Human Elongation Factor 1α Promoter Research Articles

Establishment of a megakaryoblastic cell line for conventional assessment of platelet calcium signaling

Platelet function tests utilizing agonists or patient serum are generally performed to assess platelet activation ex vivo. However, inter-individual differences in platelet reactivity and donor requirements make it difficult to standardize these tests. Here, we established a megakaryoblastic cell line for the conventional assessment of platelet activation. We first compared intracellular signaling pathways using CD32 crosslinking in several megakaryoblastic cell lines, including CMK, UT-7/TPO, and MEG-01 cells. We confirmed that CD32 was abundantly expressed on the cell surface, and that intracellular calcium mobilization and tyrosine phosphorylation occurred after CD32 crosslinking. We next employed GCaMP6s, a highly sensitive calcium indicator, to facilitate the detection of calcium mobilization by transducing CMK and MEG-01 cells with a plasmid harboring GCaMP6s under the control of the human elongation factor-1α promoter. Cells that stably expressed GCaMP6s emitted enhanced green fluorescent protein fluorescence in response to intracellular calcium mobilization following agonist stimulation in the absence of pretreatment. In summary, we have established megakaryoblastic cell lines that mimic platelets by mobilizing intracellular calcium in response to several agonists. These cell lines can potentially be utilized in high-throughput screening assays for the discovery of new antiplatelet drugs or diagnosis of disorders caused by platelet-activating substances.

Baculovirus-induced recombinant protein expression in human mesenchymal stromal stem cells: A promoter study

Human mesenchymal stem cells (hMSCs) are the current workhorses of regenerative medicine and gene therapy. The corresponding vectors are usually based on lentiviruses, adenoviruses, retroviruses or adeno-associated viruses, but recently they have been joined by baculoviruses, which are more widely known for their role in the development of pesticides and vaccines. Here we show that gene transfer to an immortalized human mesenchymal stroma cell line can be achieved by baculovirus transduction. We also compared the performance of five different constitutive promoters controlling GFP expression. The human elongation factor 1α promoter (EF1α) achieved the strongest GFP expression, whereas the mouse phosphoglycerate kinase 1 promoter (PGK) was the weakest. The human EF1α promoter is therefore recommended for the regulation of genes introduced into hMSC-TERTs by baculovirus vectors.

A Shot in the Bone Corrects a Genetic Disease

A Shot in the Bone Corrects a Genetic Disease

Development of a novel single step reverse genetics system for feline calicivirus

The reverse genetics system is a useful tool to generate infectious virus. Feline calicivirus (FCV), a member of the genus Vesivirus in the family Caliciviridae, has a positive sense, single-stranded RNA genome. Two reverse genetics systems have been established for FCV; however, these methods need multi-steps to produce progeny infectious virus. In this study, a novel plasmid-based single step reverse genetics system for FCV has been developed. The plasmid carries FCV F4 strain genomic sequence with an introduced silent mutation. In addition, at the 5′- and 3′-end, a human elongation factor-1α promoter and a cis-acting hepatitis delta virus ribozyme following poly-A, were added, respectively. When the plasmid was transfected into Crandell-Rees feline kidney cells, progeny FCV was generated. The reverse genetics system-derived FCV (rFCV) showed similar growth kinetics and antigenic characteristics and had identical genomic terminals to those of the original FCV F4 strain. The presence of the introduced silent mutation in the rFCV genomic cDNA supported that the progeny virus was originated from the plasmid. This novel FCV reverse genetics system is simple and can be used to evaluate the functions of the viral genome, proteins, and phenotypic characterization of FCV strains in the future.

Systemic TNFα Gene Therapy Synergizes With Liposomal Doxorubicine in the Treatment of Metastatic Cancer

Tumor necrosis factor alpha (TNFα) is a potent antitumoral cytokine, either killing tumor cells directly or affecting the tumor vasculature leading to enhanced accumulation of macromolecular drugs. Due to dose limiting side effects systemic administration of TNFα protein at therapeutically active doses is precluded. With gene vectors, tumor restricted TNFα expression can be achieved and in principle synergize with chemotherapy. Synthetic gene carriers based on polyamines were intravenously injected, which either passively accumulate within the tumor or specifically target the epidermal growth factor receptor. A single intravenous injection of TNFα gene vector promoted accumulation of liposomal doxorubicine (Doxil) in murine neuroblastoma and human hepatoma by enhancing tumor endothelium permeability. The expression of transgenic TNFα was restricted to tumor tissue. Three treatment cycles with TNFα gene vectors and Doxil significantly delayed tumor growth in subcutaneous murine Neuro2A neuroblastoma. Also tumors re-growing after initial treatment were successfully treated in a fourth cycle pointing at the absence of resistance mechanisms. Systemic Neuro2A metastases or human LS174T colon carcinoma metastases in liver were also successfully treated with this combined approach. In conclusion, this schedule opens the possibility for the efficient treatment of tumors metastases otherwise not accessible for macromolecular drug carriers.

