Expression of retroviral vectors in transgenic mice obtained by embryo infection.

Pre-implantation embryos were infected with the retroviral vector MMCV-neo, which carries the neomycin resistance (neo) gene and the v-myc gene. Three transgenic substrains (M-TKneo 1-3) were derived which stably transmit a single intact copy of the vector. In all of the substrains, expression of the neo gene from the internal thymidine kinase (TK) promoter was detected, with two of the substrains expressing the gene in all tissues analysed. In the third substrain, the vector had integrated on the X chromosome and neo expression varied between different tissues. A second series of transgenic mice were obtained with the retroviral vector SAX, in which the human adenosine deaminase cDNA (ADA) is under the control of an internal SV40 promoter. Four substrains (M-SAX 1-4) were analysed; however, no expression of the ADA cDNA was detected. In all mice, no expression was found of the genes under the control of the viral 5' long terminal repeats (LTRs). In the M-TKneo substrains the vector was hypomethylated irrespective of its expression whereas in the M-SAX mice the vector was hypermethylated. These results demonstrate for the first time that the TK promoter can apparently express a gene in all tissues of adult mice and that retroviral vectors with internal promoters may provide an alternative to DNA injection for the efficient expression of genes in transgenic mice.     

Q vectors, bicistronic retroviral vectors for gene transfer

We have developed a retroviral vector that incorporates unique features of some previously described vectors. This vector includes: 3' long terminal repeats (LTRs) of the self-inactivating class; a 5' LTR that is a hybrid of the cytomegalovirus (CMV) enhancer and the mouse sarcoma virus promoter; an internal CMV immediate early region promoter to drive expression of the transduced gene and the neomycin phosphotransferase selectable marker; an expanded multiple cloning site and an internal ribosome entry site. An SV40 ori was introduced into the vector backbone to promote high copy number replication in packaging cell lines that express the SV40 large T antigen. We demonstrate that these retroviral constructs, designated Q vectors, can be used in applications where high viral titers and high level stable or transient gene expression are desirable.     

Construction of Retroviral Vectors with Improved Safety, Gene Expression, and Versatility

Murine leukemia virus (MLV)-based retroviral vectors are the most frequently used gene delivery vehicles. However, the current vectors are still not fully optimized for gene expression and viral titer, and many genetic and biochemical features of MLV-based vectors are poorly understood. We have previously reported that the retroviral vector MFG, where the gene of interest is expressed as a spliced mRNA, is superior in the level of gene expression with respect to other vectors compared in the study. As one approach to developing improved retroviral vectors, we have systematically performed mutational analysis of the MFG retroviral vector. We demonstrated that the entire gag coding sequence, together with the immediate upstream region, could be deleted without significantly affecting viral packaging or gene expression. To our knowledge, this region is included in all currently available retroviral vectors. In addition, almost the entire U3 region could be replaced with the heterologous human cytomegalovirus immediately-early promoter without deleterious effects. We could also insert internal ribosome entry sites (IRES) and multicloning sites into MFG without adverse effects. Based on these observations, we have constructed a series of new, improved retroviral constructs. These vectors produced viral titers comparable to MFG, expressed high levels of gene expression, and stably transferred genes to the target cells. Our vectors are more convenient to use because of the presence of multicloning sites and IRESs, and they are also more versatile because they can be readily converted to various applications. Our results have general implications regarding the design and development of improved retroviral vectors for gene therapy.     

Gene transfer in bovine blastocysts using replication-defective retroviral vectors packaged with gibbon ape leukemia virus envelopes

With this work we demonstrate that murine leukemia virus (MLV)-based replication-defective retroviral vectors encapsidated with Gibbon ape leukemia virus (GaLV) envelopes are significantly more infectious to bovine embryonic trachea (EBTr) cells than vectors encapsidated with murine xenotropic envelope proteins. In a test of internal promoter activity in an MLV retroviral vector, the rat β-actin promoter was shown to be better than the herpes simplex virus type 1 thymidine kinase (TK) and human cytomegalovirus (CMV) immediate early promoters for the expression of an E. coli β-galactosidase marker gene in bovine target cells. By co-culture of bovine blastocysts and virus-producing cells, or by culture of embryos in the medium harvested from virus-producing cells, we transferred the E. coli β-galactosidase gene into trophoblasts and also into inner cell mass (ICM) cells of a bovine embryo through the infection of the MLV-based replication-defective retroviruses encapsidated with GaLV envelope proteins. The infection was confirmed by the expression of the E. coli β-galactosidase gene under a β-actin internal promoter. In addition, co-culture of ICM cells with virus-producing cells resulted in differentiation of ICM cells into embryoid bodies expressing the marker genes. © 1993 Wiley-Liss, Inc.     

