Post-transcriptional Regulatory Element Research Articles

Adenoviral-mediated, high-level, cell-specific transgene expression: a SYN1-WPRE cassette mediates increased transgene expression with no loss of neuron specificity.

Viral vectors are excellent tools for studying gene function in the brain, although a limitation has been the ability to effectively target transgene expression to specific neuronal populations. This generally cannot be overcome by the use of neuron-specific promoters, as most are too large to be used with current viral vectors and expression from these promoters is often relatively weak. We therefore developed a composite expression cassette, comprising 495 bp of the weak human SYN1 (synapsin-1) promoter and 800 bp of the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). Studies in hippocampal cultures, organotypic cultures, and in vivo showed that the 3' addition of the WPRE to the SYN1 element greatly increased enhanced green fluorescent protein expression levels with no loss of neuronal specificity. In vivo studies also showed that transgene expression was enhanced with no loss of neuronal specificity in dentate-gyrus neurons for at least 6 weeks following transfection. Therefore, unlike most powerful promoter systems, which mediate expression in neurons and glia, this SYN1-WPRE cassette can target powerful long-term transgene expression to central nervous system neurons when delivered at relatively low titers of adenovirus. Its use should therefore facilitate both gene therapy studies and investigations of neuronal gene function.

87. The Woodchuck Post-Transcriptional Regulatory Element WPRE Can Overcome Restriction in Retroviral Gene Expression in Embryonic Stem Cells

87. The Woodchuck Post-Transcriptional Regulatory Element WPRE Can Overcome Restriction in Retroviral Gene Expression in Embryonic Stem Cells

603. Towards High-Level and Cell Specific Transgene Expression: The Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) Increases Expression Mediated by the Synapsin Promoter with No Loss of Neuron Specificity

603. Towards High-Level and Cell Specific Transgene Expression: The Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE) Increases Expression Mediated by the Synapsin Promoter with No Loss of Neuron Specificity

High-Level Transduction and Gene Expression in Hematopoietic Repopulating Cells Using a Human Imunodeficiency Virus Type 1-Based Lentiviral Vector Containing an Internal Spleen Focus Forming Virus Promoter

Prolonged exposure of human hematopoietic stem cells (HSC) to growth factors for efficient transduction by murine oncoretroviral vectors has major detrimental effects on repopulating activity. In this study, we have used a vesicular stomatitis virus G envelope protein (VSV-G)-pseudotyped human immunodeficiency virus type 1 (HIV-1) lentiviral-based vector system to transduce cord blood (CB) CD34+ cells over a limited time period (< or =24 hours). Under these conditions, significant gene marking was observed in engrafted human lymphoid, myeloid, and progenitor cells in all transplanted Severe Combined Immunodeficient (SCID) mice. To enhance the level of gene expression in hematopoietic cells, we also generated a series of lentiviral vectors incorporating the spleen focus forming virus (SFFV) long terminal repeat (LTR) sequences, and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). By including the central polypurine tract (cPPT) sequence of HIV-1 we were then able to achieve high levels of transduction (over 80%) and gene expression in vivo after a single exposure to viral supernatant. These results demonstrate that lentiviral vectors are highly effective for gene transfer to human HSC, and that SFFV regulatory sequences can be successfully incorporated to enhance the long-term expression of a transgene in primary human hematopoietic cells in vivo.

Characterization of Lentiviral Vector-Mediated Gene Transfer in Adult Mouse Brain

Lentiviral vectors are promising tools for gene transfer into the central nervous system. We have characterized in detail transduction with human immunodeficiency virus type 1 (HIV-1)-derived vectors encoding enhanced green fluorescent protein (eGFP) in the adult mouse brain. Different brain regions such as the striatum, hippocampus, and the lateral ventricle were targeted. The eGFP protein was transported anterogradely in the nigrostriatal pathway, but we have found no evidence of transport of the lentiviral vector particle. The performance levels of the different generations of packaging and transfer plasmid were compared. Omission of the accessory genes from the packaging plasmid resulted in a modest decrease in transgene expression. Inclusion of the woodchuck hepatitis posttranscriptional regulatory element, on the one hand, and the central polypurine tract and termination sequences, on the other hand, in the transfer vector each resulted in a 4- to 5-fold increase in transgene expression levels. Combination of both elements enhanced expression levels more than the sum of the individual components, suggesting a synergistic effect. In the serum of mice injected with lentiviral vectors a humoral response to vector proteins was detected, but this did not compromise transgene expression. Immune response to the transgene was found only in a minority of the animals.

CRM1-dependent function of a cis-acting RNA export element.

