cells surface.
Conclusions
The packaged recombinant retrovirus based on pcDNA3.1(-)-envm-preS2 had a better hepatocellular tropism mediated by pHSA. The efficiency of this system in gene transfer might provide an optimal system for hepatocyte gene therpy.
引文
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