An Adeno-Associated Virus Vector Efficiently and Specifically Transduces Mouse Skeletal Muscle

Expression of a therapeutic gene in the skeletal muscle is a practical strategy to compensate a patients' insufficient circulating factor. Its clinical application requires a muscle-targeting vector capable of inducing a continuous high-level transgene expression. We modified an adeno-associated virus serotype 2 (AAV2) vector expressing luciferase from the mouse muscle creatine kinase gene promoter-enhancer (Ckm). First, AAVS1 insulator was inserted into the vector genome for transcriptional enhancement. This increased transduction of mouse quadriceps muscle by 11-fold at 4weeks after intramuscular injection. Second, two capsid modifications were combined (21F capsid): incorporation of a segment of AAV1 capsid to produce a hybrid capsid and substitution of a tyrosine with a phenylalanine. Use of 21F capsid increased muscle transduction further by 18-fold, resulting in 200-fold higher efficacy than that of the unmodified vector. Compared with a vector having human elongation factor 1α promoter which showed similar efficacy in the muscle, this vector having Ckm transduced non-muscle organs less efficiently after intravenous administration. The AAV2 vector composed of the modified genome and capsid provides a backbone to develop a clinical vector expressing a therapeutic gene in the muscle.

Lentiviral vectors incorporating a human elongation factor 1α promoter for the treatment of canine leukocyte adhesion deficiency

Canine leukocyte adhesion deficiency (CLAD) provides a unique large animal model for testing new therapeutic approaches for the treatment of children with leukocyte adhesion deficiency (LAD). In our CLAD model, we examined two different fragments of the human elongation factor 1alpha (EF1alpha) promoter (EF1alphaL, 1189 bp and EF1alphaS, 233 bp) driving the expression of canine CD18 in a self-inactivating (SIN) lentiviral vector. The EF1alphaS vector resulted in the highest levels of canine CD18 expression in CLAD CD34(+) cells in vitro. Subsequently, autologous CD34(+) bone marrow cells from four CLAD pups were transduced with the EF1alphaS vector and infused following a non-myeloablative dose of 200 cGy total-body irradiation. None of the CLAD pups achieved levels of circulating CD18(+) neutrophils sufficient to reverse the CLAD phenotype, and all four animals were euthanized because of infections within 9 weeks of treatment. These results indicate that the EF1alphaS promoter-driven CD18 expression in the context of a RRLSIN lentiviral vector does not lead to sufficient numbers of CD18(+) neutrophils in vivo to reverse the CLAD phenotype when used in a non-myeloablative transplant regimen in dogs.

Enhanced transgene expression in the mouse skeletal muscle infected by the adeno‐associated viral vector with the human elongation factor 1α promoter and a human chromatin insulator

Efficient and continuous expression of a therapeutic transgene is a key factor for improving the efficacy of gene therapy. Some insulators are known to contribute to continuous high-level expression of a therapeutic transgene. Using the human AAVS1 insulator (DHS) found in the AAVS1 DNase I hypersensitive site, chicken beta-globin insulator (cHS4) and sea urchin arylsufatase insulator (Ars), we newly constructed three recombinant adeno-associated virus vectors (rAAV) and examined their capability of transducing the mouse quadriceps muscle. DHS increased transgene expression from the human elongation factor 1alpha promoter (EF) by 1000-fold, up to the high level achieved by the human cytomegalovirus immediate early promoter/enhancer (CMV), which comprises an extremely strong promoter for driving a transgene. cHS4 enhanced the expression by 100-fold, whereas Ars did not. The enhanced expression was maintained for at least 24 weeks. Vector copy numbers were similar with and without DHS or cHS4; thus, the enhancement is most likely due to up-regulated transcription. Neither DHS, nor cHS4 affected transgene expression from CMV. DHS enhanced expression from the human muscle creatine kinase promoter/enhancer by 100-fold in mice, as did DHS from EF. Although DHS was unable to further enhance high expression from the strong viral enhancer/promoter, it enhanced low expression from the human promoters by 100- to 1000-fold. Thus, DHS may be useful for constructing rAAVs that express a therapeutic transgene from less efficient, tissue specific promoters.

