Selection of genetically modified cell population using hapten-specific antibody/receptor chimera

Efficient selection of the genetically modified cell population is a critical step to obtain the cells with desired properties. In this study, we propose an antigen-mediated genetically modified cell amplification (AMEGA) system employing an antibody/receptor chimera that triggers a growth signal in response to a non-toxic hapten dimer. An anti-fluorescein single-chain Fv fused to the extracellular D2 domain of erythropoietin receptor and transmembrane/intracellular domains of gp130 was expressed together with a model transgene, enhanced green fluorescent protein (EGFP) downstream of IRES sequence, by retroviral infection to IL-3-dependent Ba/F3 cells. Addition of fluorescein dimers connected by various oligo-DNA linkers induced selective growth of transfectants, thus leading to efficient expansion of EGFP-positive cell population. Also, digestion of the oligonucleotides by specific restriction endonuclease completely suppressed cell growth. Because these hapten dimers are not harmful for normal cells, the approach will be especially useful for reversible in vitro or in vivo expansion of genetically modified cell population employed for cell therapy and tissue engineering.     

Construction of a novel expression system on a human artificial chromosome

Efficient regulation of transgene would greatly facilitate the analysis of gene function in biological systems for basic research and clinical applications. The tetracycline-regulatable system (TRS) has proven to be a promising tool for such purposes. Despite their widespread application, a number of challenges are still associated with the use of TRS, including clonal variability in the regulation and copy number. We have recently constructed a novel human artificial chromosome (HAC) called 21DeltaqHAC. By housing a TRS-based DNA-PKcs expression cassette in this HAC, we were able to circumvent the problems associated with conventional TRS-based vectors. We achieved tight control of DNA-PKcs expression and rescued the radiosensitive phenotype of DNA-PKcs-deficient CHO cells. The combined use of HAC and the TRS serves as a model for controllable and fixed copy number expression vectors. Our study also demonstrates the suitability of the HAC to accommodate multi-subunit constructs such as that of the TRS.     

Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression

Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid^tet-O (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy.     

Bypassing antibiotic selection: positive screening of genetically modified cells with an antigen-dependent proliferation switch

While antibiotic selection has been routinely used for the selection of genetically modified cells, administration of cytotoxic drugs often leads to deleterious effects not only to inert cells but also to transfected or transduced ones. In this study, we propose an Antigen-MEdiated Genetically modified cell Amplification (AMEGA) system employing antibody/receptor chimeras without antibiotic selection. Based on a rational design where the extracellular domains of dimeric erythropoietin receptor (EpoR) or gp130 were substituted with heterodimeric V_H/V_L regions of anti-hen egg lysozyme (HEL) antibody and EpoR D2 domains, the genes encoding the chimeras as well as a model transgene, enhanced green fluorescent protein (EGFP), were retrovirally infected into IL-3-dependent Ba/F3 cells followed by direct HEL selection in the absence of IL-3. After a single round of selection, EGFP-positive cells were selectively amplified, resulting in a population of almost 100% positive cells. The AMEGA without antibiotic selection will not harm normal cells, which will be especially useful for increasing the efficacy for stem cell-based gene therapy.     

Construction of a novel human artificial chromosome vector for gene delivery

Potential problems of conventional transgenes include insertional disruption of the host genome and unpredictable, irreproducible expression of the transgene by random integration. Alternatively, human artificial chromosomes (HACs) can circumvent some of the problems. Although several HACs were generated and their mitotic stability was assessed, a practical way for introducing exogenous genes by the HACs has yet to be explored. In this study, we developed a novel HAC from sequence-ready human chromosome 21 by telomere-directed chromosome truncation and added a loxP sequence for site-specific insertion of circular DNA by the Cre/loxP system. This 21HAC vector, delivered to a human cell line HT1080 by microcell fusion, bound centromere proteins A, B, and C and was mitotically stable during long-term culture without selection. The EGFP gene inserted in the HAC vector expressed persistently. These results suggest that the HAC vector provides useful system for functional studies of genes in isogenic cell lines.     

