An enhancer/promoter combination strengthens the expression of blood-coagulation factor VIII in non-viral expression vectors

We explored the potential of fusion of hepatic locus control region 1 (HCR-1) with HCR-2 to express B-domain-deleted human factor VIII (FVIII) in four cell lines. B-domain-deleted human FVIII expression was controlled by HCR-1/HCR-2, followed by liver specific and ubiquitous promoters. Chimera enhancer HCR-1/HCR-2, followed by cytomegalovirus (CMV) promoter, gave 2-fold more FVIII expression in all cell lines (105.6 +/- 2.8 for Hek-293, 68.8 +/- 3.8 for HepG2, 34.8 +/- 1.3 for CHO, and 27.2 +/- 1.6 ng x mL(-1) x 10(6) cells(-1) for L.N.) when compared to the vector with CMV alone (54.8 +/- 3.3 for Hek-293, 32.4 +/- 1.2 for HepG2, 18.6 +/- 1.1 for CHO, and 10.1 +/- 1.7 ng x mL(-1) x 10(6) cells(-1) for L.N.). Elongation factor 1-alpha gene and human CMV promoters were more efficient than the promoters from the human alpha-1-antitrypsin gene, and fviii was less efficient in hepatic cell lines. HCR-1/HCR-2, followed by strong promoters, increases FVIII expression in vitro. Our results underscore the importance of cis sequences for enhancing in vitro FVIII expression; this may be helpful for designing new strategies to improve heterologous expression systems.     

Optimisation of the Factor VIII yield in mammalian cell cultures by reducing the membrane bound fraction

In vivo, clotting Factor VIII (FVIII) circulates in plasma bound to von Willebrand factor (vWF), and the vWF:FVIII complex prevents binding of FVIII to phosphatidylserine (PS). Activation of FVIII by thrombin releases FVIII from vWF, and subsequently FVIII binds to PS exposed on activated platelets and forms the tenase complex together with clotting Factor IX. In vitro, during serum free production of recombinant FVIII (rFVIII), production cells also expose PS, and since vWF is not present to hinder interaction of secreted rFVIII with PS, rFVIII is partly associated with the cell membrane of the production cells. Recently, we showed that as much as 90% of secreted rFVIII is bound to transiently transfected production cells during serum free conditions. In this study, we investigated the effect of including vWF in the serum free medium, and demonstrate that addition of vWF results in release of active membrane bound rFVIII to the culture medium. Moreover, the attachment of rFVIII to cell membranes of un-transfected HEK293 cells was studied in the presence of compounds that competes for interactions between rFVIII and PS. Competitive assays between iodinated rFVIII (¹²⁵I-rFVIII) and annexin V or ortho-phospho-l-serine (OPLS) demonstrated that annexin V and OPLS were able to reduce the membrane bound fraction of rFVIII by 70% and 30%, respectively. Finally, adding OPLS to CHO cells stably expressing FVIII increased the yield by 50%. Using this new knowledge, the recovery of rFVIII could be increased considerably during serum free production of this therapeutic protein.     

Two independent domains of factor VIII co-expressed using recombinant vaccinia viruses have procoagulant activity

Using recombinant DNA technology, the NH2 and COOH terminal domains of the human Factor VIII molecule were co-expressed in baby hamster kidney 21 (BHK21) cells using the vaccinia virus system. Procoagulant activity was detectable in cell supernatants, thus suggesting that the central portion present in the FVIII protein (domain B) is not required for FVIII function.     

