Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.

Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDC-TRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 self-differentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated a simpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

The innovative evolution of cancer gene and cellular therapies.

We provide an overview of the latest developments in cancer gene therapy--from the bench to early-stage clinical trials. We describe the most recent work of worldwide teams including experienced scientists and clinicians, reflecting the recent emergence of gene therapy from the 'Valley of Death'. The treatment efficacy of clinical gene therapy has now been shown in a number of diseases including cancer and we are observing a renewed interest by big pharmaceutical and biotechnology companies most obviously demonstrated by Amgen's acquisition of Biovex for up to USD$1 billion. There is an opportunity to be cautiously hopeful regarding the future of gene therapy in the clinic and we review here some of the most recent progress in the field.

Selection for Evi1 activation in myelomonocytic leukemia induced by hyperactive signaling through wild-type NRas.

Activation of NRas signaling is frequently found in human myeloid leukemia and can be induced by activating mutations as well as by mutations in receptors or signaling molecules upstream of NRas. To study NRas-induced leukemogenesis, we retrovirally overexpressed wild-type NRas in a murine bone marrow transplantation (BMT) model in C57BL/6J mice. Overexpression of wild-type NRas caused myelomonocytic leukemias ∼3 months after BMT in the majority of mice. A subset of mice (30%) developed malignant histiocytosis similar to mice that received mutationally activated NRas(G12D)-expressing bone marrow. Aberrant Ras signaling was demonstrated in cells expressing mutationally active or wild-type NRas, as increased activation of Erk and Akt was observed in both models. However, more NRas(G12D) were found to be in the activated, GTP-bound state in comparison with wild-type NRas. Consistent with observations reported for primary human myelomonocytic leukemia cells, Stat5 activation was also detected in murine leukemic cells. Furthermore, clonal evolution was detected in NRas wild-type-induced leukemias, including expansion of clones containing activating vector insertions in known oncogenes, such as Evi1 and Prdm16. In vitro cooperation of NRas and Evi1 improved long-term expansion of primary murine bone marrow cells. Evi1-positive cells upregulated Bcl-2 and may, therefore, provide anti-apoptotic signals that collaborate with the NRas-induced proliferative effects. As activation of Evi1 has been shown to coincide with NRAS mutations in human acute myeloid leukemia, our murine model recapitulates crucial events in human leukemogenesis.

Lentiviral vectors for induction of self-differentiation and conditional ablation of dendritic cells.

Development of lentiviral vectors (LVs) in the field of immunotherapy and immune regeneration will strongly rely on biosafety of the gene transfer. We demonstrated previously the feasibility of ex vivo genetic programming of mouse bone marrow precursors with LVs encoding granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), which induced autonomous differentiation of long-lived dendritic cells (DCs), referred to as self-differentiated myeloid-derived antigen-presenting-cells reactive against tumors (SMART-DCs). Here, LV biosafety was enhanced by using a DC-restricted and physiological promoter, the major histocompatibility complex (MHC) II promoter, and including co-expression of the herpes simplex virus-thymidine kinase (sr39HSV-TK) conditional suicide gene. Tricistronic vectors co-expressing sr39HSV-TK, GM-CSF and IL-4 transcriptionally regulated by the MHCII promoter or the ubiquitous cytomegalovirus (CMV) promoter were compared. Despite the different gene transfer effects, such as the kinetics, levels of transgene expression and persistency of integrated vector copies, both vectors induced highly viable SMART-DCs, which persisted for at least 70 days in vivo and could be ablated with the pro-drug Ganciclovir (GCV). SMART-DCs co-expressing the tyrosine-related protein 2 melanoma antigen administered subcutaneously generated antigen-specific, anti-melanoma protective and therapeutic responses in the mouse B16 melanoma model. GCV administration after immunotherapy did not abrogate DC vaccination efficacy. This demonstrates proof-of-principle of genetically programmed DCs that can be ablated pharmacologically.

Permanent, lowered HLA class I expression using lentivirus vectors with shRNA constructs: Averting cytotoxicity by alloreactive T lymphocytes.

