艾滋病的靶向基因治疗
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摘要
免疫缺陷病毒HIV／SIV胞膜蛋白Env gp120／gp41的受体或抗体分子与毒素蛋白相融合而形成的靶向性毒素，能够选择性杀伤被HIV／SIV感染后于表面表达Env gp120的细胞。但是，毒素蛋白作为异源分子，具有很强的免疫原性。CD4和绿脓杆菌毒素PE40的融合蛋白在临床试验中产生了剂量限制性的肝细胞毒性，并引起抗体的产生，从而在再次使用时会被抗体中和而失效。为避免毒素的免疫原性而对毒素进行的各种修饰均未达到理想的效果。本研究采用一种新的技术路线，利用CD4分子胞外V1V2区、化学趋化因子受体CXCR4和CCR5作为结合HIV／S1Vgp120的靶向性分子，将其与人的组蛋白H1相融合，通过H1与DNA的结合作用，在CD4、CXCR4和CCR5的引导下将毒素基因表达型质粒靶向性导入被HIV／SIV感染的细胞，以期通过毒素基因在靶细胞内部的表达并合成出毒素蛋白而发挥杀伤被HIV／SIV感染的细胞的目的。该方法在理论上能够完全避免毒素的免疫原性，减小毒素对正常细胞的毒副作用。
本实验利用PCR技术以携带PE40(PEⅡ和Ⅲ结构域)基因的质粒为模板扩增出PE结构域Ⅲ(PE domainⅢ)的编码序列，同时将PEⅢC端的REDLK残基突变为KDEL，得到强效突变毒素PEⅢ_(mut)的基因片段。将该突变基因插入真核细胞表达载体pcDNA3.1(-)mychisA中，构建了毒素基因的真核表达重组质粒pA-PEⅢ_(mut)。为了更好地将毒素基因限制在HIV感染的细胞中表达，另将毒素基因置于HIV 5′LTR下游构建了真核细胞表达质粒pYL-PEⅢ_(mut)，以使毒素基因的表达受HIV TAR／Tat的调控，当细胞内有病毒Tat蛋白存在时毒素基因才能表达。该带有毒素基因的表达型重组质粒即使被误导入正常细胞，因该细胞未受HIV的感染而无Tat蛋白的存在，毒素基因不能表达，细胞将不会被杀伤。
利用RT-PCR方法，从正常中国人外周血淋巴细胞cDNA中获得CD4V1V2、CXCR4和CCR5的完整编码序列，进一步通过PCR扩增分别去掉CXCR4和CCR5羧基端胞内区的34和52个氨基酸，同时在C端引入长为9个氨基酸残基的流感病毒血凝素HA标签以便于蛋白检测。利用PCR技术从正常中国人外周血淋巴细胞基因组DNA中获得组蛋白H1C端86个氨基酸的编码序列，该序列中含有35个赖氨酸。利用P．pastoris酵母表达载体pPIC9、pHIL-S1及pPIC9K构建了表达靶向蛋白CD4V1V2、CD4V1V2-H1_(86)、CXCR4、CXCR4-H1_(86)、CCR5及CCR5-H1_(86)的一系列表达型重组质粒。各表达质粒线性化后通过电转或原生质体法分别转化入酵母株GS115、KM71和SMD1168中。经小规模诱导培养，从pPIC9重组体的KM71转化株中筛选出高表达CD4V1V2、CD4V1V2-H186的菌株。CD4V1V2和CD4V1V2-H186的表达量分别达1.4g／L和428mg／L，其中CD4V1V2在培养上清总蛋白中的含量大于90％，而CD4V1V2-H1_(86)在培养上清总蛋白中的含量为7.5％左右。由SMD1168表达的CD4V1V2-H1_(86)约为370mg／L，占培养上清的6％。从pPIC9和pHIL-S1重组质粒的KM71和GS115酵母转化子中未筛选出高表达CXCR4和CCR5各相关融合蛋白的酵母菌株。进一步利用G418的生长抑制作用，对生长于含1.25mg／ml和1.75mg／ml G418的培养基上的KM71／pPIC9K-C4H和KM71／pPIC9K-C5H转化子进行筛选，得到CXCR4-H1_(86)和CCR5-H1_(86)的高表达株。分泌至上清的CXCR4-H1_(86)-HA和CCR5-H1_(86)-HA分别占培养上清总蛋白的29％和15.5％左右，相应含量为454mg／L和167mg／L。
选择CD4V1V2-H1_(86)和CCR5-H1_(86)-HA的高表达菌株进行摇瓶大规模培养诱导，从SDS-PAGE凝胶中回收目的蛋白，所得靶向蛋白CD4V1V2-H1_(86)和CCR5-H1_(86)-HA的纯度较高。进一步通过酸性丙酮的沉淀除去蛋白质中的SDS。利用CD4V1V2-H1_(86)和CCR5-H1_(86)-HA作为靶向蛋白，pA-PEⅢ_(mut)作为毒素基因表达型重组质粒进行细胞试验。根据蛋白质与DNA的电荷摩尔数比为1：0.9制备转染用复合物，同时按1μl／1μgDNA的量加入脂质体Lipofectin。分别以1μg、2μg和3μg质粒DNA的剂量转染被SIV感染的人T细胞系Hut-78细胞，同时用未受感染的Hut-78正常细胞作为对照。结果显示，靶向蛋白及单独使用的毒素基因表达质粒本身对正常细胞及被SIV感染的细胞均无毒副作用，靶向蛋白能介导毒素基因选择性进入被SIV感染的细胞。细胞试验所得的数据表明，CD4V1V2-H1_(86)介导的毒素基因在3μg的DAN剂量条件下发挥作用，对被SIV感染的Hut-78(Hut-78／SIV)细胞的杀伤率约为37％；而CCR5-H1_(86)-HA和CD4V1V2-H1_(86)与CCR5-H1_(86)-HA共同介导的毒素基因在1μg的质粒DNA剂量条件下即发挥细胞毒作用。CCR5-H1_(86)-HA单独介导时，在1μg、2μg和3μg质粒DNA的剂量条件下，被杀伤的Hut-78／SIV细胞介于50～60％之间；CCR5-H1_(86)-HA与CD4V1V2-H_(86)共同介导时，在1μg、2μg和3μg的质粒DNA剂量条件下，被杀伤的Hut-78／SIV细胞分别约为66％、62％和94％。