High Level Production of a Lysosomal Enzyme from HSC-Derived Erythroid Cells for Therapeutic Correction of Hurler Syndrome in Mice.

Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)- mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Restricting transgene expression to late erythroblasts and red blood cells (RBC) may reduce the risk of activating oncogenes in HSC and its offspring in all lineages. In this study, we sought to evaluate the feasibility of redirecting a fraction of robust protein-synthesis machinery in maturing erythroid cells for production of alpha-L-iduronidase (IDUA), the lysosomal enzyme missing in Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). We first utilized a murine erythroid leukemia (MEL) cell line to compare IDUA expression and release from LVs using human elongation factor 1α promoter (EF), LTR of SFFV (SF), or a erythroid specific hybrid promoter (IHK) containing human ALAS2 intron 8 erythroid specific enhancer, HS40 core element from human alpha LCR and human ankyrin-1 promoter. We have previously shown highly erythroid-specific expression in vivo using this hybrid promoter, which is sustained in secondary transplantation recipient mice and resists proviral silencing (Moreau-Gaudry et al. Blood, 2001; Mohamedali et al, Mol Ther 2004). MEL cells were introduced to differentiation with hexamethylene bis-acetamide, and progressive erythroid differentiation was confirmed by morphologic evaluation in cytospins. Relatively low levels of expression from IHK (5% of SF, and 8% of EF) were observed in non-induced MEL cells; however, it increased steadily during induction and reached a similarly high expression level as those from the SF promoter. Expression from EF promoter reduced significantly, and the levels from SF promoter remained unchanged with erythroid differentiation. A similar pattern was found in IDUA activities released from stably transduced MEL cells during induction. We then evaluated in vivo the systemic IDUA production in an enzyme-deficient MPS I mouse model using erythroid-specific LV. Lineage-negative bone marrow cells (Lin−) were isolated with 92–97% purity by lineage depletion using immunomagnetic cell sorting. After a short pre-stimulation period, Lin− cells were transduced twice with LV-IHK-IDUA-ires-GFP for a total multiplicity of infection (MOI) at 2–20, resulting in up to 71% transduction efficiency. Four-months after transplantation of Lin- cells transduced at high MOI, sustained and higher-than normal plasma IDUA levels were achieved in all treated MPS I mice (1.5 to 8-fold of normal levels) with GFP transgene expression detected in up to 7% of Ter119+ blood cells. To evaluate transgene expression pattern during erythroid differentiation in bone marrow, we defined erythroblast subpopulations by immunostaining with CD71 and Ter119 for enrichment in proerythroblasts (I), basophilic erythroblasts (II), polychromatophilic erythroblasts (III), and orthrochromatic erythroblasts and reticulocytes (IV). GFP expression was predominantly detected in population III (9–17.3%), followed by the most differentiated population IV. To determine transduction efficiency in HSC, spleen colony-forming-cell assay was performed that showed 15–25% GFP+ spleen colonies in 2o BMT recipients. Moreover, long-term systemic metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in liver and spleen, with relatively moderate improvement in brain. These results demonstrate for the first time that a lysosomal enzyme can be produced and secreted successfully and steadily at superphysiological levels in circulation by erythroid cells using tissue-specific LV, with phenotypic correction of Hurler Syndrome in mice. This data warrants further evaluation for the potential application of erythroid-specific vectors in treating non-RBC related diseases.

Overexpression of a transcription factor LYL1 induces T- and B-cell lymphoma in mice

LYL1, a member of the class II basic helix-loop-helix transcription factors, is aberrantly expressed in a fraction of human T-cell acute lymphoblastic leukemia. Here, we generated transgenic mice ubiquitously overexpressing LYL1 using a construct expressing full-length cDNA driven by a human elongation factor 1alpha promoter. Four independent lines exhibiting high LYL1 expression were established. Of these transgenic mice, 96% displayed loss of hair with a short kinked tail. Furthermore, 30% of them developed malignant lymphoma, with an average latent period of 352 days. In these mice, histological examination revealed tumor cell infiltration in multiple organs and immunohistochemical analysis showed that the infiltrated tumor cells were either CD3 or CD45R/B220-positive; fluorescence-activated cell sorter analysis indicated that each tumor consisted either of mainly CD4, CD8 double-positive T cells or mature B cells; the clonality of LYL1-induced lymphoma was confirmed by T-cell receptor rearrangement and immunoglobulin heavy-chain gene rearrangement analyses. Mammalian two-hybrid analysis and luciferase assay suggested that excess LYL1 blocked the dimerization of E2A and thus inhibited the regulatory activity of E2A on the CD4 promoter. Reverse transcription-polymerase chain reaction results showed that the expression of certain E2A/HEB target genes was downregulated. Taken together, our results provide direct evidence that aberrant expression of LYL1 plays a role in lymphomagenesis.