Transplantation of many tissues requires histocompatibility matching of human leukocyte antigens (HLA) to prevent graft rejection, to reduce the level of immunosuppression needed to maintain graft survival, and to minimize the risk of graft-versus-host disease, particularly in the case of bone marrow transplantation. However, recent advances in fields of gene delivery and genetic regulation technologies have opened the possibility of engineering grafts that display reduced levels of HLA expression. Suppression of HLA expression could help to overcome the limitations imposed by extensive HLA polymorphisms that restrict the availability of suitable donors, necessitate the maintenance of large donor registries, and complicate the logistics of procuring and delivering matched tissues and organs to the recipient. Accordingly, we investigated whether knockdown of HLA by RNA interference (RNAi), a ubiquitous regulatory system that can efficiently and selectively inhibit the expression of specific gene products, would enable allogeneic cells to evade immune recognition. For efficient and stable delivery of short hairpin-type RNAi constructs (shRNA), we employed lentivirus-based gene transfer vectors, which provide a delivery system that can achieve integration into genomic DNA, thereby permanently modifying transduced graft cells. Our results show that lentivirus-mediated delivery of shRNA targeting pan-Class I and allele-specific HLA can achieve efficient and dose-dependent reduction in surface expression of HLA in human cells, associated with enhanced resistance to alloreactive T lymphocyte-mediated cytotoxicity, while avoiding MHC-non-restricted killing. We hypothesize that RNAi-induced silencing of HLA expression has the potential to create histocompatibility-enhanced, and, eventually, perhaps "universally" compatible cellular grafts.

Immunotherapy with acute leukemia cells modified into antigen-presenting cells: ex vivo culture and gene transfer methods.

Adult patients with acute leukemia have, in general, a poor prognosis, with long-term, disease-free survival achieved in only approximately one-third of cases. One of the proposed mechanisms for this poor overall response is the inability of the immune system to detect and eliminate residual malignant leukemia cells, which subsequently serve as a source of leukemic relapse. This review discusses the rationale of immunotherapy for acute leukemia and presents in vitro and in vivo model systems that were devised for pre-B acute lymphocytic leukemia (ALL) and acute myeloid leukemia (AML). New advances in the ex vivo manipulation of acute leukemia cells are presented, which attempt to modify these cells into functional antigen-presenting cells. These cells can then be used as autologous vaccines at the time of minimal residual disease after standard chemotherapy, to stimulate host immune responses against their own leukemia cells. The various approaches toward this aim include incubation of leukemia cells with cytokines or growth factors and gene manipulation of these cells. In particular, ex vivo culture of ALL cells with CD40 ligand, incubation of AML cells with granulocyte-macrophage colony-stimulating factor and interleukin-4 (GM-CSF/IL-4) and lentiviral transduction of ALL and AML cells for expression of immunomodulators (CD80 and GM-CSF) are current approaches under investigation for the development of autologous acute leukemia cell vaccines.

Transduction of acute myeloid leukemia cells with third generation self-inactivating lentiviral vectors expressing CD80 and GM-CSF: effects on proliferation, differentiation, and stimulation of allogeneic and autologous anti-leukemia immune responses.

Acute myeloid leukemia (AML) patients treated with available therapies achieve remission in approximately 60% of cases, but the long-term event-free survival is less than 30%. Use of immunotherapy during remission is a potential approach to increase survival. We propose to develop cell vaccines by genetic modification of AML cells with CD80, an essential T cell costimulator that is lacking in the majority of AML cases, and GM-CSF, to induce proliferation and activation of professional antigen-presenting cells. Here, we evaluated third generation self inactivating (SIN) lentiviral vectors, which have the potential advantage of improved safety. CD80 and GM-CSF expression by these vectors was higher than that reported with second generation vectors (Stripecke et al, Blood 2000; 96: 1317-1326). In some cases, endogenous GM-CSF expression by transduced AML cells induced phenotypic changes consistent with the maturation of leukemia blasts into antigen-presenting cells. Further, in all cases studied, GM-CSF expression was associated with higher proliferation and cell viability. Allogeneic and autologous mixed lymphocyte reactions performed with transduced irradiated AML cells expressing CD80 and/or GM-CSF demonstrated that expression of either transgene enhanced T cell activation. These pre-clinical data demonstrate the potential feasibility of third generation SIN vectors for use in AML immunotherapy.

Receptor-specific targeting mediated by the coexpression of a targeted murine leukemia virus envelope protein and a binding-defective influenza hemagglutinin protein.

