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.     

Engineering Viral Promoters for Gene Transfer to Human Neuroblasts

1. The strength and activity of several viral promoters in human neuroblasts were evaluated in vitro. 2. Several luciferase reporter gene contructs under the control of different viral promoters (HIV-1 LTR, HTLV-I LTR, MMTV LTR, RSV LTR, CMV, SV40), in the presence or in the absence of the viral SV40 enhancer, were transfected into two well-established human neural cell lines, including one derived from human embryonic olfactory cells (B4) and one derived from an adrenal neuroblastoma (SH-SY-5Y). The epithelial cell line HeLa was used as a control.3. The enzymatic activity of luciferase was evaluated after normalization with an internal control. The results indicated that in the context of the reporter gene constructs, the CMV promoter alone was, overall, the most active in any tested cell line. However, addition of the SV40 enhancer to the CMV promoter abolished luciferase activity in SH-SY-5Y cells while significantly increasing luciferase expression in the CNS derived B4 fetal neuroblasts.4. The results suggest that gene therapeutic vectors aimed to promote enzymatic activity through gene transfer into undifferentiated human neural cells are feasible. However, since differences in promoter activity in neuroectodermal-derived cells are very relevant, gene construct variants should be considered to optimize the system.     

Activity of the simian virus 40 early promoter-enhancer in herpes simplex virus type 1 vectors is dependent on its position, the infected cell type, and the presence of Vmw175.

We have studied some of the parameters governing the expression of a foreign promoter-reporter gene construct incorporated into herpes simplex virus (HSV) type 1. These include the genetic background of the parental virus, the site of transgene insertion within the HSV genome, and the infected cell type. The genetic background of the vector constructs denoted delta 3 was an HSV type 1 mutant deleted for nearly the entire coding portion of Vmw175 (ICP4), the product of the essential immediate-early gene IE3. For vectors denoted +, the IE3 deletion had been repaired by marker rescue. We used as a reporter gene the bacterial chloramphenicol acetyltransferase (CAT) gene, driven by the simian virus 40 (SV40) early promoter and enhancer region. The SV40-cat hybrid gene was inserted either into the HSV thymidine kinase (TK) locus to create the vectors TKScat delta 3 and TKScat+ or into an intergenic site within the BamHI z fragment of the short unique portion of the viral genome to create the vectors GScat delta 3 and GScat+. In Vero and BHK cells infected with TKScat delta 3, CAT activity was first detected at 10 h postinfection and continued to accumulate until 36 h postinfection. In cells of primate origin infected with the replication-competent vector TKScat+, or in primate cells which complement the IE3 deficiency and which were infected with TKScat delta 3, CAT activity was significantly lower than in cells of rodent origin. However, levels of CAT were increased in the presence of cycloheximide, suggesting that the low production of CAT in primate cells was due to repression of SV40-cat hybrid gene expression. In contrast with results with TKScat delta 3 and TKScat+, CAT activity was not detectable in any of the tested cell types infected with GScat delta 3 or GScat+ except under conditions of cycloheximide reversal. These results show that while HSV gene products expressed in the presence of Vmw175 inhibited SV40-cat expression in the tk locus in a cell-type-specific manner, HSV gene products expressed in the presence or absence of Vmw175 inhibited SV40-cat expression in the BamHI z locus independently of cell type.     

Quantitative analysis of luciferase activity of viral and hybrid promoters in bovine preimplantation embryos

This study was conducted to investigate quantitatively the luciferase activity of gene constructs with viral and hybrid enhancers and promoters in bovine preimplantation embryos by using firefly luciferase reporter genes. In Experiment I, to examine the stability of the luciferase, bioluminescence intensity of bovine embryos injected with the luciferase gene driven by the SV40 early promoter and enhancer (SVEluc) was measured with a luminometer at 2 days after microinjection. The results indicated that the bioluminescence could be analysed at any time within 30 min because the luciferase activity was constant during the measurement period from 5 to 30 min. In Experiment II, the luciferase expression of fertilized oocytes injected with four gene constructs (TKEluc, TK6WEluc, SVEluc, and Miwluc) was analysed by using a photon imaging system at 2 or 6 days following microinjection. The results from Experiment II indicated that the reporter gene governed by the Miw promoter (RSV LTR and chicken β-actin promoter) was expressed more intensively in bovine morulae and blastocysts than three other gene constructs. In Experiment III, the effect of SV40 enhancer was investigated when fused downstream to the luciferase cDNA of the Miwluc vector. The results showed that SV40 enhancer further activated the luciferase activity of the Miw promoter in bovine preimplantation embryos. It was concluded, therefore, that the Miw promoter together with the SV40 enhancer would confer the strongest expression of the firefly luciferase reporter gene among the gene constructs tested in preimplantation bovine embryos. Mol. Reprod. Dev. 49:368–373, 1998. © 1998 Wiley-Liss, Inc.     

Gene activation mediated by protein kinase C in human macrophage and teratocarcinoma cells expressing aminoglycoside phosphotransferase activity.