Hepatocyte-specific gene expression from integrated lentiviral vectors

BACKGROUND: For many applications, efficient gene therapy will require long-term, organ-specific therapeutic gene expression. Lentiviral vectors based on HIV-1 are promising gene delivery vehicles due to their ability to integrate transgenes into non-dividing cells. Many experimental vectors express transgenes under the control of the cytomegalovirus (CMV) immediate-early gene promoter. Although this promoter directs strong gene expression in vitro, it may be shut off rapidly in vivo. This study explores the potential of HIV-1-based vectors to transduce hepatocytes and compares gene expression from different promoters in integrated vectors. METHODS: HIV-1-based vector plasmids expressing the green fluorescent protein (GFP) under the control of the CMV promoter, the alpha-1 antitrypsin gene promoter or promoters derived from the hepatitis B virus (HBV) genome were used to compare expression in transfected and transduced cell lines. RESULTS: Hepatocyte cell lines differed strikingly in their transfectability. Transduction with replication-deficient HIV-1-based vector particles incorporating the different promoter elements was uniformly effective in hepatocyte and non-hepatocyte lines. However, in hepatocytes, only the CMV, alpha-1 antitrypsin and HBV core but not HBV surface promoters were able to produce GFP expression. Addition of the HBV enhancer 2 element improved the transducing ability of the HBV surface promoter and suppressed expression in non-hepatocytes increasing specificity for hepatocytes. CONCLUSIONS: Integrated lentiviral vectors can be used to direct transgene expression in liver cells both promiscuously and specifically. Promoters derived from the alpha-1 antitrypsin gene or HBV are alternatives to the CMV promoter. Inclusion of the HBV enhancer 2 permits strong liver-specific gene expression in vitro.     

Retroviral vectors encoding ADA regulatory locus control region provide enhanced T-cell-specific transgene expression

Background

Murine retroviral vectors have been used in several hundred gene therapy clinical trials, but have fallen out of favor for a number of reasons. One issue is that gene expression from viral or internal promoters is highly variable and essentially unregulated. Moreover, with retroviral vectors, gene expression is usually silenced over time. Mammalian genes, in contrast, are characterized by highly regulated, precise levels of expression in both a temporal and a cell-specific manner. To ascertain if recapitulation of endogenous adenosine deaminase (ADA) expression can be achieved in a vector construct we created a new series of Moloney murine leukemia virus (MuLV) based retroviral vector that carry human regulatory elements including combinations of the ADA promoter, the ADA locus control region (LCR), ADA introns and human polyadenylation sequences in a self-inactivating vector backbone.

Methods

A MuLV-based retroviral vector with a self-inactivating (SIN) backbone, the phosphoglycerate kinase promoter (PGK) and the enhanced green fluorescent protein (eGFP), as a reporter gene, was generated. Subsequent vectors were constructed from this basic vector by deletion or addition of certain elements. The added elements that were assessed are the human ADA promoter, human ADA locus control region (LCR), introns 7, 8, and 11 from the human ADA gene, and human growth hormone polyadenylation signal. Retroviral vector particles were produced by transient three-plasmid transfection of 293T cells. Retroviral vectors encoding eGFP were titered by transducing 293A cells, and then the proportion of GFP-positive cells was determined using fluorescence-activated cell sorting (FACS). Non T-cell and T-cell lines were transduced at a multiplicity of infection (MOI) of 0.1 and the yield of eGFP transgene expression was evaluated by FACS analysis using mean fluorescent intensity (MFI) detection.

Results

Vectors that contained the ADA LCR were preferentially expressed in T-cell lines. Further improvements in T-cell specific gene expression were observed with the incorporation of additional cis-regulatory elements, such as a human polyadenylation signal and intron 7 from the human ADA gene.