The entry of retroviral vectors into cells requires two events: binding to a cell surface receptor and the subsequent fusion of viral and cellular membranes. The host range of a vector is therefore determined largely by the receptor specificity of the fusion protein contained in the outer viral envelope. Previous attempts to generate targeted retroviral vectors have included the addition of targeting ligands to the murine leukemia virus envelope protein (MuLV Env). Although such proteins frequently display modified cell-binding characteristics, the interaction with the targeted receptors fails to trigger virus-cell fusion. Here, we report the use of a binding-defective but fusion-competent hemagglutinin (HA) protein to complement the fusion defect in a chimeric MuLV Env targeted to the Flt-3 receptor. Retroviral vectors containing both proteins showed enhanced transduction of cells expressing Flt-3, which was abrogated by preincubating the target cells with soluble Flt-3 ligand. Furthermore, the fusion function of HA was absolutely required. These data demonstrate that it is possible to separate the binding and fusion events of retroviral entry, using two separate proteins, and suggest that varying the binding protein component in this scheme may allow a general strategy for targeting retroviral vectors.

Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage- colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses.

Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage-colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (10(8)-10(9) infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80(+)). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80(+)). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/10(6) cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/10(6) cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4-immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses. (Blood. 2000;96:1317-1326)

Combination of CD80 and granulocyte-macrophage colony-stimulating factor coexpression by a leukemia cell vaccine: preclinical studies in a murine model recapitulating Philadelphia chromosome-positive acute lymphoblastic leukemia.

Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL) is a highly aggressive malignancy caused by the bcr-abl translocation oncogene. To explore alternative treatments for Ph+ ALL we tested gene-modified cell vaccines in the BALB/c-derived BM185 leukemia model. We compared the efficacy of BM185 cell vaccine expressing CD80 alone or in combination with IL-2 or GM-CSF. Mice injected with viable BM185 leukemia cells modified to express CD80 and GM-CSF (BM185/CD80+GM-CSF) showed the highest leukemia rejection rates. Cell vaccines consisting of irradiated BM185/CD80+GM-CSF cells administered subcutaneously stimulated a potent cytotoxic T lymphocyte (CTL) response against parental BM185. Histological examination of the vaccination site showed a large concentration of immune cells. Administration of the BM185/CD80+GM-CSF cell vaccine before intravenous challenge with parental cells caused strong inhibition of leukemia development. Vaccination after subcutaneous challenge with BM185 cells caused efficient elimination of leukemia promoting 40-60% long-term survival rates. The immunization efficacy of the BM185/CD80+ GM-CSF cell vaccine was directly correlated with the percentage of cells expressing the transgenes. In all, this preclinical study shows that leukemia cell vaccines coexpressing CD80 and GM-CSF can potentially be explored for immunotherapy in Ph+ ALL patients.

Immune response to green fluorescent protein: implications for gene therapy.

Green fluorescent protein (GFP) is a widely used intracellular reporter molecule to assess gene transfer and expression. A potential use for GFP is as a co-expressed marker, to select and enrich gene-modified cells by flow cytometry. Processed peptides derived from GFP and presented by the major histocompatibility complex on the cell surface could potentially induce T cell immune responses against GFP+ cells. Thus, clinical application of GFP is premature, since in vivo studies on its immunogenicity are lacking. Therefore, we investigated immune responses against EGFP (enhanced-GFP) in two transplantable murine models: the BALB/c (H-2d) BM185 pre-B leukemia and the C57BL/6 (H-2b) EL-4 T cell lymphoma. BM185 and EL-4 cell lines modified to express high levels of EGFP showed drastic reduction of disease development when transplanted into immunocompetent mice. BM185/ EGFP did lead to rapid development of disease in immunodeficient Nu/Nu mice. Mice surviving BM185/EGFP leukemia challenge developed high cytotoxic T lymphocyte (CTL) responses against EGFP-expressing cells. Furthermore, immune stimulation against BM185/EGFP cells could also be induced by immunization with EGFP+ transduced dendritic cells. The effects of the co-expression of EGFP and immunomodulators (CD80 plus GM-CSF) were also investigated as an irradiated leukemia vaccine. EGFP co-expression by the vaccine did not interfere with the development of CTLs against the parental leukemia or with the anti-leukemia response in vivo. These results indicate that the immune response against EGFP may interfere with its applicability in gene insertion/replacement strategies but could potentially be employed for leukemia cell vaccines.

Stable transduction of quiescent CD34(+)CD38(-) human hematopoietic cells by HIV-1-based lentiviral vectors.

