FLT3配基在人骨髓基质细胞系中的基因转移与表达

目的：研究逆转录病毒介导的FL在骨髓基质细胞系HFCL中的表达。方法：采用脂质体法将重组质粒pLF-SN/HFCL和空载体pLXSN/HFCL转染包装细胞PA317,G418筛选抗性克隆,用抗性克隆上清液感染HFCL。RT－PCR和基因组DNA－PCR检测外源基因mRNA水平的表达及染色体的整合,小鼠CFU－GM集落法检测FL生物学活性。结果：在mRNA水平上有FL的表达,染色体基因组中整合有标记neo基因和FL基因。活性测试结果显示转染的骨髓基质细胞分泌FL。结论：提示骨髓基质细胞可作为基因治疗的靶细胞。     

Excision of specific DNA-sequences from integrated retroviral vectors via site-specific recombination.

Vectors for gene transfer and gene therapy were developed which combine the advantages of the integrase and recombinase systems. This was achieved by inserting two loxP sites for specific DNA excision into an MESV based retroviral vector. We show that this 'retroviral lox system' allows the infection of cells and the expression of transferred genes. In addition, we constructed an efficient retrovirus-based expression system for a modified Cre recombinase. Functional tests for DNA excision from integrated retroviral lox vectors were performed by the use of a negative selectable marker gene (thymidine kinase). Cre expression in cells infected with retroviral lox vectors and subsequent BrdU selection for cells in which site-specific recombination has occurred results in large numbers of independent cell clones. These results were confirmed by detailed molecular analysis. In addition we developed retroviral suicide vectors in which the enhancer/promoter elements of both LTRs were replaced by lox sequences. We show that lox-sequences located in the LTRs of retroviral vectors are stable during retroviral replication. Potential applications of this system would be the establishment of revertants of retrovirus-infected cells by controlled excision of nearly the complete proviral DNA.     

Stable and strictly controlled expression of LTR-flanked autoregulated expression cassettes upon adenoviral transfer

An autoregulatory bidirectional expression cassette encoding all components necessary for regulated gene expression in a one-step gene transfer was evaluated for use in adenoviral vectors. Adenoviral vectors transducing this cassette provide about 1000-fold regulation. Regulation could be further improved by integrating the cassette as a retroviral vector into the adenoviral backbone. Moreover, with these adeno/retroviral hybrid vectors, the frequency of chromosomal integration is enhanced and about 1% of infected cells show stable chromosomal integration of the autoregulated cassette. In these stably transduced cells high regulation capacity is maintained. To elucidate the molecular mechanism underlying this unexpected observation we investigated the regulation capacity of these cassettes in a viral and non-viral vector background after stable integration into the host's DNA. While naked cassettes show regulated expression that is strongly influenced by the chromosomal surrounding sequences the regulatory capacity of LTR flanked cassettes is highly comparable amongst different cell clones. This strict regulation with little influence from the flanking sequences is obtained when LTR-flanked cassettes are transduced as DNA, by retroviral or by adenoviral infection.     

逆转录病毒表达系统及其在外源蛋白高效表达中的应用

逆转录病毒表达系统是一种新的重组蛋白高效表达系统,它由逆转录病毒载体,包膜蛋白载体和包装细胞系构成,在基因治疗和生物制药领域都有很大的应用潜力。逆转录病毒和宿主细胞基因组的重组倾向于发生在转录活性区;口炎疱疹病毒-G蛋白 (vesicular stomatitis virus G, VSV-G)能有效扩大逆转录病毒感染宿主细胞的范围、提高逆转录病毒的感染效率;用高滴度的重组病毒感染细胞,经过简单的筛选即可获得高表达细胞株。本文对逆转录病毒表达系统的组成、感染的特点和机制及其应用前景作了概述。     