小鼠白蛋白启动子的组织特异性功能研究

以绿色荧光蛋白(GFP)基因作为报告基因,通过对比小鼠白蛋白启动子在不同来源细胞系中启动HGFP基因的转录活性,对小鼠白蛋白启动子的组织特异性进行了研究。结果发现,小鼠白蛋白启动子在小鼠肝癌细胞系Hepa 1—6和人肝癌细胞系：HepG2均有很强的转录起始功能,荧光显微镜下可以观察到IGFP表达。Hepa 1—6细胞在转染早期的48h内,CMV的启动子和增强子序列是小鼠白蛋白启动子转录活性的4倍。G418加压筛选2周后,CMV的启动子的转录活性下降到只有小鼠白蛋白启动子活性的1／2。转染人肝癌细胞系HepG2 2周后,荧光显微镜下可以观察到GFP表达。其他的细胞如中华仓鼠卵巢细胞系CHO和人肺癌细胞系PLA 801中转染的小鼠白蛋白启动子不能启动GFP的表达,而对照CMV启动子控制下的GFP基因可在CHO和PLA 801中表达。以上结果说明,小鼠白蛋白启动子仅在肝脏来源的细胞中可以起始下游基因的转录,在其他组织来源的细胞中不能起始转录,这表明小鼠白蛋白启动子具有肝脏组织特异的转录活性,但没有种属特异性。     

CRISPR/Cas9‐mediated gene knockout for DNA methyltransferase Dnmt3a in CHO cells displays enhanced transgenic expression and long‐term stability

CHO cells are the preferred host for the production of complex pharmaceutical proteins in the biopharmaceutical industry, and genome engineering of CHO cells would benefit product yield and stability. Here, we demonstrated the efficacy of a Dnmt3a‐deficient CHO cell line created by CRISPR/Cas9 genome editing technology through gene disruptions in Dnmt3a, which encode the proteins involved in DNA methyltransferases. The transgenes, which were driven by the 2 commonly used CMV and EF1α promoters, were evaluated for their expression level and stability. The methylation levels of CpG sites in the promoter regions and the global DNA were compared in the transfected cells. The Dnmt3a‐deficent CHO cell line based on Dnmt3a KO displayed an enhanced long‐term stability of transgene expression under the control of the CMV promoter in transfected cells in over 60 passages. Under the CMV promoter, the Dnmt3a‐deficent cell line with a high transgene expression displayed a low methylation rate in the promoter region and global DNA. Under the EF1α promoter, the Dnmt3a‐deficient and normal cell lines with low transgene expression exhibited high DNA methylation rates. These findings provide insight into cell line modification and design for improved recombinant protein production in CHO and other mammalian cells.     

Improved Expression of Recombinant Human Factor IX by Co-expression of GGCX,VKOR and Furin

Recombinant human FIX concentrates (rhFIX) are essential in the treatment and prevention of bleeding in the bleeding disorder haemophilia B. However, due to the complex nature of FIX production yields are low which leads to high treatment costs. Here we report the production of rhFIX with substantially higher yield by co-expressing human FIX with GGCX (γ-glutamyl carboxylase), VKOR (vitamin K epoxide reductase) and furin (paired basic amino acid cleaving enzyme) in Chinese hamster ovary (CHO) cells. Our results show that controlled co-expression of GGCX with FIX is critical to obtain high rhFIX titre, and, that co-expression of VKOR further increased the yield of active rhFIX. Furin co-expression improved processing of the leader peptide of rhFIX but had a minor effect on yield of active rhFIX. The optimal expression level of GGCX was surprisingly low and required unusual engineering of expression vector elements. For VKOR and furin the control of expression was less critical and could be achieved by standard vector element. Using our expression vectors an rhFIX-producing clone with an expression level of up to 30 mg/L of active rhFIX was obtained. In addition an efficient single step purification method was developed to obtain pure and active rhFIX with up to 94 % yield.     

SK-HEP cells and lentiviral vector for production of human recombinant factor VIII

Hemophilia A is caused by a deficiency in coagulation factor VIII. Recombinant factor VIII can be used as an alternative although it is unavailable for most patients. Here, we describe the production of a human recombinant B-domain-deleted FVIII (rBDDFVIII) by the human cell line SK-HEP-1, modified by a lentiviral vector rBDDFVIII was produced by recombinant SK-HEP cells (rSK-HEP) at 1.5-2.1 IU/10(6) in 24 h. The recombinant factor had increased in vitro stability when compared to commercial pdFVIII. The functionality of rBDDFVIII was shown by its biological activity and by tail-clip challenge in hemophilia A mice. The rSK-HEP cells grew in a scalable system and produced active rBDDFVIII, indicating that this platform production can be optimized to meet the commercial production scale needs.     