We compared the efficiency of transduction by an HIV-1-based lentiviral vector to that by a Moloney murine leukemia virus (MLV) retroviral vector, using stringent in vitro assays of primitive, quiescent human hematopoietic progenitor cells. Each construct contained the enhanced green fluorescent protein (GFP) as a reporter gene. The lentiviral vector, but not the MLV vector, expressed GFP in nondivided CD34(+) cells (45.5% GFP+) and in CD34(+)CD38(-) cells in G0 (12.4% GFP+), 48 hr after transduction. However, GFP could also be detected short-term in CD34(+) cells transduced with a lentiviral vector that contained a mutated integrase gene. The level of stable transduction from integrated vector was determined after extended long-term bone marrow culture. Both MLV vectors and lentiviral vectors efficiently transduced cytokine-stimulated CD34(+) cells. The MLV vector did not transduce more primitive, quiescent CD34(+)CD38(-) cells (n = 8). In contrast, stable transduction of CD34(+)CD38(-) cells by the lentiviral vector was seen for over 15 weeks of extended long-term culture (9.2 +/- 5.2%, n = 7). GFP expression in clones from single CD34(+)CD38(-) cells confirmed efficient, stable lentiviral transduction in 29% of early and late-proliferating cells. In the absence of growth factors during transduction, only the lentiviral vector was able to transduce CD34(+) and CD34(+)CD38(-) cells (13.5 +/- 2.5%, n = 11 and 12.2 +/- 9.7%, n = 4, respectively). The lentiviral vector is clearly superior to the MLV vector for transduction of quiescent, primitive human hematopoietic progenitor cells and may provide therapeutically useful levels of gene transfer into human hematopoietic stem cells.

Gene delivery to human B-precursor acute lymphoblastic leukemia cells.

Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus-type 1 (HIV-1)-based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1-based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.

Immune response to Philadelphia chromosome-positive acute lymphoblastic leukemia induced by expression of CD80, interleukin 2, and granulocyte-macrophage colony-stimulating factor.

We examined the potential of generating an immune response against Philadelphia chromosome-positive acute lymphoblastic leukemia. The immunostimulatory molecules chosen for this study were the cytokines IL-2 and GM-CSF and the costimulatory ligand CD80 (B7.1). We used a murine model based on a BALB/c pre-B cell line, BM185wt, in which leukemia is induced by the p185 BCR-ABL oncogenic product, which reproduces Philadelphia chromosome-positive ALL. BM185wt cells were transduced with retroviral vectors and the transduced clones expressing mIL-2, mGM-CSF, or mCD80 were used for challenge. Expression of the immunomodulators by BM185 cells was correlated with delay in leukemia development in immunocompetent mice, but not in immunodeficient mice, indicating an immune response against the modified leukemia cells. Expression of CD80 caused leukemia rejection in 50% of the cohort, which was associated with the CD4+ and CD8+ T cell-dependent development of anti-leukemia cytotoxic T lymphocytes. Furthermore, mice surviving the BM185/CD80 challenge or preimmunized with irradiated BM185/CD80 cells developed an immune response against subsequent challenge with the parental leukemia. These studies provide evidence that immunotherapeutic approaches can be developed for the treatment of ALL.

Regulated poly(A) tail shortening in somatic cells mediated by cap-proximal translational repressor proteins and ribosome association.

The poly(A) tail plays an important role in translation initiation. We report the identification of a mechanism that operates in mammalian somatic cells, and couples mRNA poly(A) tail length with its translation state. The regulation of human ferritin L-chain mRNA by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) is subject to this mechanism: translational repression imposed by IRP binding to the IRE of ferritin L-chain mRNA induces poly(A) tail shortening. For the accumulation of mRNAs with short poly(A) tails, IRP binding to an IRE per se is not sufficient, but must cause translational repression. Interestingly, puromycin and verrucarin (general translation inhibitors that dissociate mRNAs from ribosomes) mimick the negative effect of the specific translational repressor proteins on poly(A) tail length, whereas cycloheximide and anisomycin (general translation inhibitors that maintain the association between mRNAs and ribosomes) preserve long poly(A) tails. Thus, the ribosome association of the mRNA appears to represent the critical determinant. These findings identify a novel mechanism of regulated polyadenylation as a consequence of translational control. They reveal differences in poly(A) tail metabolism between polysomal and mRNP-associated mRNAs. A possible role of this mechanism in the maintenance of translational repression is discussed.

Mitochondrial glutamate dehydrogenase from Leishmania tarentolae is a guide RNA-binding protein.