Viruses often contain cis-acting RNA elements, which facilitate the posttranscriptional processing and export of their messages. These elements fall into two classes distinguished by the presence of either viral or cellular RNA binding proteins. To date, studies have indicated that the viral proteins utilize the CRM1-dependent export pathway, while the cellular factors generally function in a CRM1-independent manner. The cis-acting element found in the woodchuck hepatitis virus (WHV) (the WHV posttranscriptional regulatory element [WPRE]) has the ability to posttranscriptionally stimulate transgene expression and requires no viral proteins to function. Conventional wisdom suggests that the WPRE would function in a CRM1-independent manner. However, our studies on this element reveal that its efficient function is sensitive to the overexpression of the C terminus of CAN/Nup214 and treatment with the antimicrobial agent leptomycin B. Furthermore, the overexpression of CRM1 stimulates WPRE activity. These results suggest a direct role for CRM1 in the export function of the WPRE. This observation suggests that the WPRE is directing messages into a CRM1-dependent mRNA export pathway in somatic mammalian cells.

Design of HIV Vectors for Efficient Gene Delivery into Human Hematopoietic Cells

Vectors derived from human immunodeficiency virus (HIV) hold promise for efficient gene delivery into human hematopoietic cells. In this study, HIV vectors containing different combinations of cis-acting elements, including the HIV central flap sequence, and the woodchuck posttranscriptional regulatory element (WPRE) in combination with two different promoters, were used to transduce primary human lymphocytes and cord blood CD34+ progenitor cells. The effect of these elements on the transduction efficiency and transgene expression was systematically evaluated. The results demonstrate that with the combination of flap, WPRE sequences, and the promoter derived from spleen focus-forming virus (SFFV), a foreign gene can be efficiently delivered into primary human T lymphocytes and cord blood CD34+ cells. The study establishes the parameters for proper vector design to efficiently deliver foreign genes into human hematopoietic cells.

Lentiviral vectors for efficient transduction of isolated primary quiescent hepatocytes

Background / Aims : Lentiviral vectors were designed to obtain efficient transduction of primary quiescent hepatocytes. Methods : A hepatitis B virus (HBV) fragment containing enhancers and posttranscriptional regulatory element was used to increase expression levels. The human immunodeficiency virus (HIV) central polypurine tract (PPT) was used to increase transduction of quiescent cells. HBV elements were incorporated downstream and the HIV PPT was incorporated upstream of green fluorescent protein expression cassettes in third generation self inactivating lentiviral vectors. Results : The HBV fragment increased mean fluorescence of transduced HepG2 hepatoma cells 4.3±1.7-fold and 2.3–6.0-fold in various other cell types. A role of HBV×protein in the function of the HBV element was excluded. The HBV element increased the number of transducing units per pg of HIV p24 twofold. The unmodified lentiviral vector transduced 5±1% of cultured quiescent primary rat hepatocytes, HBV elements increased transduction to 54±13% and increased fluorescence 2.8±0.6-fold. The PPT increased transduction to 47±11% and increased fluorescence 2.3±0.4-fold. The vector with PPT and HBV elements transduced 68±10% of hepatocytes and increased fluorescence synergistically, 17±6 fold. Conclusions : This study shows that HBV elements or HIV PPT are required for efficient transduction of primary hepatocytes.

Lentiviral vector-mediated transduction of murine CD34(-) hematopoietic stem cells.

Efficient gene transfer into murine hematopoietic stem cells (HSCs) provides a powerful tool for exploring hematopoietic stem cell biology. In this study, we evaluated the efficiency of lentiviral vector-mediated gene transfer into murine CD34(-/low)c-Kit(+)Sca-1(+)Lin(-) (CD34(-) KSL) cells that are highly enriched for HSCs. FACS-sorted CD34(-) KSL cells were transduced with the vesicular stomatitis virus G glycoprotein-pseudotyped HIV-1-based lentiviral vector containing the green fluorescent protein (GFP) gene under the control of the cytomegalovirus promoter, and then 50 transduced cells were transplanted into lethally irradiated mice. Transduction efficiency was assessed by FACS analysis for GFP expression in peripheral blood (PB) cells. FACS-sorted GFP(+) KSL bone marrow (BM) cells from primary recipients were used for secondary transplantation, and GFP expression in PB cells of reconstituted mice was analyzed by FACS. GFP expression was detected in PB cells of all primary recipients (n = 10) at an average of 40% (range 26-58%) when the lentiviral vector containing the woodchuck hepatitis virus posttranscriptional regulatory element was used. GFP(+) cells were found in multilineage cells in PB, BM, spleen, and thymus for at least 8 months posttransplantation. In secondary recipients, donor-derived GFP(+) KSL BM cells could reconstitute and GFP expression was detected in both myeloid and lymphoid cells in PB. Our results indicate that lentiviral vectors can efficiently transduce highly enriched murine HSCs and sustain long-term expression of the transgene in the multilineage differentiated progeny in reconstituted mice.