Heat-regulated production and secretion of insulin from a human artificial chromosome vector

Human artificial chromosomes (HACs) behave as independent minichromosomes and are potentially useful as a way to achieve safe, long-term expression of a transgene. In this study, we sought to elucidate the potential of HAC vectors carrying the human proinsulin transgene for gene therapy of insulin-dependent diabetes mellitus (IDDM) using non-beta-cells as a host for the vector. To facilitate the production of mature insulin in non-beta-cells and to safely regulate the level of transgene expression, we introduced furin-cleavable sites into the proinsulin coding region and utilized the heat shock protein 70 (Hsp70) promoter. We used Cre-loxP-mediated recombination to introduce the gene cassettes onto 21DeltapqHAC, a HAC vector whose structure is completely defined, present in human fibrosarcoma HT1080 cells. We observed long-term expression and stable retention of the transgene without aberrant translocation of the HAC constructs. As expected, the Hsp70 promoter allowed us to regulate gene expression with temperature, and the production and secretion of intermediates of mature insulin were made possible by the furin-cleavable sites we had introduced into proinsulin. This study can be an initial step on the application of HAC vectors on the gene delivery to non-beta-cells, which might provide a direction for future treatment for diabetes.     

Transgene expression and differentiation of baculovirus-transduced human mesenchymal stem cells

BACKGROUND: Mesenchymal stem cells (MSCs) have drawn considerable attention as vehicles for cell- or gene-based therapies, yet various problems still exist for current gene delivery vectors. On the other hand, baculovirus has emerged as a novel gene therapy vector, but its transduction of stem cells has not been reported. METHODS: A recombinant baculovirus expressing the enhanced green fluorescent protein (EGFP) was constructed to transduce human MSCs derived from umbilical cord blood (uMSCs) or bone marrow (bMSCs). RESULTS: In this study, we demonstrated for the first time that human uMSCs or bMSCs could be transduced by baculovirus with high efficiencies (up to approximately 72.8% and 41.1%, respectively) and significantly elevated transgene (enhanced green fluorescent protein, EGFP) expression upon incubation with unconcentrated virus and phosphate-buffered saline for 4 h at 25 degrees C. The transduction efficiency into bMSCs could be further increased to approximately 72.2% by lowering the cell density. The improved transgene expression was partly attributed to the enhanced virus uptake upon transduction, as determined by quantitative real-time polymerase chain reaction (Q-PCR). MSC growth was not obstructed by baculovirus transduction itself, but was somewhat hampered by EGFP expression. Nonetheless, the baculovirus-transduced cells remained capable of differentiating into adipogenic lineage. The adipogenic progenitors appeared more permissive to baculovirus transduction than the undifferentiated bMSCs, thus allowing for the maintenance and enhancement of transgene expression by repeated transduction after subculture. CONCLUSIONS: These findings implicate the potential applications of baculovirus as an alternative vector to genetically modify MSCs for ex vivo gene therapy.     

Efficiency of de novo centromere formation in human artificial chromosomes

In a comparative study, we show that human artificial chromosome (HAC) vectors based on alpha-satellite (alphoid) DNA from chromosome 17 but not the Y chromosome regularly form HACs in HT1080 human cells. We constructed four structurally similar HAC vectors, two with chromosome 17 or Y alphoid DNA (17alpha, Yalpha) and two with 17alpha or Yalpha and the hypoxanthine guanine phosphoribosyltransferase locus (HPRT1). The 17alpha HAC vectors generated artificial minichromosomes in 32-79% of the HT1080 clones screened, compared with only approximately 4% for the Yalpha HAC vectors, indicating that Yalpha is inefficient at forming a de novo centromere. The 17alpha HAC vectors produced megabase-sized, circular HACs containing multiple copies of alphoid fragments (60-250 kb) interspersed with either vector or HPRT1 DNA.The 17alpha-HPRT1 HACs were less stable than those with 17alpha only, and these results may influence the design of new HAC gene transfer vectors.     