Retroviral infection of hES cells produces random-like integration patterns

Retroviral integration provides us with a powerful tool to realize prolonged gene expressions that are often critical to gene therapy. However, the perturbation of gene regulations in host cells by viral genome integration can lead to detrimental effects, yielding cancer. The oncogenic potential of retroviruses is linked to the preference of retroviruses to integrate into genomic regions that are enriched in gene regulatory elements. To better navigate the double-edged sword of retroviral integration we need to understand how retroviruses select their favored genomic loci during infections. In this study I showed that in addition to host proteins that tether retroviral pre-integration complexes to specific genomic regions, the epigenetic architecture of host genome might strongly affect retroviral integration patterns. Specifically, retroviruses showed their characteristic integration preference in differentiated somatic cells. In contrast, retroviral infections of hES cells, which are known to display decondensed chromatin, produced random-like integration patterns lacking of strong preference for regulatory-element-rich genomic regions. Better identification of the cellular and viral factors that determine retroviral integration patterns will facilitate the design of retroviral vectors for safer use in gene therapy.     

人TNF-α双顺反子逆转录病毒载体的构建及其在成纤维细胞中的表达

利用脑炎心肌炎病毒的内核糖体进入位点连接人ＴＮＦ－αｃＤＮＡ和选择基因ＮｅｏＲ基因,使ＴＮＦ－α及ＮｅｏＲ基因均受控于病毒ＬＴＲ启动子,将两基因同时转录至同一ｍＲＮＡ,从而构建成人ＴＮＦ－α双顺反子逆转录病毒载体ｐＧＣＥＮ／ＴＮＦ－α．在ＬｉｐｏｆｅｃｔＡＭＩＮＥ介导下将其导入包装细胞ＰＡ３１７,Ｇ４１８筛选得单克隆,病毒滴度为１０６ＣＦＵ／ｍｌ重组病毒分泌的细胞株．经ＰＣＲ证明外源基因已整合至细胞基因组,Ｎｏｒｔｈｅｒｎ印迹显示出单一ＬＲＴ转录本．持续Ｇ４１８筛选能明显促进目的基因ＴＮＦ－α的表达．用重组病毒上清感染小鼠成纤维细胞ＮＩＨ３Ｔ３,Ｇ４１８筛选获得的混合抗性克隆持续高表达ＴＮＦ－α,４０Ｇｙγ线照射后能维持高效表达至７ｄ．实验结果表明,含ＩＲＥＳ的双顺反子逆转录病毒载体将是一个很好的基因转移载体．     

Recombinant adeno-associated virus-mediated high-efficiency, transient expression of the murine cationic amino acid transporter (ecotropic retroviral receptor) permits stable transduction of human HeLa cells by ecotropic retroviral vectors.

Adeno-associated virus has a broad host range, is nonpathogenic, and integrates into a preferred location on chromosome 19, features that have fostered development of recombinant adeno-associated viruses (rAAV) as gene transfer vectors for therapeutic applications. We have used an rAAV to transfer and express the murine cationic amino acid transporter which functions as the ecotropic retroviral receptor, thereby rendering human cells conditionally susceptible to infection by an ecotropic retroviral vector. The proportion of human HeLa cells expressing the receptor at 60 h varied as a function of the multiplicity of infection (MOI) with the rAAV. Cells expressing the ecotropic receptor were efficiently transduced with an ecotropic retroviral vector encoding a nucleus-localized form of beta-galactosidase. Cells coexpressing the ecotropic receptor and nucleus-localized beta-galactosidase were isolated by fluorescence-activated cell sorting, and cell lines were recovered by cloning at limiting dilution. After growth in culture, all clones contained the retroviral vector genome, but fewer than 10% (3 of 47) contained the rAAV genome and continued to express the ecotropic receptor. The ecotropic receptor coding sequences in the rAAV genome were under the control of a tetracycline-modulated promoter. In the presence of tetracycline, receptor expression was low and the proportion of cells transduced by the ecotropic retroviral vector was decreased. Modulation of receptor expression was achieved with both an episomal and an integrated form of the rAAV genome. These data establish that functional gene expression from an rAAV genome can occur transiently without genome integration.     