Adeno-Associated Virus: A Ubiquitous Commensal of Mammals

Adeno-Associated Virus: A Ubiquitous Commensal of Mammals

Widespread Correction of Lysosomal Storage Following Intrahepatic Injection of a Recombinant Adeno-associated Virus in the Adult MPS VII Mouse

Mucopolysaccharidosis type VII is a lysosomal storage disease caused by deficiency of the acid hydrolase β-glucuronidase. MPS VII mice develop progressive lysosomal accumulation of glycosaminoglycans within multiple organs, including the brain. Using this animal model, we investigated whether gene transfer mediated by a recombinant adeno-associated virus (rAAV) type 2 vector is capable of reversing the progression of storage in adult mice. We engineered an rAAV2 vector to carry the murine β-glucuronidase cDNA under the transcriptional direction of the human elongation factor-1α promoter. Intrahepatic administration of this vector in adult MPS VII mice resulted in stable hepatic β-glucuronidase expression (473 ± 254% of that found in wild-type mouse liver) for at least 1 year postinjection. There was widespread distribution of vector genomes and β-glucuronidase within extrahepatic organs. The level of enzyme activity was sufficient to reduce lysosomal storage within the liver, spleen, kidney, heart, lung, and brain. Within selected regions of the brain, neuronal, glial, and perivascular cells had histopathologic evidence of reduced storage. Also, brain α-galactosidase and β-hexosaminidase enzyme levels, secondarily elevated by the storage abnormality, were normalized. These data demonstrate that peripheral administration of an rAAV2 vector in adult MPS VII mice can lead to transgene expression levels sufficient for improvements in both the peripheral and the central manifestations of this disease.

In Vitro and In Vivo Characterization of a Dual-Function Green Fluorescent Protein-HSV1-Thymidine Kinase Reporter Gene Driven by the Human Elongation Factor 1α Promoter

Toward the goal of monitoring activity of native mammalian promoters with molecular imaging techniques, we stably transfected DU145 prostate carcinoma cells with a fusion construct of enhanced green fluorescent protein (EGFP) and wild-type herpes simplex virus-1 thymidine kinase (HSV1-TK) as a reporter gene driven by the promoter for human elongation factor 1 alpha (EF-1 alpha-EGFP-TK). Using this model system, expression of EGFP was quantified by flow cytometry and fluorescence microscopy, while the HSV1-TK component of the reporter was quantified with 8-[3H]ganciclovir (8-[3H]GCV). As analyzed by flow cytometry, passage of EGFP-TK-DU145 transfected cells (ETK) in vitro resulted in populations of cells with high and low expression of EGFP over time. High and low ETK cells retained 23-fold and 5-fold more GCV, respectively, than control. While differences in uptake and retention of GCV corresponded to relative expression of the reporter gene in each subpopulation of cells as determined by both flow cytometry (EGFP) and quantitative RT-PCR, the correlation was not linear. Furthermore, in high ETK cells, net retention of various radiolabeled nucleoside analogues varied; the rank order was 8-[3H]GCV < 9-(4-fluoro-3-hydroxymethylbutyl)guanine ([18F]FHBG) approximately 8-[3H]penciclovir (8-[3H]PCV) < 2'-fluoro-2'-deoxy-5-iodouracil-beta-D-arabinofuranoside (2-[14C]FIAU). Xenograft tumors of ETK cells in vivo accumulated 2.5-fold more 8-[3H]GCV per gram of tissue and showed greater fluorescence from EGFP than control DU145 cells, demonstrating that the reporter gene functioned in vivo. These data extend previous reports by showing that a human promoter can be detected in vitro and in vivo with a dual-function reporter exploiting optical and radiotracer techniques.