细胞试验表明，本研究所设计的靶向蛋白能有效地介导毒素基因表达质粒靶向性转入被SIV感染的细胞中，所构建的毒素基因表达质粒pA-PEⅢ_(mut)能表达毒素蛋白PEⅢ_(mut)。该毒素突变型能强烈杀伤靶细胞。由此推测，利用靶向蛋白CXCR4-H1_(86)-HA和HIV LTR调控的毒素基因表达质粒pYL-PEⅢ，同样能有效地选择性杀伤被HIV感染的细胞。
本研究为毒素药物的应用提供了一个新的途径。该技术路线不仅适用于艾滋病的治疗，还可通过靶向蛋白的更换，用于癌症或其它病毒感染疾病或自身免疫疾病等的治疗，由此开辟一条对上述诸疾病可能有良好治疗效果的新途径。
Targeted toxins usually are hybrid toxins containing protein toxins linked to binding doamins such as antibodies or protein ligands and can specially kill cells expressing the corresponding surface antigens or receptors. During the past decade, several types of anti-HIV hybrid toxins are constructed by substituting the normal cell binding region of the native toxin with extracellular regions of CD4 or Fab regions of anti-Env antibodies. Most cytotoxic domains have been derived from natural protein toxins such as Pseudomonas aeruginosa exotoxin A(PE), ricin and diphtheria toxin. In vitro data suggested that Env-targeted toxins displayed selective toxicity toward HIV-infected cells. However, the high hopes for the therapeutic potential of Env-targeted toxins such as CD4-PE40 were dashed in Phase I trials with individuals infected by HIV. The toxin leaded to dose-limiting hepatoxicity and immunogenicity. Up to now, such kind of targeting fusion protein can not be used to clinical treatment for interrelated diseases. In this case, it's necessary to search a safe and effective application of cytotoxins for clinical treatment.It's known that CD4 and one of chemokine coreceptors, CCR5 or CXCR4, constitute receptor complex which is bound by HIV/SIV Env gp120. While CXCR4 is used by T cell line-tropic HIV-1 strains, CCR5 is used by macrophage-tropic isolates and SIV strains. CCR5 and CXCR4 belong to the family of seven-transmembrane GTP-binding protein-coupled receptors. Studies have demonstrated that multiple elements distributed throughout the extracellular segments of coreceptor appeared to contribute to the HIV entry. However, the extracellular domain V1V2 of CD4 can bind gp120 effectively.Based on the knowledge, a novel alternative Env-targeting therapeutic strategy was developed. CD4 extrocellular region V1V2, chemokine receptors CCR5 and CXCR4 were used here as targeting agents to direct a gene encoding cytotoxic molecule into HIV/SIV-infected cells, which display HIV Env gpl20 on their surface. It's expected that the toxin be expressed in host cells and then kill these cells.PE domain III with responsibility for ADP-ribosylation is used here as the toxin. Encoding sequence for toxin PE domainIII was generated by PCR amplification from plasmid containing gene for PE40 (PE domainll and PE domainIII). To enhance the toxicity of PEIII, 5 amino acid residues of REDLK in C terminus of PEIII was replaced with 4 residues of KDEL during PCR and thus gene called PEIII_(mut) was engineered. Two mammalian expression plasmids containing PEIII_(mut) gene(pA-PEIII_(mut) and pLY-PEIII_(mut)) were engineered, in which PEⅢ_(mut) were regulated by CMV promoter and HIV LTR respectively.
Encoding sequences for CD4V1V2, CXCR4 and CCR5 were obtained by RT-PCR from Chinese lymphocyte cDNAs and inserted into plasmid pBSKS. Introcellular parts of CCR5 and CXCR4 were further removed and hemagglutinin tag (HA tag) was added to the C terminus of truncated chemokine receptors via PCR amplificaton. Human histone H1 was adopted to conjoin DNA and targeting agents. To minimize the size of H1, only the 86 amino acid region of its C terminus containing 35 lysine residues was chosen. Corresponding encoding sequence was obtained from Chinese peripheral blood genome DNA via PCR amplification. The fusion genes for targeting protein composed of chemokine receptor and H1_(86) were then engineered.
Series of expression vectors containing genes for targeting proteins were constructed based on methylotrophic yeast vector pPIC9, pHIL-S1 and pPIC9K. These vectors were respectively linearized at SalI, SacI or BgⅢsites and then transferred into Pichia pastoris strains of GS115, KM71 and SMD1168 via spheroplast transformation or electroporation. Upon induction with 1％methanol, the genes of CD4V1V2 and CD4V1V2-H1_(86) cloned in pPIC9 vectors were overexpressed in KM71 strain. CD4V1V2 and CD4V1V2-H1_(86) were secreted into the culture medium by means of theα- mating factor signal sequence and the yields were 1.4g/L and 428mg/L respectively, in which CD4V1V2 and CD4V1V2-H1_(86) accounted for about 90％and 7.5％of total proteins in culture supernatant respectively. As for SMD1168 strain, expression level of CD4V1V2-H1_(86) is about 6％of total proteins in supernatant, which is corresponding to 370 mg/L. However, none of pPIC9 or pHIL-S1 based recombinants showed high expression of chemokine receptor-related protein. Using G418 growth inhibition screen, high expression of CXCR4-H1_(86) and CCR5-H1_(86) were obtained from KM71 strains transformed with pPIC9K-based recombinants. The yields of CXCR4-H1_(86)-HA and CCR5-H1_(86)-HA secreted in to culture media were about 457mg/L and 167mg/L respectively, in which CXCR4-H1_(86)-HA and CCR5-H1_(86)-HA reached 29.3％and 15.2％of total supernatant proteins.