Generation of a conditionally self-eliminating HAC gene delivery vector through incorporation of a tTAVP64 expression cassette

Human artificial chromosome (HAC)-based vectors represent an alternative technology for gene delivery and expression with a potential to overcome the problems caused by virus-based vectors. The recently developed alphoid^tetO-HAC has an advantage over other HAC vectors because it can be easily eliminated from cells by inactivation of the HAC kinetochore via binding of chromatin modifiers, tTA or tTS, to its centromeric tetO sequences. This provides a unique control for phenotypes induced by genes loaded into the HAC. The alphoid^tetO-HAC elimination is highly efficient when a high level of chromatin modifiers as tetR fusion proteins is achieved following transfection of cells by a retrovirus vector. However, such vectors are potentially mutagenic and might want to be avoided under some circumstances. Here, we describe a novel system that allows verification of phenotypic changes attributed to expression of genes from the HAC without a transfection step. We demonstrated that a single copy of tTA^VP64 carrying four tandem repeats of the VP16 domain constitutively expressed from the HAC is capable to generate chromatin changes in the HAC kinetochore that are not compatible with its function. To adopt the alphoid^tetO-HAC for routine gene function studies, we constructed a new TAR-BRV- tTA^VP64 cloning vector that allows a selective isolation of a gene of interest from genomic DNA in yeast followed by its direct transfer to bacterial cells and subsequent loading into the loxP site of the alphoid^tetO-HAC in hamster CHO cells from where the HAC may be MMCT-transferred to the recipient human cells.     

人类人工染色体构建及其作为基因治疗载体的价值

人类人工染色体(HAC)作为基因治疗载体将解决基因治疗存在的一些关键问题。本文探讨了在不完全了解着丝粒、复制起始点、端粒等人类染色体基本功能单位的情况下构建HAC的三种策略。利用染色体基本功能单位在细胞内构建成功的第一代HAC,解决了HAC构建的一些难题,同时也带来了某些新的问题。HAC作为基因治疗载体具有很多优势,但第一代HAC离它作为基因治疗载体还相距很远。为此,作者正在进行解决这些问题的尝试。     

Growth promotion of genetically modified hematopoietic progenitors using an antibody/c-Mpl chimera

Thrombopoietin is a potent cytokine that exerts proliferation of hematopoietic stem cells (HSCs) through its cognate receptor, c-Mpl. Therefore, mimicry of c-Mpl signaling by a receptor recognizing an artificial ligand would be attractive to attain specific expansion of genetically modified HSCs. Here we propose a system enabling selective expansion of genetically modified cells using an antibody/receptor chimera that can be activated by a specific antigen. We constructed an antibody/c-Mpl chimera, in which single-chain Fv (ScFv) of an anti-fluorescein antibody was tethered to the extracellular D2 domain of the erythropoietin receptor and transmembrane/cytoplasmic domains of c-Mpl. When the chimera was expressed in interleukin (IL)-3-dependent pro-B cell line Ba/F3, genetically modified cells were selectively expanded in the presence of fluorescein-conjugated BSA (BSA-FL) as a specific antigen. Furthermore, highly purified mouse HSCs transduced with the retrovirus carrying antibody/c-Mpl chimera gene proliferated in vitro in response to BSA-FL, and the cells retained in vivo long-term repopulating abilities. These results demonstrate that the antibody/c-Mpl chimera is capable of signal transduction that mimics wild-type c-Mpl signaling.     