The treatment of hemophilia A: from protein replacement to AAV-mediated gene therapy

Factor VIII (FVIII) is an essential component in blood coagulation, a deficiency of which causes the serious bleeding disorder hemophilia A. Recently, with the development of purification level and recombinant techniques, protein replacement treatment to hemophiliacs is relatively safe and can prolong their life expectancy. However, because of the possibility of unknown contaminants in plasma-derived FVIII and recombinant FVIII, and high cost for hemophiliacs to use these products, gene therapy for hemophilia A is an attractive alternative to protein replacement therapy. Thus far, the adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. It is likely that hemophilia will be the first genetic disease to be cured by somatic cell gene therapy.     

人类不同类型细胞中X-盒结合蛋白1转录活性研究

人类X-盒结合蛋白1(X-box binding protein1,XBP1)作为一种重要的转录因子,在细胞中涉及了广泛的信号调控过程。为进一步研究XBP1的生物学功能,首先利用生物信息学技术确定XBP1基因的启动子区域和2个缺失突变体的基因序列,聚合酶链反应扩增XBP1启动子和2个缺失突变体的基因序列,分别克隆至真核报告载体pCAT3-Basic中,构建3个报告载体p1-XBP1p、p2-XBP1p和p3-XBP1p,确定启动子活性最强的序列,并以该序列分别转染正常人肝细胞L02、人肝母细胞瘤细胞HepG2、人肝癌细胞SMMC-7721、人类红白血病细胞K562、人皮肤成纤维细胞HSF和人贮脂细胞Lipocyte Ito Cell 6种不同类型的细胞后(FuGENE 6 transfection reagents),CAT-ELISA方法检测氯霉素乙酰转移酶(CAT)在不同细胞系中的表达活性。每一组CAT结果反应了XBP1启动子转录活性的大小,其中p3-XBP1p在HepG2细胞中活性最强,是pCAT3-Basic的12.4倍,其次是K562和SMMC-7721,分别是10.9倍和10.0倍;在L02细胞中CAT酶活性低于上述3种异常细胞,在HSF和Ito细胞中CAT酶活性低或没有活性。运用适时荧光PCR方法和免疫印迹分别从mRNA水平和蛋白水平检测了XBP1在不同细胞中的表达情况,结果均显示XBP1在HepG2、K562和SMMC-7721细胞中的转录和表达强于L02、HSF和Ito细胞,在HSF细胞和L02细胞中转录和表达较低,在Ito细胞中几乎检测不到XBP1的表达,与CAT-ELISA检测结果一致。因此,XBP1启动子的转录活性在不同细胞中是具有差异的,而XBP1启动子转录活性的大小直接调控下游基因XBP1的表达,导致不同细胞中XBP1的表达丰度也不相同,XBP1启动子的转录活性和表达与细胞类型、细胞周期和组织特异性密切相关。本研究发现XBP1启动子的ATG上游-227bp～66bp区域与XBP1的转录活性密切相关,属于XBP1启动子的核心区域;进一步比较XBP1基因核心启动子区在不同细胞中转录活性的差别和XBP1基因表达丰度的差异,为揭示真核细胞中XBP1的转录调控机制奠定基础。     

靶向XBP1S的RNA干扰质粒对细胞增殖的影响

目的 构建靶向人XBP1S的siRNA真核表达载体（pSUPER-XBP1S）并观察其对人HeLa细胞和HepG2细胞增殖能力的影响.方法 设计并合成针对XBP1S基因的siRNA,退火成互补双链后克隆至真核表达载体pSUPER构建重组质粒,并将其转染入HeLa细胞和HepG2细胞中.采用RT-PCR检测转染前后XBP1S在HeLa细胞和HepG2细胞中的转录,Western印迹检测转染前后XBP1S蛋白的表达;MTT法、细胞计数检测重组质粒对HeLa细胞和HepG2细胞增殖能力的影响.结果 重组质粒能有效地抑制HeLa细胞和HepG2细胞中XBP1S基因的转录和表达;转染HeLa细胞和HepG2细胞后,细胞增殖抑制率及细胞增殖数与对照组比较,差异有统计学意义（P〈0.05）.结论 成功构建了靶向人XBP1S的siRNA表达载体pSUPER-XBP1S,并且有效的抑制了HeLa细胞和HepG2细胞中XBP1S的转录和表达,有效抑制了细胞的增殖能力.     