Phage phiC31 integrase-mediated genomic integration of the common cytokine receptor gamma chain in human T-cell lines

BACKGROUND: X-linked severe combined immunodeficiency (SCID-X1, X-SCID) is a life-threatening disease caused by a mutated common cytokine receptor gamma chain (gammac) gene. Although ex vivo gene therapy, i.e., transduction of the gammac gene into autologous CD34(+) cells, has been successful for treating SCID-X1, the retrovirus vector-mediated transfer allowed dysregulated integration, causing leukemias. Here, to explore an alternative gene transfer methodology that may offer less risk of insertional mutagenesis, we employed the phiC31 integrase-based integration system using human T-cell lines, including the gammac-deficient ED40515(-). METHODS: A phiC31 integrase and a neo(r) gene expression plasmid containing the phiC31 attB sequence were co-delivered by electroporation into Jurkat cells. After G418 selection, integration site analyses were performed using linear amplification mediated-polymerase chain reaction (LAM-PCR). ED40515(-) cells were also transfected with a gammac expression plasmid containing attB, and the integration sites were determined. IL-2 stimulation was used to assess the functionality of the transduced gammac in an ED40515(-)-derived clone. RESULTS: Following co-introduction of the phiC31 integrase expression plasmid and the plasmid carrying attB, the efficiency of integration into the unmodified human genome was assessed. Several integration sites were characterized, including new integration sites in intergenic regions on chromosomes 13 and 18 that may be preferred in hematopoietic cells. An ED40515(-) line bearing the integrated gammac gene exhibited stable expression of the gammac protein, with normal IL-2 signaling, as assessed by STAT5 activation. CONCLUSIONS: This study supports the possible future use of this phiC31 integrase-mediated genomic integration strategy as an alternative gene therapy approach for treating SCID-X1.     

Retroviral integration profiles: their determinants and implications for gene therapy

Retroviruses have often been used for gene therapy because of their capacity for the long-term expression of transgenes via stable integration into the host genome. However, retroviral integration can also result in the transformation of normal cells into cancer cells, as demonstrated by the incidence of leukemia in a recent retroviral gene therapy trial in Europe. This unfortunate outcome has led to the rapid initiation of studies examining various biological and pathological aspects of retroviral integration. This review summarizes recent findings from these studies, including the global integration patterns of various types of retroviruses, viral and cellular determinants of integration, implications of integration for gene therapy and retrovirus-mediated infectious diseases, and strategies to shift integration to safe host genomic loci. A more comprehensive and mechanistic understanding of retroviral integration processes will eventually make it possible to generate safer retroviral vector platforms in the near future.     

Recombinant adeno-associated virus mediates a high level of gene transfer but less efficient integration in the K562 human hematopoietic cell line.

We tested the ability of a recombinant adeno-associated virus (rAAV) vector to express and integrate exogenous DNA into human hematopoietic cells in the absence of selection. We developed an rAAV vector, AAV-tNGFR, carrying a truncated rat nerve growth factor receptor (tNGFR) cDNA as a cell surface reporter under the control of the Moloney murine leukemia virus (MoMuLV) long terminal repeat. An analogous MoMuLV-based retroviral vector (L-tNGFR) was used in parallel, and gene transfer and expression in human hematopoietic cells were assessed by flow cytometry and DNA analyses. Following gene transfer into K562 cells with AAV-tNGFR at a multiplicity of infection (MOI) of 13 infectious units (IU), 26 to 38% of cells expressed tNGFR on the surface early after transduction, but the proportion of tNGFR expressing cells steadily declined to 3.0 to 3.5% over 1 month of culture. At an MOI of 130 IU, nearly all cells expressed tNGFR immediately posttransduction, but the proportion of cells expressing tNGFR declined to 62% over 2 months of culture. The decline in the proportion of AAV-tNGFR-expressing cells was associated with ongoing losses of vector genomes. In contrast, K562 cells transduced with the retroviral vector L-tNGFR expressed tNGFR in a constant fraction. Integration analyses on clones showed that integration occurred at different sites. Integration frequencies were estimated at about 49% at an MOI of 130 and 2% at an MOI of 1.3. Transduction of primary human CD34+ progenitor cells by AAV-tNGFR was less efficient than with K562 cells and showed a declining percentage of cells expressing tNGFR over 2 weeks of culture. Thus, purified rAAV caused very high gene transfer and expression in human hematopoietic cells early after transduction, which steadily declined during cell passage in the absence of selection. Although the efficiency of integration was low, overall integration was markedly improved at a high MOI. While prolonged episomal persistence may be adequate for gene therapy of nondividing cells, a very high MOI or improvements in basic aspects of AAV-based vectors may be necessary to improve integration frequency in the rapidly dividing hematopoietic cell population.     