To assay the selective cytotoxicity of complex composed of targeting proteins and toxin gene expression plasmid, SIV infected/uninfected human T-cell line Hut-78 cells were used. Targeting proteins of CD4V1V2- H1+(86)and CCR5-H1_(86) were purified from SDS-PAGE gel and the SDS in which was removed by acid acetone. And then, transfection complexes were made according to the charge ratios of CD4V1V2-H1_(86), CCR5-H1_(86) and recombinant pA-PEⅢ_(mut)(1: 0.9). A small quantity of Lipofectin (1μg lipofectin perμg DNA) was added in transfection mixture to enhance the efficiency of targeting protein-mediated transfection. The assay In vitro showed that CD4V1V2-H1_(86), CCR5-H1_(86), pA-PEⅢ_(mut) and pcDNA3.1(-)- MycHisA have no toxicity to SIV-infected/uninfected Hut-78 cells when they are used alone in transfection. The complex composed of the plasmid carrying toxin gene PEⅢ_(mut) and targeting proteins CD4V1V2-H186 or/and CCR-H186 can not enter into uninfected Hut-78 cells and thus have no toxicity to normal cells; while the complex can specifically kill Hut-78 cells infected with SIV. About 31.7％SIV-infected cells were killed when 3μg pAEⅢ_(mut) was delivered by corresponding quantity of CD4V1V2-H1_(86). 50％～60％cytotoxicity was observed when toxin gene was transferred with CCR5-H1_(86)-HA. Higher killing effect was achieved when CCR5-H1_(86)-HA and CD4V1V2-H1_(86) were used together to deliver toxin gene to SIV- infected cells. About 94％SIV-infected cells were killed when 1.54×10~5cells (containing 48.7％SIV-infected cells) were transfected with 3μg pAEⅢ_(mut) and corresponding quantity of protein CD4V1V2-H1_(86) and CCR5-H1_(86). It's expected that CXCR4-H1_(86) and pYL-PEⅢ_(mut) can also effectively target HIV-infected cells and have selective killing function on these cells.
In vitro results indicated that targeting protein, consisting of a HIV/SIV Env targeting ligand and a DNA-bingding moiety, can effectively mediate selective transfection of toxin gene. The approach developed here has great potential for HIV therapy due to safety and non-immunogenicity. It could be extended for the efficient therapy of cancer, other viral infections and autoimmunodiseases, as long as the targeting protein is changed by interrelated others.
本实验利用PCR技术以携带PE40(PEⅡ和Ⅲ结构域)基因的质粒为模板扩增出PE结构域Ⅲ(PE domainⅢ)的编码序列，同时将PEⅢC端的REDLK残基突变为KDEL，得到强效突变毒素PEⅢ_(mut)的基因片段。将该突变基因插入真核细胞表达载体pcDNA3.1(-)mychisA中，构建了毒素基因的真核表达重组质粒pA-PEⅢ_(mut)。为了更好地将毒素基因限制在HIV感染的细胞中表达，另将毒素基因置于HIV 5′LTR下游构建了真核细胞表达质粒pYL-PEⅢ_(mut)，以使毒素基因的表达受HIV TAR／Tat的调控，当细胞内有病毒Tat蛋白存在时毒素基因才能表达。该带有毒素基因的表达型重组质粒即使被误导入正常细胞，因该细胞未受HIV的感染而无Tat蛋白的存在，毒素基因不能表达，细胞将不会被杀伤。