Conclusion

These studies suggest that the combination of an authentically regulated ADA gene in a murine retroviral vector, together with additional locus-specific regulatory refinements, will yield a vector with a safer profile and greater efficacy in terms of high-level, therapeutic, regulated gene expression for the treatment of ADA-deficient severe combined immunodeficiency.     

Transient gene expression mediated by integrase-defective retroviral vectors

Nonintegrating retroviral vectors were produced from a Moloney murine leukemia virus (MoMLV)-based retroviral vector system by introducing a point mutation into the integrase (IN) gene of the packaging plasmid. The efficacy of IN-defective retroviral vectors was measured through the transient expression of ZsGreen or luciferase in human cell lines. The IN-defective retroviral vectors could transduce target cells efficiently, but their gene expression was transient and lower than that seen with the integrating vectors. IN-defective retroviral vector gene expression decreased to background levels in fewer than 10 days. Southern blot analysis of transduced K562 cells confirmed the loss of a detectable vector sequence by 15 days. The residual integration activity of the IN-defective vector was 1000- to 10,000-fold lower than that of the integrating vector. These results demonstrate that the IN-defective retroviral vectors can provide a useful tool for efficient transient gene expression targeting of primary hematopoietic stem cells and lymphoid cells.     

A murine leukemia virus (MuLV) long terminal repeat derived from rhesus macaques in the context of a lentivirus vector and MuLV gag sequence results in high-level gene expression in human T lymphocytes

We constructed human immunodeficiency virus type 1 (HIV-1) vectors that will allow higher levels of gene expression in T cells. Gene expression under the control of an internal cytomegalovirus (CMV) immediate-early promoter in a self-inactivating lentiviral vector (CSCG) is 4- to 15-fold lower in T-cell lines (SUPT1 and CEMX174) than in non-lymphoid-cell lines (HeLa and 293T). This is in contrast to a Moloney murine leukemia virus (MoMLV)-based retrovirus vector (SRalphaLEGFP). We therefore replaced the internal CMV promoter of CSCG with three different murine oncoretroviral long terminal repeat (LTR) promoters-murine sarcoma virus (MSV), MoMLV (MLV), and the LTR (termed Rh-MLV) that is derived from the ampho-mink cell focus-forming (AMP/MCF) retrovirus in the serum of one rhesus macaque monkey that developed T-cell lymphoma following autologous transplantation of enriched bone marrow stem cells transduced with a retrovirus vector preparation containing replication-competent viruses (E. F. Vanin, M. Kaloss, C. Broscius, and A. W. Nienhuis, J. Virol. 68:4241-4250, 1994). We found that the combination of Rh-MLV LTR and a partial gag sequence of MoMLV (Deltagag(871-1612)) in CS-Rh-MLV-E gave the highest level of enhanced green fluorescent protein (EGFP) gene expression compared with MLV, MSV LTR, phosphoglycerate kinase, and CMV promoters in T-cell lines, as well as activated primary T cells. Interestingly, there was a further two- to threefold increase in EGFP expression (thus, 10-fold-higher expression than with CMV) when the Rh-MLV promoter and Deltagag(871-1612) were used in a self-inactivating-vector setting that has a further deletion in the U3 region of the HIV-1 LTR. These hybrid vectors should prove useful in gene therapy applications for T cells.     

High recombination rate of an Epstein-Barr virus-simian virus 40 hybrid shuttle vector in human cells

The stability of an Epstein-Barr virus (EBV)-simian virus 40 (SV40) hybrid shuttle vector, the p205-GTI plasmid, was analyzed in human cells during EBV- or SV40-type replication mode. When the p205-GTI plasmid was maintained as an episomal EBV vector in the human 293 cell line, no rearrangement was detected. To induce the SV40 replication mode, cells containing the episomal p205-GTI plasmid were either transfected with vectors carrying the T antigen gene or infected with SV40. Surprisingly, we observed both production and amplification of different classes of recombinant molecules. Particular types of modifications were found in most of the recombinants. The most striking rearrangement was a duplication of the promoter and enhancer regions of SV40 which was inserted in the thymidine kinase (TK) promoter. This recombination process involved a few bases of homology, and one of the recombination junctions implicated the GC boxes which constitute the essential components of the TK and SV40 early promoters. Our results suggest that a combination of a low level of base homology and a specific DNA sequence function (promoter and enhancer sites) leads to a very high level of recombinational activity during T-antigen-dependent plasmid replication.     

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.     

