Human immunodeficiency virus tat gene transfer to the murine central nervous system using a replication-defective herpes simplex virus vector stimulates transforming growth factor beta 1 gene expression.

The high incidence of neurological disorders in patients afflicted with acquired immunodeficiency syndrome (AIDS) may result from human immunodeficiency virus type 1 (HIV-1) induction of chemotactic signals and cytokines within the brain by virus-encoded gene products. Transforming growth factor beta1 (TGF-beta1) is an immunomodulator and potent chemotactic molecule present at elevated levels in HIV-1-infected patients, and its expression may thus be induced by viral trans-activating proteins such as Tat. In this report, a replication-defective herpes simplex virus (HSV)-1 tat gene transfer vector, dSTat, was used to transiently express HIV-1 Tat in glial cells in culture and following intracerebral inoculation in mouse brain in order to directly determine whether Tat can increase TGF-beta1 mRNA expression. dSTat infection of Vero cells transiently transfected by a panel of HIV-1 long terminal repeat deletion mutants linked to the bacterial chloramphenicol acetyltransferase reporter gene demonstrated that vector-expressed Tat activated the long terminal repeat in a trans-activation response element-dependent fashion independent of the HSV-mediated induction of the HIV-1 enhancer, or NF-kappaB domain. Northern blot analysis of human astrocytic glial U87-MG cells transfected by dSTat vector DNA resulted in a substantial increase in steady-state levels of TGF-beta1 mRNA. Furthermore, intracerebral inoculation of dSTat followed by Northern blot analysis of whole mouse brain RNA revealed an increase in levels of TGF-beta1 mRNA similar to that observed in cultured glial cells transfected by dSTat DNA. These results provided direct in vivo evidence for the involvement of HIV-1 Tat in activation of TGF-beta1 gene expression in brain. Tat-mediated stimulation of TGF-beta1 expression suggests a novel pathway by which HIV-1 may alter the expression of cytokines in the central nervous system, potentially contributing to the development of AIDS-associated neurological disease.

Eradication of established intracranial rat gliomas by transforming growth factor beta antisense gene therapy.

Like human gliomas, the rat 9L gliosarcoma secretes the immunosuppressive transforming growth factor beta (TGF-beta). Using the 9L model, we tested our hypothesis that genetic modification of glioma cells to block TGF-beta expression may enhance their immunogenicity and make them more suitable for active tumor immunotherapy. Subcutaneous immunizations of tumor-bearing animals with 9L cells genetically modified to inhibit TGF-beta expression with an antisense plasmid vector resulted in a significantly higher number of animals surviving for 12 weeks (11/11, 100%) compared to immunizations with control vector-modified 9L cells (2/15, 13%) or 9L cells transduced with an interleukin 2 retroviral vector (3/10, 30%) (P < 0.001 for both comparisons). Histologic evaluation of implantation sites 12 weeks after treatment revealed no evidence of residual tumor. In vitro tumor cytotoxicity assays with lymph node effector cells revealed a 3- to 4-fold increase in lytic activity for the animals immunized with TGF-beta antisense-modified tumor cells compared to immunizations with control vector or interleukin 2 gene-modified tumor cells. These results indicate that inhibition of TGF-beta expression significantly enhances tumor-cell immunogenicity and supports future clinical evaluation of TGF-beta antisense gene therapy for TGF-beta-expressing tumors.

Specific activation in jun-transformed avian fibroblasts of a gene (bkj) related to the avian beta-keratin gene family.

We have analyzed differential gene expression in normal versus jun-transformed avian fibroblasts by using subtracted nucleic acid probes and differential nucleic acid hybridization techniques for the isolation of cDNA clones. One clone corresponded to a gene that was strongly expressed in a previously established quail (Coturnix japonica) embryo fibroblast line (VCD) transformed by a chimeric jun oncogene but whose expression was undetectable in normal quail embryo fibroblasts. Furthermore, the gene was expressed in quail or chicken fibroblast cultures that were freshly transformed by retroviral constructs carrying various viral or cellular jun alleles and in chicken fibroblasts transformed by the avian retrovirus ASV17 carrying the original viral v-jun allele. However, its expression was undetectable in a variety of established avian cell lines or freshly prepared avian fibroblast cultures transformed by other oncogenes or a chemical carcinogen. The nucleotide and deduced amino acid sequences of the cDNA clone were not identical to any sequence entries in the data bases but revealed significant similarities to avian beta-keratin genes; the highest degree of amino acid sequence identity was 63%. The gene, which we termed bkj, may represent a direct or indirect target for jun function.

Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.

Previous studies in transgenic mice and cultured cells have indicated that the major enhancer function for erythroid cell expression of the globin genes is provided by the heterodimeric basic-leucine zipper transcription factor NF-E2. Globin gene expression within cultured mouse erythroleukemia cells is highly dependent on NF-E2. To examine the requirement for this factor in vivo, we used homologous recombination in embryonic stem cells to generate mice lacking the hematopoietic-specific subunit, p45 NF-E2. The most dramatic aspect of the homozygous mutant mice was an absence of circulating platelets, which led to the death of most animals due to hemorrhage. In contrast, the effect of loss of NF-E2 on the erythroid lineage was surprisingly mild. Although neonates exhibited severe anemia and dysmorphic red-cell changes, probably compounded by concomitant bleeding, surviving adults exhibited only mild changes consistent with a small decrease in the hemoglobin content per cell. p45 NF-E2-null mice responded to anemia with compensatory reticulocytosis and splenomegaly. Globin chain synthesis was balanced, and switching from fetal to adult globins progressed normally. Although these findings are consistent with the substitution of NF-E2 function in vivo by one or more compensating proteins, gel shift assays using nuclear extracts from p45 NF-E2-null mice failed to reveal novel complexes formed on an NF-E2 binding site. Thus, regulation of globin gene transcription through NF-E2 binding sites in vivo is more complex than has been previously appreciated.