DNA Vaccines Expressing a Fusion Product of Outer Surface Proteins A and C fromBorrelia burgdorferiInduce Protective Antibodies Suitable for Prophylaxis but Not for Resolution of Lyme Disease

DNA vaccines encoding the outer surface protein A (OspA) of Borrelia burgdorferi have been shown to induce protective humoral responses capable of preventing but not curing infection in mice. Subsequent studies showed that an established infection or disease could be resolved by passive transfer of antibodies to OspC. In the present study, DNA vaccines encoding either the OspC antigen alone or fused to OspA and under the transcriptional control of the human elongation factor 1alpha promoter were evaluated for their protective and/or curative potential. In contrast to ospA-containing plasmids, none of the six constructs with ospC alone were immunogenic in vivo, independent of whether they contained promoter or leader sequences from ospA and/or ospC, or alternatively, the signal sequence of the human tissue plasminogen activator. Solely, a DNA vaccine encoding an OspA-OspC fusion product led to expression of the respective polypeptide chain in transfected cells in vitro and to the induction of OspA- and OspC-specific antibodies in vivo. Immune sera raised against the OspA-OspC fusion product conveyed full protection against subsequent infection, most probably via OspA-specific antibodies, but were unable to resolve infection.

Coexpression of factor VIII heavy and light chain adeno-associated viral vectors produces biologically active protein.

We are interested in using recombinant adeno-associated viral vectors in the treatment of hemophilia A. Because of the size constraints of recombinant adeno-associated viral vectors, we delivered the heavy and light chains of the human factor 8 (hFVIII) cDNA independently by using two separate vectors. Recombinant AAV vectors were constructed that utilized the human elongation factor 1alpha promoter, a human growth factor polyadenylation signal, and the cDNA sequences encoding either the heavy or light chain of hFVIII. Portal vein injections of each vector alone, a combination of both vectors, or a hFIX control vector were performed in C57BL/6 mice. An ELISA specific for the light chain of hFVIII demonstrated very high levels (2-10 microgram/ml) of protein expression in animals injected with the light chain vector alone or with both vectors. We utilized a chromogenic assay in combination with an antibody specific to hFVIII to determine the amount of biologically active hFVIII in mouse plasma. In animals injected with both the heavy and light chain vectors, greater than physiological levels (200-400 ng/ml) of biologically active hFVIII were produced. This suggests that coexpression of the heavy and light chains of hFVIII may be a feasible approach for treatment of hemophilia A.

Inducible high-level expression vector for mammalian cells, pEF-LAC carrying human elongation factor 1α promoter and lac operator

We have constructed an inducible high-level expression vector, pEF-LAC. pEF-LAC has a modified human polypeptide chain elongation factor 1α (EF-1α) promoter containing three lactose operator sequences. Using the cat reporter gene, we characterized the transcriptional activity of pEF-LAC. In the transient transfection of NIH3T3 and BaF3 cells, the transcriptional activity of pEF-LAC was higher than that of the original human elongation factor 1α promoter, simian virus 40 (SV40) promoter, and Rous sarcoma virus (RSV) long terminal repeat (LTR). Cotransfection of the lactose repressor expression plasmid effectively suppressed the promoter activity of pEF-LAC, and the activity was fully recovered by addition of isopropyl β- d-thiogalactopyranoside (IPTG). Even in the stable transfection of Rat-1 cells, the promoter activity of the integrated pEF-LAC was much higher than that of the RSV-LTR and regulated in an IPTG-dependent manner. These results suggest that pEF-LAC is a useful vector for the inducible high-level expression of the cloned gene in a variety of mammalian cells.

Use of the human elongation factor 1α promoter as a versatile and efficient expression system

We have characterized the promoter region of the human elongation factor 1α-encoding gene ( EF-1α) and developed a versatile expression system which has a wide host range and a high efficiency of gene expression. To identify the promoter region of the EF-1α gene necessary for efficient gene expression, we constructed four pEF-CAT plasmids that have the bacterial cat gene fused to four different sites of the human EF-1α gene: ( i) ligated to the end of the TATA box (pEF220-CAT); ( ii) ligated in exon 1 (pEF204-CAT and pEF233-CAT), and ( iii) ligated in exon 2 (pEF321-CAT). All the pEF-CAT plasmids were highly expressed in all the cell types tested, including fibroblasts and lymphoid cells. Plasmid pEF321-CAT, which contains the first exon and the first intron, gave the highest level of cat expression. Plasmids pEF204- and pEF233-CAT, which contain part of the first exon but do not contain the first intron, were less efficient in cat expression than was pEF321-CAT. Plasmid pEF220-CAT, which lacks both the first exon and the first intron, was the least efficient. Plasmid pEF321-CAT was several- to 100-fold more efficient in cat expression than plasmid pSV2-CAT depending on the recipient cell types. The promoter of pEF321 plasmid also directed the stable expression of the bacterial neo gene more efficiently than the promoter of the simian virus 40 (SV40) early gene or the long terminal repeat of Rous sarcoma virus. Using this system, the SV40 early gene and the cDNA encoding human CD4 were also expressed efficiently.