Glucose 6-phosphate dehydrogenase expression is less prone to variegation when driven by its own promoter

The ability to transfer permanently genes into mammalian cells makes retroviruses suitable vectors for the ultimate purpose of treating inherited genetic disease. However, expression of the retrovirally transferred genes is variable (position effect and expression variegation) because retroviruses are highly susceptible to the influence of the host genome sequences which flank the integration site. We have investigated this phenomenon with respect to the human housekeeping enzyme, glucose 6-phosphate dehydrogenase (hG6PD). We have constructed retroviral vectors in which the hG6PD cDNA is driven by either of two conventional retroviral promoters and enhancers from the Moloney Murine Leukemia Virus (MMLV) and the Myeloproliferative Sarcoma Virus (MPSV) long terminal repeats (LTR) or by the hG6PD own promoter replacing most of enhancer and promoter LTR (GRU5). We have compared the activity of retrovirally transferred hG6PD driven by these promoters after retroviral integration in bulk cultures and in individual clones of murine fibroblasts. The level of hG6PD expressed by the hG6PD promoter of GRU5-G6PD was significantly lower than that expressed by conventional retroviral vectors. However, analysis of the single copy clones showed less variation of expression with GRU5-G6PD (coefficient of variation, CV, 35.5%) than with conventional vectors (CV, 58.9%). Thus we have several vectors competent for reliable transfer and expression of hG6PD. The hG6PD promoter provides reproducible expression of hG6PD and limits the variability of expression. This decreased variability is important in order to help ensuring a consistent level of delivery of the needed gene product in future therapeutic protocols.     

Inhibition of retroviral pathogenesis by RNA interference

BACKGROUND: RNA interference (RNAi) is a newly discovered cellular defense system that is known to suppress replication of genomic parasites in model organisms. It has been widely conjectured that RNAi may also serve as an antiviral system in vertebrates. RESULTS: Retroviral infection could be initiated by electroporation of cloned Rous sarcoma virus (RSV) proviral DNA into the developing chick neural tube. Coelectroporation of proviral DNA and short double-stranded RNAs matching sequences of avain retroviruses, which were designed to induce RNAi against RSV, inhibited viral replication. Replication of RSV after electroporation resulted in disruption of embryonic development and early death, but this, too, could be suppressed by RNAi against the RSV genome. RNAi could also inhibit the growth of RSV and HIV in cell culture. Analysis of the step of the retroviral life cycle that is inhibited by RNAi revealed that it primarily prevented accumulation of the viral RNAs synthesized late during infection. RNA genomes introduced in viral particles early during infection were less sensitive. CONCLUSIONS: RNAi can block retroviral infection in vertebrates. The tissue electroporation method described here should allow RNAi to be used widely to study gene function and control of infection in vertebrate animals.     

A pro-inflammatory role of deubiquitinating enzyme cylindromatosis (CYLD) in vascular smooth muscle cells

Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.     

High-efficiency identification of genes by functional analysis from a retroviral cDNA expression library.