Enhancement of green fluorescent protein expression in adeno-associated virus with the woodchuck hepatitis virus post-transcriptional regulatory element.

AbstractSince the advent of recombinant DNA technology, maximization of exogenous gene expression has been an important issue for molecular biologists. Efforts at enhancing transgene expression have mostly been directed at improving the efficiency of delivery and increasing levels of transcription and translation. Less progress has been made in the application of post-transcriptional methods for improving gene expression. Here is described the use of an element derived from the woodchuck hepatitis virus (WHV) that possesses the ability to enhance the expression of heterologous genes post-transcriptionally. Green fluorescent protein (GFP) is frequently employed as a fusion protein to enable the detection, visualization, and quantification of molecules under study. Problems of expression are often encountered when using this strategy, making the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) a useful addition to vectors designed to express fusion proteins. This chapter discusses general issues of cloning and placement of the WPRE when designing such vectors.KeywordsGreen Fluorescent ProteinGreen Fluorescent Protein ExpressionEnhance Green Fluorescent Protein ExpressionGreen Fluorescent Protein Fusion ProteinHEPES Buffer SalineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Role of polypyrimidine tract binding protein in the function of the hepatitis B virus posttranscriptional regulatory element.

The hepatitis B virus posttranscriptional regulatory element (PRE) is an RNA element that increases the expression of unspliced mRNAs, apparently by facilitating their export from the nucleus. We have identified a cellular protein that binds to the PRE as the polypyrimidine tract binding protein (PTB), which shuttles rapidly between the nucleus and the cytoplasm. Mutants of the PRE with mutations in PTB binding sites show markedly decreased activity, while cells that stably overexpress PTB show increased PRE-dependent gene expression. Export of PTB from the nucleus, like PRE function, is blocked by a mutant form of Ran binding protein 1 but not by leptomycin B. Therefore, PTB is important for PRE activity and appears to function as an export factor for PRE-containing mRNAs.

Remote delivery of rAAV-GFP to the rat brainstem through the recurrent laryngeal nerve.

To demonstrate that a recombinant adeno-associated viral vector (rAAV) carrying the gene for green fluorescent protein (GFP) could be delivered to the rat brainstem by remote injection into the recurrent laryngeal nerve. rAAV-GFP is a serotype 2 adeno-associated vector containing the cDNA of GFP and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under the control of the CAG promoter (obtained from Matthew During, PhD, Thomas Jefferson Medical College). Five microliters or 10 microL of 1.4 x 109 particles/microL of rAAV-GFP were injected into the right recurrent laryngeal nerve of adult Sprague-Dawley rats. Rats were killed and perfused at 3 (n = 3) and 11 weeks (n = 3). Brainstems were removed and cryosectioned. Fluorescent in-situ hybridization (FISH) was performed on cryosections from animals killed at 3 weeks using a cDNA probe for woodchuck polyribosomal enzyme within the rAAV vector. In a third group (n = 2), Fluoro-Gold (Fluorochrome, Inc., Denver, CO) was injected into the right thyroarytenoid muscle for comparison of neuronal uptake distribution. These rats were killed and perfused at 3 weeks. The presence of GFP was noted in neurons throughout the medulla of all rat brainstems after unilateral rAAV-GFP injection at both 3 and 11 weeks. In contrast to the Fluoro-Gold, GFP was noted bilaterally and outside of the nucleus ambiguus. FISH confirmed the presence of virus within neurons expressing GFP at 3 weeks. Remote delivery of rAAV-GFP to the rat brainstem is possible through injection into the recurrent laryngeal nerve. This has important therapeutic implications for the future treatment of recurrent laryngeal nerve injury and neurodegenerative diseases.

Characterization of nuclear RNases that cleave hepatitis B virus RNA near the La protein binding site.