To identify specific proteins interacting with guide RNAs (gRNAs) in mitochondrial ribonucleoprotein complexes from Leishmania tarentolae, fractionated and unfractionated mitochondrial extracts were subjected to UV cross-linking with added labeled gRNA and also with [alpha-32P]UTP-labeled endogenous RNA. An abundant 110-kDa protein (p110) localized in the T-V complex, which sediments in glycerol gradients at the leading edge of the 10S terminal uridylyltransferase peak, was found to interact with both types of labeled RNAs. The p110 protein was gel isolated and subjected to microsequence analysis, and the gene was cloned. The sequence revealed significant similarity with mitochondrial glutamate dehydrogenases. A polyclonal antiserum was raised against a recombinant fragment of the p110 gene and was used to demonstrate a stable and specific gRNA-binding activity by coimmunoprecipitation and competitive gel shift analyses. Complex formation was strongly inhibited by competition with poly(U) or by deletion or substitution of the gRNA 3' oligo(U) tail. Also, addition of a 3' oligo(U) tail to an unrelated transcript was sufficient for p110 binding. Both the gRNA-binding activity of the p110 protein and in vitro gRNA-independent and gRNA-dependent uridine insertion activities in the mitochondrial extract were inhibited by high concentrations of dinucleotides.

Proteins binding to 5' untranslated region sites: a general mechanism for translational regulation of mRNAs in human and yeast cells.

We demonstrate that a bacteriophage protein and a spliceosomal protein can be converted into eukaryotic translational repressor proteins. mRNAs with binding sites for the bacteriophage MS2 coat protein or the spliceosomal human U1A protein were expressed in human HeLa cells and yeast. The presence of the appropriate binding protein resulted in specific, dose-dependent translational repression when the binding sites were located in the 5' untranslated region (UTR) of the reporter mRNAs. Neither mRNA export from the nucleus to the cytoplasm nor mRNA stability was demonstrably affected by the binding proteins. The data thus reveal a general mechanism for translational regulation: formation of mRNA-protein complexes in the 5' UTR controls translation initiation by steric blockage of a sensitive step in the initiation pathway. Moreover, the findings establish the basis for novel strategies to study RNA-protein interactions in vivo and to clone RNA-binding proteins.

Translational repression by the human iron-regulatory factor (IRF) in Saccharomyces cerevisiae.

The regulation of the synthesis of ferritin and erythroid 5-aminolevulinate synthase in mammalian cells is mediated by the interaction of the iron regulatory factor (IRF) with a specific recognition site, the iron responsive element (IRE), in the 5' untranslated regions (UTRs) of the respective mRNAs. A new modular expression system was designed to allow reconstruction of this regulatory system in Saccharomyces cerevisiae. This comprised two components: a constitutively expressed reporter gene (luc; encoding luciferase) preceded by a 5' UTR including an IRE sequence, and an inducibly expressed cDNA encoding human IRF. Induction of the latter led to the in vivo synthesis of IRF, which in turn showed IRE-binding activity and also repressed translation of the luc mRNA bearing an IRE-containing 5' UTR. The upper stem-loop region of an IRE, with no further IRE-specific flanking sequences, sufficed for recognition and repression by IRF. Translational regulation of IRE-bearing mRNAs could also be demonstrated in cell-free yeast extracts. This work defines a minimal system for IRF/IRE translational regulation in yeast that requires no additional mammalian-specific components, thus providing direct proof that IRF functions as a translational repressor in vivo. It should be a useful tool as the basis for more detailed studies of eukaryotic translational regulation.

The human U1 snRNP-specific U1A protein inhibits polyadenylation of its own pre-mRNA.

Human, mouse, and Xenopus mRNAs encoding the U1 snRNP-specific U1A protein contain a conserved 47 nt region in their 3' untranslated regions (UTRs). In vitro studies show that human U1A protein binds to two sites within the conserved region that resemble, in part, the previously characterized U1A-binding site on U1 snRNA. Overexpression of human U1A protein in mouse cells results in down-regulation of endogenous mouse U1A mRNA accumulation. In vitro and in vivo experiments demonstrate that excess U1A protein specifically inhibits polyadenylation of pre-mRNAs that contain the conserved 3' UTR from human U1A mRNA. Thus, U1A protein regulates the production of its own mRNA via a mechanism that involves pre-mRNA binding and inhibition of polyadenylation.