Novel Tat-encoding bicistronic human immunodeficiency virus type 1-based gene transfer vectors for high-level transgene expression

We describe bicistronic single-exon Tat (72-amino-acid Tat [Tat72])- and full-length Tat (Tat86)-encoding gene transfer vectors based on human immunodeficiency virus type 1 (HIV-1). We created versions of these vectors that were rendered Rev independent by using the constitutive transport element (CTE) from Mason-Pfizer monkey virus (MPMV). Tat72-encoding vectors performed better than Tat86-expressing vectors in gene transfer experiments. CTE-containing vectors, produced in a Rev-independent packaging system, had gene transfer efficiencies nearly equivalent to those produced using a combination RNA transport (CTE and Rev-Rev response element)-based packaging system. The Tat72-encoding vectors could be efficiently transduced into a variety of cell types, showed higher levels of transgene expression than vectors with the simian cytomegalovirus immediate-early or the simian virus 40 early promoter, and provide an alternative to HIV-1 vectors with internal promoters.     

High level expression of proteins using sequences from the ferritin heavy chain gene locus

An expression vector has been generated using a gene highly expressed under conditions found in a typical fed-batch bioreactor process. The ferritin heavy chain (HC) gene exhibits higher levels of expression in the late stages of a fed-batch bioreactor than in the early stages. This property was considered advantageous for an expression vector, since the maximal cell density would coincide with maximal expression. The rat ferritin HC genomic region was isolated and converted into an expression vector where large segments of 5' and 3' flanking regions were included in an attempt to recreate the same high level of expression in stably transfected cells. Expression from the resulting ferritin HC vector was compared to vectors containing the commonly used strong promoters, CMV IE, and SV40 early promoter/enhancer, in the generation of stable transfectants. The ferritin HC vector was able to generate cell lines with significantly higher expression levels than those under the control of the viral promoters.     

Differential expression of mammalian or viral promoter-driven gene in adherent versus suspension cells

Although expression vectors using viral and mammalian promoters constitutively express genes of interest in adherent cells, few studies have examined whether the function of these vectors in suspended cells, such as in over-agar or soft agar assay (an in vitro cell transformation assay), is as robust as when they are in adherent cells. The selection of appropriate expression vector to optimally express genes in suspended cells would be useful in determining whether these genes play a critical role in maintaining colony formation or cell transformation. To compare promoter-driven expression vector function in adherent versus suspension cells, we performed transient transfection assays using viral (simian virus 40 [SV40] and cytomegalovirus [CMV]) and mammalian (beta-actin) promoters fused to luciferase or beta-galactosidase reporter gene. Over-agar assay was used to suspend cells on top of agar, which allowed cell retrieval and analysis. We found that beta-actin and SV40 promoters exhibited suppressed gene expression of 70 and 56%, respectively, in cells suspended on agar compared with those attached on plates. The suppressed response by the exogenous beta-actin promoter in suspension was consistent with the response of the endogenous beta-actin promoter activity because the steady-state level of beta-actin messenger ribonucleic acid in suspended cells was significantly reduced by 50% relative to that expressed in attached cells. In contrast to SV40 promoter, CMV promoter activity was not decreased in cells suspended in over-agar when compared with adherent cells. These studies show that regardless of mammalian or viral vectors, one cannot assume that all expression vectors behave similarly in both suspension and adherent state.     

Dramatic Increase in Expression of a Transgene by Insertion of Promoters Downstream of the Cargo Gene

For expression of genes in mammalian cells, various vectors have been developed using promoters including CMV, EF-1α, and CAG promoters and have been widely used. However, such expression vectors sometimes fail to attain sufficient expression levels depending on the nature of cargo genes and/or on host cell types. In the present study, we aimed to develop a potent promoter system that enables high expression levels of cargo genes ubiquitously in many different cell types. We found that insertion of an additional promoter downstream of a cargo gene greatly enhanced the expression levels. Among the constructs we tested, C-TSC cassette (C: CMV-RU5′ located upstream; TSC: another promoter unit composed of triple tandem promoters, hTERT, SV40, and CMV, located downstream of the cDNA plus a polyadenylation signal) had the most potent capability, showing far higher efficiency than that of potent conventional vector systems. The results indicate that the new expression system is useful for production of recombinant proteins in mammalian cells and for application as a gene therapeutic measure.