Hepatitis B virus (HBV) RNA is downregulated by inflammatory cytokines induced in the liver by adoptively transferred HBV-specific cytotoxic T lymphocytes (CTLs) and during murine cytomegalovirus (MCMV) infections of the livers of HBV transgenic mice. The disappearance of HBV RNA is tightly associated with the cytokine-induced proteolytic cleavage of a previously defined HBV RNA-binding protein known as La autoantigen. La binds to a predicted stem-loop structure at the 5' end of the posttranscriptional regulatory element of HBV RNA between nucleotides 1243 and 1333. In the present study, we searched for nuclear RNase activities that might be involved in HBV RNA decay. Nuclear extracts derived from control livers and CTL-injected and MCMV-infected livers were analyzed for the ability to cleave HBV RNA. Endonucleolytic activity that cleaved HBV RNA at positions 1269 to 1270 and 1271 to 1272, immediately 5' of the stem-loop bound by the La protein (positions 1272 to 1293), was detected. Furthermore, we provide evidence that the cytokine-dependent downregulation of HBV RNA following MCMV infection is temporally associated with the upregulation of the endonucleolytic activity herein described. Collectively, these results suggest a model in which the steady-state HBV RNA content is controlled by the stabilizing influence of La and the destabilizing influence of nuclear RNase activities.

Identification of a novel posttranscriptional regulatory element by using a rev- and RRE-mutated human immunodeficiency virus type 1 DNA proviral clone as a molecular trap.

Human immunodeficiency virus (HIV) and all other lentiviruses utilize the essential viral protein Rev, which binds to RRE RNA, to export their unspliced and partially spliced mRNAs from the nucleus. We used a rev- and RRE-defective HIV type 1 (HIV-1) molecular clone in complementation experiments to establish a method for the rapid isolation of posttranscriptional regulatory elements from the mammalian genome by selecting for rescue of virus replication. Viruses rescued by this method contained a novel element with homology to rodent intracisternal A-particle (IAP) retroelements. A functional element was contained within a 247-nucleotide fragment named RNA transport element (RTE), which was able to promote replication of the Rev- and RRE-defective HIV-1 in both human lymphoid cell lines and primary lymphocytes, demonstrating its potent posttranscriptional function. RTE was functional in many cell types, indicating that the cellular factors that recognize RTE are widely expressed and evolutionarily conserved. RTE also promoted RNA export from Xenopus oocyte nuclei. RTE-mediated RNA transport was CRM1 independent, and RTE did not show high affinity for binding to mRNA export factor TAP/NXF1. Since CRM1 and TAP/NXF1 are critical export receptors associated with the two recognized mRNA export pathways, these results suggest that RTE functions via a distinct export mechanism. Taken together, our results identify a novel posttranscriptional control element that uses a conserved cellular export mechanism.

High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors

Recent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus–based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1α and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1α promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1α promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3′ long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.

High-level transgene expression in human hematopoietic progenitors and differentiated blood lineages after transduction with improved lentiviral vectors

AbstractRecent experiments point to the great value of lentiviral vectors for the transduction of human hematopoietic stem cells (hHSCs). Vectors used so far, however, have been poorly satisfying in terms of either biosafety or efficiency of transgene expression. Herein is described the results obtained with human immunodeficiency virus–based vectors optimized in both of these aspects. It is thus shown that vectors containing the EF1α and, to a lesser extent, the phosphoglycerate kinase (PGK) promoter, govern high-level gene expression in human hematopoietic progenitors as well as derived hematopoietic lineages of therapeutic relevance, such as erythrocytes, granulocytes, monocytes, dendritic cells, and megakaryocytes. EF1α promoter-containing lentiviral vectors can also induce strong transgene expression in primary T lymphocytes isolated from peripheral blood. A self-inactivating design did not affect the performance of EF1α promoter-based vectors but significantly reduced expression from the PGK promoter. This negative effect could nevertheless be largely rescued by inserting the post-transcriptional regulatory element of woodchuck hepatitis virus upstream of the vector 3′ long terminal repeat. These results have important practical implications for the genetic treatment of lymphohematologic disorders as well as for the study of hematopoiesis via the lentivector-mediated modification of hHSCs.

Lentiviral vectors for enhanced gene expression in human hematopoietic cells.