利用RT-PCR方法，从正常中国人外周血淋巴细胞cDNA中获得CD4V1V2、CXCR4和CCR5的完整编码序列，进一步通过PCR扩增分别去掉CXCR4和CCR5羧基端胞内区的34和52个氨基酸，同时在C端引入长为9个氨基酸残基的流感病毒血凝素HA标签以便于蛋白检测。利用PCR技术从正常中国人外周血淋巴细胞基因组DNA中获得组蛋白H1C端86个氨基酸的编码序列，该序列中含有35个赖氨酸。利用P．pastoris酵母表达载体pPIC9、pHIL-S1及pPIC9K构建了表达靶向蛋白CD4V1V2、CD4V1V2-H1_(86)、CXCR4、CXCR4-H1_(86)、CCR5及CCR5-H1_(86)的一系列表达型重组质粒。各表达质粒线性化后通过电转或原生质体法分别转化入酵母株GS115、KM71和SMD1168中。经小规模诱导培养，从pPIC9重组体的KM71转化株中筛选出高表达CD4V1V2、CD4V1V2-H186的菌株。CD4V1V2和CD4V1V2-H186的表达量分别达1.4g／L和428mg／L，其中CD4V1V2在培养上清总蛋白中的含量大于90％，而CD4V1V2-H1_(86)在培养上清总蛋白中的含量为7.5％左右。由SMD1168表达的CD4V1V2-H1_(86)约为370mg／L，占培养上清的6％。从pPIC9和pHIL-S1重组质粒的KM71和GS115酵母转化子中未筛选出高表达CXCR4和CCR5各相关融合蛋白的酵母菌株。进一步利用G418的生长抑制作用，对生长于含1.25mg／ml和1.75mg／ml G418的培养基上的KM71／pPIC9K-C4H和KM71／pPIC9K-C5H转化子进行筛选，得到CXCR4-H1_(86)和CCR5-H1_(86)的高表达株。分泌至上清的CXCR4-H1_(86)-HA和CCR5-H1_(86)-HA分别占培养上清总蛋白的29％和15.5％左右，相应含量为454mg／L和167mg／L。
选择CD4V1V2-H1_(86)和CCR5-H1_(86)-HA的高表达菌株进行摇瓶大规模培养诱导，从SDS-PAGE凝胶中回收目的蛋白，所得靶向蛋白CD4V1V2-H1_(86)和CCR5-H1_(86)-HA的纯度较高。进一步通过酸性丙酮的沉淀除去蛋白质中的SDS。利用CD4V1V2-H1_(86)和CCR5-H1_(86)-HA作为靶向蛋白，pA-PEⅢ_(mut)作为毒素基因表达型重组质粒进行细胞试验。根据蛋白质与DNA的电荷摩尔数比为1：0.9制备转染用复合物，同时按1μl／1μgDNA的量加入脂质体Lipofectin。分别以1μg、2μg和3μg质粒DNA的剂量转染被SIV感染的人T细胞系Hut-78细胞，同时用未受感染的Hut-78正常细胞作为对照。结果显示，靶向蛋白及单独使用的毒素基因表达质粒本身对正常细胞及被SIV感染的细胞均无毒副作用，靶向蛋白能介导毒素基因选择性进入被SIV感染的细胞。细胞试验所得的数据表明，CD4V1V2-H1_(86)介导的毒素基因在3μg的DAN剂量条件下发挥作用，对被SIV感染的Hut-78(Hut-78／SIV)细胞的杀伤率约为37％；而CCR5-H1_(86)-HA和CD4V1V2-H1_(86)与CCR5-H1_(86)-HA共同介导的毒素基因在1μg的质粒DNA剂量条件下即发挥细胞毒作用。CCR5-H1_(86)-HA单独介导时，在1μg、2μg和3μg质粒DNA的剂量条件下，被杀伤的Hut-78／SIV细胞介于50～60％之间；CCR5-H1_(86)-HA与CD4V1V2-H_(86)共同介导时，在1μg、2μg和3μg的质粒DNA剂量条件下，被杀伤的Hut-78／SIV细胞分别约为66％、62％和94％。
细胞试验表明，本研究所设计的靶向蛋白能有效地介导毒素基因表达质粒靶向性转入被SIV感染的细胞中，所构建的毒素基因表达质粒pA-PEⅢ_(mut)能表达毒素蛋白PEⅢ_(mut)。该毒素突变型能强烈杀伤靶细胞。由此推测，利用靶向蛋白CXCR4-H1_(86)-HA和HIV LTR调控的毒素基因表达质粒pYL-PEⅢ，同样能有效地选择性杀伤被HIV感染的细胞。
本研究为毒素药物的应用提供了一个新的途径。该技术路线不仅适用于艾滋病的治疗，还可通过靶向蛋白的更换，用于癌症或其它病毒感染疾病或自身免疫疾病等的治疗，由此开辟一条对上述诸疾病可能有良好治疗效果的新途径。
Targeted toxins usually are hybrid toxins containing protein toxins linked to binding doamins such as antibodies or protein ligands and can specially kill cells expressing the corresponding surface antigens or receptors. During the past decade, several types of anti-HIV hybrid toxins are constructed by substituting the normal cell binding region of the native toxin with extracellular regions of CD4 or Fab regions of anti-Env antibodies. Most cytotoxic domains have been derived from natural protein toxins such as Pseudomonas aeruginosa exotoxin A(PE), ricin and diphtheria toxin. In vitro data suggested that Env-targeted toxins displayed selective toxicity toward HIV-infected cells. However, the high hopes for the therapeutic potential of Env-targeted toxins such as CD4-PE40 