Lentiviral vectors encoding human MUC1-specific,MHC-unrestricted single-chain TCR and a fusion suicide gene: potential for universal and safe cancer immunotherapy

MUC1 tumor antigen is a target for immunotherapy of most human adenocarcinomas and some hematological malignancies. Expression of a MUC1-specific, MHC-unrestricted single-chain T cell receptor (scTCR) on cells of both innate and adaptive immune system through reconstitution of lethally irradiated mice by retroviral vector-transduced bone marrow cells, had been shown to effectively control the growth of MUC1⁺ tumors independent of their MHC type, suggesting that this receptor is a good candidate for broadly applicable gene therapy/immunotherapy. However, the translational application of this immuno-gene therapy modality was discouraged by the progressive transgene silencing in reconstituted T and B cells, as well as the potential of tumorogenesis intrinsic to oncoretroviral vectors. To overcome these problems and facilitate the future clinical use of this receptor, we have constructed a panel of novel self-inactivating lentiviral vectors (LVs) which harbor two independent internal promoters, one driving expression of the scTCR gene and the other of a fusion suicide gene, the HSV-TK–EGFP fusion gene, allowing the transduced cells to be destroyable by the pro-drug ganciclovir. Despite the large size of insert, these vectors were efficiently packaged into high titer virus that transferred the expression of transgene in both T cell lines and primary T cells. Sustained expression was maintained in a T cell line for over 4 months in vitro, suggesting its efficient resistance to transgene silencing. Both scTCR and HSV-TK–EGFP genes were functional in the transduced cells, as evidenced by their specific recognition of MUC1⁺ tumors and efficient eradication by ganciclovir.     

Non-integrating lentiviral vectors based on the minimal S/MAR sequence retain transgene expression in dividing cells

Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.     

APRT缺陷型CHO细胞系的建立及其重组蛋白表达能力评价北大核心CSCD

中国仓鼠卵巢(Chinese hamster ovary,CHO)细胞是生产复杂重组药物蛋白的首选宿主细胞,腺嘌呤磷酸核糖转移酶(adenine phosphoribosyltransferase,APRT)催化腺嘌呤与磷酸核糖缩合形成腺苷一磷酸,是嘌呤生物合成步骤中的关键酶。采用基因编辑技术敲除CHO细胞中aprt基因,验证获得的APRT缺陷型CHO细胞系的生物学特性;构建两种真核表达载体:对照载体(含有目的基因增强型绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和弱化载体(含有启动子和起始密码子突变的aprt弱化表达盒及EGFP),分别转染APRT缺陷型和野生型CHO细胞并筛选获得稳定转染的细胞池;重组CHO细胞传代培养60代并用流式细胞术检测EGFP表达的平均荧光强度,并比较不同实验组重组蛋白EGFP的表达稳定性。PCR扩增和测序结果表明,CHO细胞aprt基因成功敲除;获得的APRT缺陷型CHO细胞系在细胞形态、生长增殖、倍增时间等生物学特性方面与野生CHO细胞无显著差异。目的蛋白瞬时表达结果表明,与野生型CHO细胞相比,转染对照载体和弱化载体的APRT缺陷型CHO细胞系中EGFP的表达分别提高了42%±6%和56%±9%;特别是长期传代培养时,转染弱化载体的APRT缺陷型细胞中EGFP表达量显著高于野生型CHO细胞(P<0.05);构建的基于APRT缺陷型CHO细胞系能够明显提高重组蛋白的长期表达稳定性。研究结果为建立高效稳定的CHO细胞表达系统提供了一种有效的细胞工程策略。     

带Tat标记质粒载体的构建及其转导融合蛋白进入细胞的研究

采用点突变技术构建了带 6×His、Tat和Flag多个标记的pET HTF的质粒载体 ,利用基因重组技术构建pET HTF EGFP融合蛋白载体 .酶切和DNA测序证明 ,所构建的pET HTF和pET HTF EGFP载体正确 .BL2 1(DE3)表达融合蛋白 ,用Ni2 + 分离柱纯化His Tat Flag EGFP蛋白 ,并加入培养的NIH3T3细胞 .荧光显微镜观察显示 ,His Tat Flag EGFP融合蛋白进入细胞 .带His、Tat和Flag标记的质粒载体pET 14b HTF表达的融合蛋白能够进入细胞 ,该载体为进行蛋白质功能研究和基因治疗研究提供了一个重要工具     