The bacterial neomycin phosphotransferase gene driven by the Moloney mouse leukemia virus long terminal repeat (LTR) or SV40 early region promoter was introduced into the human promonocyte-macrophage cell line, U937, and into the pluripotential human embryonic teratocarcinoma cell line, NT2/D1. Clonally derived cell lines capable of growing in 2-4 mg/ml of the aminoglycoside antibiotic, G418 (Geneticin), were established and transfected with pHIVCat, a plasmid expressing the bacterial chloramphenicol acetyl transferase (CAT) activity under the control of the human immunodeficiency virus (HIV-1) LTR. All of the G418 resistant (neo(r)) U937 cell lines and 10 of 14 neo(r) NT2/D1 cell lines exhibited reduced basal levels of CAT expression or impaired responses to activation of the HIV-1 LTR by phorbol 12-myristate 13-acetate (PMA) when compared to the parental lines. Other differences included inhibition of tat activation of the HIV-1 LTR and increased sensitivity of U937 cells to human tumor necrosis factor alpha. The expression of other eukaryotic promoters including the HTLV-1 LTR, SV40 ori sequences, and the human beta-actin gene promoter was similarly affected. However, differentiation of the neo(r) U937 cells into macrophages was neither delayed nor impaired. Because PMA is an activator of protein kinase C (PKC) and a potent inducer of HIV-1 directed gene expression, the amounts, sensitivity to G418, and cytosol to membrane translocation of this enzyme were determined in the wild type and neo(r) U937 cells. G418 at concentrations too low to affect cell growth (12-150 micrograms/ml) inhibited PMA-induced transactivation responses in wild type cells but did not inhibit PKC-dependent protein phosphorylation in vitro. PKC activities in the wild type and neo(r) cells were similar in absolute amounts and in the cytosol-membrane distribution of the enzyme. In contrast with wild type cells, however, all of the cytosolic Ca(2+)-phospholipid-dependent form of PKC disappeared from the neo(r) cells within 30 min after PMA induction. The results suggested that, depending upon the cell type, gene cotransfer using aminoglycoside resistance as a selectable marker may seriously perturb important cellular control mechanisms such as the PKC pathway leading to activation of gene expression.     

Activity of the human telomerase catalytic subunit (hTERT) gene promoter could be increased by the SV40 enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80 approximately 90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Activity of the Human Telomerase Catalytic Subunit (hTERT) Gene Promoter Could Be Increased by the SV40 Enhancer

Telomerase is a ribonucleoprotein complex of which the function is to add telomeric repeats to chromosomal ends. Telomerase consists of two essential components, the telomerase RNA template (hTR) and the catalytic subunit (hTERT). hTERT is expressed only in cells and tissues positive for telomerase activity, i.e., tumor and fetal cells. The aim of this study is to test the increased telomerase promoter activity for cancer gene therapy in adenovirus vector. We cloned the hTERT promoter in place of the SV40 promoter in the pGL3-contol vector to be increased by the SV40 enhancer sequences, resulting in strong expression of luc+ only in telomerase positive cancer cells. Then we transfected the constructed plasmid into a normal human cell line and several cancer cell lines. Through these experiments, we identified the selective and increased expression of the luciferase gene controlled by the hTERT promoter and the SV40 enhancer in the telomerase positive cancer cell lines. To investigate the possibility of utilizing the hTERT promoter and the SV40 enhancer in targeted cancer gene therapy, we constructed an adenovirus vector expressing HSV-TK controlled by the hTERT promoter and the SV40 enhancer for the induction of specific telomerase positive cancer cell death. NSCLC cells infected by Ad-hT-TK-enh were more significantly suppressed and induced apoptosis than those infected by Ad-hT-TK. Telomerase is activated in 80～90% of cancers, so adenovirus with increasing telomerase promoter activity might be used for targeted cancer gene therapy using suicide genes. These results show that the hTERT promoter and the SV40 enhancer might be used for targeted cancer gene therapy.     

Negative regulatory element associated with potentially functional promoter and enhancer elements in the long terminal repeats of endogenous murine leukemia virus-related proviral sequences.

Three series of recombinant DNA clones were constructed, with the bacterial chloramphenicol acetyltransferase (CAT) gene as a quantitative indicator, to examine the activities of promoter and enhancer sequence elements in the 5' long terminal repeat (LTR) of murine leukemia virus (MuLV)-related proviral sequences isolated from the mouse genome. Transient CAT expression was determined in mouse NIH 3T3, human HT1080, and mink CCL64 cultured cells transfected with the LTR-CAT constructs. The 700-base-pair (bp) LTRs of three polytropic MuLV-related proviral clones and the 750-bp LTRs of four modified polytropic proviral clones, in complete structures either with or without the adjacent downstream sequences, all showed very little or negligible activities for CAT expression, while ecotropic MuLV LTRs were highly active. The MuLV-related LTRs were divided into three portions and examined separately. The 3' portion of the MuLV-related LTRs that contains the CCAAC and TATAA boxes was found to be a functional promoter, being about one-half to one-third as active as the corresponding portion of ecotropic MuLV LTRs. A MboI-Bg/II fragment, representing the distinct 190- to 200-bp inserted segment in the middle, was found to be a potential enhancer, especially when examined in combination with the simian virus 40 promoter in CCL64 cells. A PstI-MboI fragment of the 5' portion, which contains the protein-binding motifs of the enhancer segment as well as the upstream LTR sequences, showed moderate enhancer activities in CCL6 cells but was virtually inactive in NIH 3T3 cells and HT1080 cells; addition of this fragment to the ecotropic LTR-CAT constructs depressed CAT expression. Further analyses using chimeric LTR constructs located the presence of a strong negative regulatory element within the region containing the 5' portion of the enhancer and the immediate upstream sequences in the MuLV-related LTRs.