were dashed in Phase I trials with individuals infected by HIV. The toxin leaded to dose-limiting hepatoxicity and immunogenicity. Up to now, such kind of targeting fusion protein can not be used to clinical treatment for interrelated diseases. In this case, it's necessary to search a safe and effective application of cytotoxins for clinical treatment.It's known that CD4 and one of chemokine coreceptors, CCR5 or CXCR4, constitute receptor complex which is bound by HIV/SIV Env gp120. While CXCR4 is used by T cell line-tropic HIV-1 strains, CCR5 is used by macrophage-tropic isolates and SIV strains. CCR5 and CXCR4 belong to the family of seven-transmembrane GTP-binding protein-coupled receptors. Studies have demonstrated that multiple elements distributed throughout the extracellular segments of coreceptor appeared to contribute to the HIV entry. However, the extracellular domain V1V2 of CD4 can bind gp120 effectively.Based on the knowledge, a novel alternative Env-targeting therapeutic strategy was developed. CD4 extrocellular region V1V2, chemokine receptors CCR5 and CXCR4 were used here as targeting agents to direct a gene encoding cytotoxic molecule into HIV/SIV-infected cells, which display HIV Env gpl20 on their surface. It's expected that the toxin be expressed in host cells and then kill these cells.PE domain III with responsibility for ADP-ribosylation is used here as the toxin. Encoding sequence for toxin PE domainIII was generated by PCR amplification from plasmid containing gene for PE40 (PE domainll and PE domainIII). To enhance the toxicity of PEIII, 5 amino acid residues of REDLK in C terminus of PEIII was replaced with 4 residues of KDEL during PCR and thus gene called PEIII_(mut) was engineered. Two mammalian expression plasmids containing PEIII_(mut) gene(pA-PEIII_(mut) and pLY-PEIII_(mut)) were engineered, in which PEⅢ_(mut) were regulated by CMV promoter and HIV LTR respectively.
Encoding sequences for CD4V1V2, CXCR4 and CCR5 were obtained by RT-PCR from Chinese lymphocyte cDNAs and inserted into plasmid pBSKS. Introcellular parts of CCR5 and CXCR4 were further removed and hemagglutinin tag (HA tag) was added to the C terminus of truncated chemokine receptors via PCR amplificaton. Human histone H1 was adopted to conjoin DNA and targeting agents. To minimize the size of H1, only the 86 amino acid region of its C terminus containing 35 lysine residues was chosen. Corresponding encoding sequence was obtained from Chinese peripheral blood genome DNA via PCR amplification. The fusion genes for targeting protein composed of chemokine receptor and H1_(86) were then engineered.