Accumulated data indicate that current generation lentiviral vectors, which generally utilize an internal human cytomegalovirus (CMV) immediate early region enhancer-promoter to transcribe the gene of interest, are not yet optimized for efficient expression in human hematopoietic stem/progenitor cells (HSPCs). As a first step toward this goal, we constructed self-inactivating derivatives of the HIV-1-based transfer vector pHR' containing the enhanced green fluorescent protein (GFP) gene as reporter and the Woodchuck hepatitis virus posttranscriptional regulatory element (WPRE). GFP expression was driven by a variety of strong viral and cellular promoters, including the murine stem cell virus (MSCV) long terminal repeat (LTR), a Gibbon ape leukemia virus (GALV) LTR, the human elongation factor 1alpha (EF1alpha) promoter, the composite CAG promoter (consisting of the CMV immediate early enhancer and the chicken beta-actin promoter), and the human phosphoglycerate kinase 1 (PGK) promoter. In contrast to results obtained in human embryonic kidney 293T cells and fibrosarcoma HT1080 cells, in which the CMV promoter expressed GFP at the highest levels, significantly higher levels of GFP expression (3- to 5-fold) were achieved with the MSCV LTR, the EF1alpha promoter, and the CAG promoter in the human HSPC line KG1a. Removal of the WPRE indicated that it stimulated GFP expression from all of the vectors in KG1a cells (up to 3-fold), although it only marginally improved the performance of the intron-containing EF1alpha and CAG promoters (<1.5-fold stimulation). The vectors using the MSCV LTR, the GALV LTR, and the PGK promoter were the most efficient at transducing primary human CD34+ cord blood progenitors under the conditions employed. High-level GFP expression in the NOD/SCID xenograft model was demonstrated with the pHR' derivative bearing the MSCV LTR. These new lentiviral vector backbones provide a basis for the rational design of improved delivery vehicles for human HSPC gene transfer applications.

Identification of an RNA element that confers post-transcriptional repression of connective tissue growth factor/hypertrophic chondrocyte specific 24 (ctgf/hcs24) gene: similarities to retroviral RNA-protein interactions.

The repressive effect of the 3'-untranslated region (3'-UTR) in human connective tissue growth factor/ hypertrophic chondrocyte specific 24 (ctgf/hcs24) mRNA on gene expression had been demonstrated in our previous study. Here, we identified a minimal RNA element in the 3'-UTR, which acts as a cis-acting element of structure-anchored repression (CAESAR). Deletion analyses of the 3'-UTR led us to minimize the element of 84 bases at the junction of the coding region and the 3'-UTR. The minimized RNA segment is predicted, and actually capable of forming a stable secondary structure in vitro. Mutational analyses disclosed a significant relationship between the predicted structure and repressive effect. The utility of CAESAR as a post-transcriptional regulatory element was represented by the fact that steady-state mRNA levels were not affected by CAESAR linked in cis, while protein levels from such a chimeric gene were markedly reduced. Of note, the CAESAR sequence exerted no effect, when it was placed upstream of the promoter. Finally, RNA gel electromobility-shift analyses demonstrated a nuclear factor that interacts with the folded CAESAR. Taken together, it was uncovered that CAESAR of ctgf is a novel post-transcriptional structured RNA regulatory element, probably acting through direct interactions with a nuclear factor as observed in retroviral RNA elements with certain proteins.

Influence of promoter and WHV post-transcriptional regulatory element on AAV-mediated transgene expression in the rat brain.

Recombinant adeno-associated viruses (rAAVs) can transduce several tissues, including the brain. However, in brain the duration of gene expression in different areas is variable, which has been ascribed to viral (CMV) promoter silencing in some regions over time. We have compared expression of enhanced green fluorescent protein (EGFP) in the nigrostriatal pathway of rats mediated by rAAVs containing the CMV or platelet-derived growth factor-beta chain (PDGF-beta) promoter. In addition, we studied the effects of the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) on transgene expression in vivo. The rAAV vectors containing the neuron-specific PDGF-beta chain promoter transduced significantly more dopaminergic neurons than titer-matched vectors carrying the CMV promoter. Moreover, the WPRE further increased EGFP expression, and a rAAV vector incorporating both the PDGF-beta chain promoter and the WPRE resulted in efficient EGFP expression in dopaminergic neurons and their projections in the striatum for at least 41 weeks after virus injection. Our results emphasize the importance of a strong tissue-specific promoter in achieving optimal transgene expression, not only in long-term but also in short-term studies where viral titers may be limiting. Furthermore, they suggest that incorporation of the WPRE into rAAVs, and possibly other types of vectors, is useful to enhance transgene expression in vivo.

AAV and RT-PCR: true or false?

Recombinant AAV vectors are capable of long-term gene expression with low toxicity in vivo (1). Therapeutic and sustained protein expression and correction of animal models of disease are now being reported, and human clinical trials are in progress. Several laboratories have evaluated AAV-mediated transduction by a variety of methods, including the technique of RT-PCR (2–13). However, there is a potential flaw with RT-PCR analysis in this context: Specifically, a false-positive signal can be obtained from contaminating AAV single-stranded DNA (ssDNA).