Retroviral gene transfer efficiently delivers genes of interest stably into target cells, and expression cDNA cloning has been shown to be highly successful. Considering these two advantages, we now report a method by which one can identify genes stimulating cell growth through functional analysis. The first step requires the construction of a retroviral cDNA expression library and the optimization of transfection of vector DNA into virus packaging cells. The second step involves the cocultivation of target cells with libraries of retrovirus-producing cells, resulting in the amplification of target cells transduced with a gene(s) stimulating cell growth. Under standardized conditions of transfection, we detected an average of 4,000 independent clones per dish, among which expression of a retroviral beta-galactosidase gene at an abundance of 0.2% could be detected. Next, we demonstrated the augmentation of the sensitivity of the assay by retroviral infection and functional analysis. We did this by cocultivating factor-dependent (FD) cells with dishes of GP/E cells transfected with plasmids containing various molar ratios of pN2-IL3 DNA and retroviral library cDNA and by determining the highest dilution of pN2-IL3 which still resulted in the conversion of FD cells to factor independence. The retroviral interleukin-3 gene at an abundance as low as 0.001% could be detected. Indeed, we were able to detect from FD cells the development of factor-independent colonies with different phenotypes after retroviral transfer of cDNAs from an immortalized hemopoietic stem cell line. Thus, the combination of a standardized high-efficiency DNA transfection and retrovirus-mediated gene transfer should facilitate the identification of genes capable of conferring to target FD cells a detectable new function or phenotype. By scaling up the size of the experiment realistically during screening, the assay can detect cDNA at an abundance of lower than 0.0001%.     

Targeting rare populations of murine antigen-specific T lymphocytes by retroviral transduction for potential application in gene therapy for autoimmune disease

CD4+ T cells are important mediators in the pathogenesis of autoimmunity and would therefore provide ideal candidates for lymphocyte-based gene therapy. However, the number of Ag-specific T cells in any single lesion of autoimmunity may be quite low. Successful gene transfer into autoantigen-specific CD4+ T cells would serve as an ideal vehicle for site-targeted gene therapy if it were possible to transduce preferentially the small number of autoantigen-specific T cells. In this study we have demonstrated that retroviral infection of CD4+ lymphocytes from either autoantigen-stimulated TCR transgenic mice, or Ag-activated immunized nontransgenic mice, with a retroviral vector (pGCIRES), resulted in the transduction of only the limited number of Ag-reactive CD4+ T cells. In contrast, polyclonal activation of the same cultures resulted in transduction of non-antigen-specific lymphocytes. Transduction of Ag-reactive CD4+ T cells with pGCIRES retrovirus encoding the regulatory genes IL-4 (IL4) and soluble TNF receptor (STNFR) resulted in stable integration and long-term expression of recombinant gene products. Moreover, expression of the pGCIRES marker protein, GFP, directly correlated with the expression of the upstream regulatory gene. Retroviral transduction of CD4+ T cells targeted specifically Ag-reactive cells and was cell cycle-dependent and evident only during the mitosis phase. These studies suggest that retroviral transduction of autoantigen-specific murine CD4+ T cells, using the pGCIRES retroviral vector, may provide a potential method to target and isolate the low frequency of autoantigen-specific murine CD4+ T cells, and provides a rational approach to gene therapy in animal models of autoimmunity.     

A comprehensive study of optimal conditions for naked plasmid DNA transfer into skeletal muscle by electroporation

Efficient gene transfer is a key factor in gene therapy. Reducing the damage caused by gene transfer to muscle by electroporation is very important for its clinical application. Extensive investigation of optimal conditions for gene transfer by electroporation is required. The parameters used for electroporation, including plasmid concentration; injection volume; the plasmid dose of the injection; the concentration of saline media; the size of plasmid DNA; the age of the mice; the lag time between plasmid injection and electroporation; and the effect of repeated gene transfer by electroporation, were systematically investigated in the present study. The efficiencies of gene transfer by electroporation in normal and rodent models of diabetes were also evaluated. We found that electroporation used for non-viral gene transfer could be repeated in the same place in the muscle, but the expression efficiency was closely related to the muscle damage. Increasing pulse times could enhance the efficiency of gene transfer with a lower strength of electric field. It was better to use a higher plasmid concentration than to use a larger dose of plasmid and repeated injection to achieve a high level of transgene expression. Optimal conditions varied in different animal models, being milder for diabetic mice than for normal mice, and it was also shown that the conditions that worked well on these small rodents were not necessarily suitable for larger animals. Our results provide a comprehensive view of the factors that affect the efficiency of gene transfer into skeletal muscle by electroporation.