Series of expression vectors containing genes for targeting proteins were constructed based on methylotrophic yeast vector pPIC9, pHIL-S1 and pPIC9K. These vectors were respectively linearized at SalI, SacI or BgⅢsites and then transferred into Pichia pastoris strains of GS115, KM71 and SMD1168 via spheroplast transformation or electroporation. Upon induction with 1％methanol, the genes of CD4V1V2 and CD4V1V2-H1_(86) cloned in pPIC9 vectors were overexpressed in KM71 strain. CD4V1V2 and CD4V1V2-H1_(86) were secreted into the culture medium by means of theα- mating factor signal sequence and the yields were 1.4g/L and 428mg/L respectively, in which CD4V1V2 and CD4V1V2-H1_(86) accounted for about 90％and 7.5％of total proteins in culture supernatant respectively. As for SMD1168 strain, expression level of CD4V1V2-H1_(86) is about 6％of total proteins in supernatant, which is corresponding to 370 mg/L. However, none of pPIC9 or pHIL-S1 based recombinants showed high expression of chemokine receptor-related protein. Using G418 growth inhibition screen, high expression of CXCR4-H1_(86) and CCR5-H1_(86) were obtained from KM71 strains transformed with pPIC9K-based recombinants. The yields of CXCR4-H1_(86)-HA and CCR5-H1_(86)-HA secreted in to culture media were about 457mg/L and 167mg/L respectively, in which CXCR4-H1_(86)-HA and CCR5-H1_(86)-HA reached 29.3％and 15.2％of total supernatant proteins.
To assay the selective cytotoxicity of complex composed of targeting proteins and toxin gene expression plasmid, SIV infected/uninfected human T-cell line Hut-78 cells were used. Targeting proteins of CD4V1V2- H1+(86)and CCR5-H1_(86) were purified from SDS-PAGE gel and the SDS in which was removed by acid acetone. And then, transfection complexes were made according to the charge ratios of CD4V1V2-H1_(86), CCR5-H1_(86) and recombinant pA-PEⅢ_(mut)(1: 0.9). A small quantity of Lipofectin (1μg lipofectin perμg DNA) was added in transfection mixture to enhance the efficiency of targeting protein-mediated transfection. The assay In vitro showed that CD4V1V2-H1_(86), CCR5-H1_(86), pA-PEⅢ_(mut) and pcDNA3.1(-)- MycHisA have no toxicity to SIV-infected/uninfected Hut-78 cells when they are used alone in transfection. The complex composed of the plasmid carrying toxin gene PEⅢ_(mut) and targeting proteins CD4V1V2-H186 or/and CCR-H186 can not enter into uninfected Hut-78 cells and thus have no toxicity to normal cells; while the complex can specifically kill Hut-78 cells infected with SIV. About 31.7％SIV-infected cells were killed when 3μg pAEⅢ_(mut) was delivered by corresponding quantity of CD4V1V2-H1_(86). 50％～60％cytotoxicity was observed when toxin gene was transferred with CCR5-H1_(86)-HA. Higher killing effect was achieved when CCR5-H1_(86)-HA and CD4V1V2-H1_(86) were used together to deliver toxin gene to SIV- infected cells. About 94％SIV-infected cells were killed when 1.54×10~5cells (containing 48.7％SIV-infected cells) were transfected with 3μg pAEⅢ_(mut) and corresponding quantity of protein CD4V1V2-H1_(86) and CCR5-H1_(86). It's expected that CXCR4-H1_(86) and pYL-PEⅢ_(mut) can also effectively target HIV-infected cells and have selective killing function on these cells.
In vitro results indicated that targeting protein, consisting of a HIV/SIV Env targeting ligand and a DNA-bingding moiety, can effectively mediate selective transfection of toxin gene. The approach developed here has great potential for HIV therapy due to safety and non-immunogenicity. It could be extended for the efficient therapy of cancer, other viral infections and autoimmunodiseases, as long as the targeting protein is changed by interrelated others.
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