The relationship of N-linked glycosylation and heavy chain-binding protein association with the secretion of glycoproteins

The relationship of N-linked glycosylation and association with heavy chain binding protein (BiP) to the secretion of Factor VIII (FVIII), von Willebrand Factor (vWF), and tissue plasminogen activator (tPA) was studied in Chinese hamster ovary (CHO) cells. FVIII has a heavily glycosylated region containing 20 clustered potential N-linked glycosylation sites. A significant proportion of FVIII was detected in a stable complex with BiP and not secreted. Deletion of the heavily glycosylated region resulted in reduced association with BiP and more efficient secretion. Tunicamycin treatment of cells producing this deleted form of FVIII resulted in stable association of unglycosylated FVIII with BiP and inhibition of efficient secretion. vWF contains 17 potential N-linked glycosylation sites scattered throughout the molecule. vWF was transiently associated with BiP and efficiently secreted demonstrating that CHO cells are competent to secrete a highly glycosylated protein. tPA, which has three utilized N-linked glycosylation sites, exhibited low level association with BiP and was efficiently secreted. Disruption of N-linked glycosylation of tPA by either site-directed mutagenesis or tunicamycin treatment resulted in reduced levels of secretion and increased association with BiP. This effect was enhanced by high levels of tPA expression. The glycosylation state and extent of association with BiP could be correlated with secretion efficiency.     

Stable and high-level production of recombinant Factor IX in human hepatic cell line

Hemophilia B is a genetic disease of the coagulation system that affects one in 30,000 males worldwide. Recombinant human Factor IX (rhFIX) has been used for hemophilia B treatment, but the amount of active protein generated by these systems is inefficient, resulting in a high-cost production of rhFIX. In this study, we developed an alternative for rhFIX production. We used a retrovirus system to obtain two recombinant cell lines. We first tested rhFIX production in the human embryonic kidney 293 cells (293). Next, we tested a hepatic cell line (HepG2) because FIX is primarily expressed in the liver. Our results reveal that intracellular rhFIX expression was more efficient in HepG2/rhFIX (46%) than in 293/rhFIX (21%). The activated partial thromboplastin time test showed that HepG2/rhFIX expressed biologically active rhFIX 1.5 times higher than 293/rhFIX (P = 0.016). Recovery of rhFIX from the HepG2 by reversed-phase chromatography was straightforward. We found that rhFIX has a pharmacokinetic profile similar to that of FIX purified from human plasma when tested in hemophilic B model. HepG2/rhFIX cell line produced the highest levels of rhFIX, representing an efficient in vitro expression system. This work opens up the possibility of significantly reducing the costs of rhFIX production, with implications for expanding hemophilia B treatment in developing countries.     

Mapping and partial characterization of proteases expressed by a CHO production cell line

The proteolytic activities expressed by a Chinese hamster ovary (CHO) cell line in the cultivation supernatant during the production of recombinant factor VIII were mapped with a broad spectrum protease assay and a series of different types of protease inhibitors. The destabilizing effect on the product emanated from a metalloproteinase, which could be effectively blocked by chelating agents to lead to product stabilization. Amino acid sequences of the isolated metalloproteinase were found to have sequence homology with matrix metalloproteinases (MMPs) MMP3, MMP10, and MMP12. Several species with metalloproteinase activity were characterized and found to be related to each other. The results indicate that an MMP pro-enzyme of >/=200 kDa was released from the CHO cells during the production phase. The enzyme expressed collagenase/gelatinase activity when activated. Due to autoproteolysis, a number of smaller, less specific MMPs were formed with the smallest form, a 19.4 kDa protein, being the most active. These results may be of particular relevance for other production processes using CHO cells for the expression of recombinant proteins.