Production of an infectious Herpesvirus saimiri-based episomally maintained amplicon system

Viral vectors have a number of obstacles to overcome for effective gene therapy, including immune stimulation, packaging potential and cell tropism. Herpesvirus saimiri (HVS) has many favourable traits including, a large packaging capability, wide cell tropism, and the ability to episomally persist as an artificial chromosome. To further develop HVS as a gene therapy vector we aim to produce a safe disabled HVS-based recombinant viral system for gene therapy applications. An HVS recombinant viral amplicon was constructed with a transgene packaging potential of 50 kb. The recombinant HVS genome was shown to be replication disabled and used to generate a stable cell line, OMKHVS Delta Bam, in which the modified genome persists as a non-integrated episome. To assess whether the modified genome could be packaged into a virus-like particle (HVSampVLP), OMKHVS Delta Bam was infected with replication competent virus or transfected with a defective helper virus. The resultant HVSampVLPs were able to infect SW480 tumour cells, delivering the recombinant disabled genome, which persisted as a non-integrated episome in the dividing cell population. This study forms the basis of a replication disabled HVS amplicon system for use in gene therapy applications.     

An efficient gene transfer system for hematopoietic cell line using transient and stable vectors

The hematopoietic system represents an interesting model for gene transfer protocols. Here, we have evaluated the efficiency of a gene transfer system using the polycationic compound SuperFect (Qiagen) and the K562 hematopoietic cell line. Transient and stable vectors carrying the enhanced green fluorescent protein (EGFP) reporter gene were employed. The stable vector was constructed based on Epstein-Barr virus sequences such as EBV oriP (origin of replication) and EBNA (EBV nuclear antigen)-1, both for DNA replication. The transfection efficiency of the viable cells was estimated by flow cytometry at approximately 98% for transient and stable vectors. Transiently transfected cells presented optimal EGFP expression until day 2 when fluorescence started to decrease. In contrast, stable transfectants continuously expressed the marker gene product for 10 weeks in the presence of G418. Our results represent an efficient gene transfer method for K562 hematopoietic cells and may be used as an alternative approach for further gene transfer studies involving hematopoietic cells.     

Genetically targeted adenovirus vector directed to CD40-expressing cells

The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L). The tumor necrosis factor-like domain of CD40L retains its functional tertiary structure upon incorporation into this chimera and allows the virus to use CD40 as a surrogate receptor for cell entry. The ability of the modified Ad vector to infect CD40-positive dendritic cells and tumor cells with a high efficiency makes this virus a prototype of choice for the derivation of therapeutic vectors for the genetic immunization and targeted destruction of tumors.     

Reporter-linked monitoring of transgene expression in living cells using the ecdysone-inducible promoter system

Inducible promoter systems such as the ecdysone-inducible system or the tetracycline-regulated expression systems have proven to be powerful tools in studying gene function. In practice, such systems have met with the difficulty that either the vector expressing the transactivator gene or the vector carrying the response element are frequently silenced by flanking genomic sequences after stable integration. In order to identify those cells in a heterogeneous population in which a transgene is expressed from an ecdysone-inducible promoter, we have created the vector p2ER-EGFP/mcs that contains two ecdysone-inducible expression cassettes in tandem. Using two reporter genes, lacZ and green fluorescent protein (EGFP), we demonstrate that the expression of both genes can be co-induced from a very low baseline in CHO cells expressing the modified ecdysone receptor and the retinoid X receptor. The expression of EGFP and lacZ from vector p2ER-EGFP/lacZ follows the same Muristerone A concentration-dependence as that of EGFP from vector pER-EGFP, indicating that the juxtaposition of the two inducible promoters in vector p2ER-EGFP/mcs does not cause cross interference between them. We suggest that this modification of the ecdysone-inducible promoter system will allow for the visual control of the induced expression of other genes by Muristerone A.