Fate of a redundant gamma-globin gene in the atelid clade of New World monkeys: implications concerning fetal globin gene expression.

Conclusive evidence was provided that gamma 1, the upstream of the two linked simian gamma-globin loci (5'-gamma 1-gamma 2-3'), is a pseudogene in a major group of New World monkeys. Sequence analysis of PCR-amplified genomic fragments of predicted sizes revealed that all extant genera of the platyrrhine family Atelidae [Lagothrix (woolly monkeys), Brachyteles (woolly spider monkeys), Ateles (spider monkeys), and Alouatta (howler monkeys)] share a large deletion that removed most of exon 2, all of intron 2 and exon 3, and much of the 3' flanking sequence of gamma 1. The fact that two functional gamma-globin genes were not present in early ancestors of the Atelidae (and that gamma 1 was the dispensible gene) suggests that for much or even all of their evolution, platyrrhines have had gamma 2 as the primary fetally expressed gamma-globin gene, in contrast to catarrhines (e.g., humans and chimpanzees) that have gamma 1 as the primary fetally expressed gamma-globin gene. Results from promoter sequences further suggest that all three platyrrhine families (Atelidae, Cebidae, and Pitheciidae) have gamma 2 rather than gamma 1 as their primary fetally expressed gamma-globin gene. The implications of this suggestion were explored in terms of how gene redundancy, regulatory mutations, and distance of each gamma-globin gene from the locus control region were possibly involved in the acquisition and maintenance of fetal, rather than embryonic, expression.

Modifications of RNA processing modulate the expression of hemoglobin genes.

The developmental changes in hemoglobin gene expression known as "switching" involve both the sequential activation and silencing of the individual globin genes. We postulated that in addition to changes in transcription, posttranscriptional mechanisms may be involved in modulating globin gene expression. We studied globin RNA transcripts in human adult erythroid cells (hAEC to analyze the mechanism of silencing of the embryonic epsilon-globin gene in the adult stage and in K562 erythroleukemic cells to analyze the inactive state of their adult beta-globin genes. In hAEC, which express primarily the beta-globin gene, quantitative PCR analysis shows that beta-mRNA exon levels are high and comparable among the three exons; the RNA transcripts corresponding to exons of the gamma-globin gene are low, with slight differences among the three exons. Although epsilon-globin is not expressed, epsilon-globin RNA transcripts are detected, with exon I levels comparable to that of gamma-globin exon I and much higher than epsilon-exons II and III. As expected, in K562 cells that express high levels of epsilon- and gamma-globin, epsilon- and gamma-mRNA levels are high, with comparable levels of exons I, II, and III. In K562 cells beta-mRNA levels are very low but beta-exon I levels are much higher than that of exons II or III. Moreover, all or most of the globin transcripts for the highly expressed globin genes in both cell types (gamma and beta in hAEC, epsilon and gamma in K562 cells) found in the cytoplasm or nucleus are correctly processed. The globin transcripts that are detected both in the cytoplasm and nucleus of cells without expression of the corresponding protein are largely unspliced (containing one or two intervening sequences). These studies suggest that in addition to changes in transcription rates, changes in completion or processing of globin RNA transcripts may contribute to the developmental regulation of the hemoglobin phenotype.

beta2 knockout mice develop parenchymal iron overload: A putative role for class I genes of the major histocompatibility complex in iron metabolism.

Hemochromatosis (HC) is an inherited disorder of iron absorption, mapping within the human major histocompatibility complex (MHC). We have identified a multigene system in the murine MHC that contains excellent candidates for the murine equivalent of the human HC locus and implicate nonclassical class I genes in the control of iron absorption. This gene system is characterized by multiple copies of two head-to-head genes encoded on opposite strands and driven by one common regulatory motif. This regulatory motif has a striking homology to the promoter region of the beta-globin gene, a gene obviously involved in iron metabolism and hence termed beta-globin analogous promoter (betaGAP). Upstream of the betaGAP sequence are nonclassical class I genes. At least one of these nonclassical class I genes, Q2, is expressed in the gastrointestinal tract, the primary site of iron absorption. Also expressed in the gastrointestinal tract and downstream of the betaGAP motif is a second set of putative genes, termed Hephaestus (HEPH). Based on these observations, we hypothesized that the genes that seem to be controlled by the betaGAP regulatory motifs would be responsible for the control of Fe absorption. As a test of this hypothesis, we predicted that mice which have altered expression of class I gene products, the beta2-microglobulin knockout mice, [beta2m(-/-)], would develop Fe overload. This prediction was confirmed, and these results indicate beta2m-associated proteins are involved in the control of intestinal Fe absorption.

Preselection of retrovirally transduced bone marrow avoids subsequent stem cell gene silencing and age-dependent extinction of expression of human β-globin in engrafted mice

Transcriptional silencing of genes transferred into hematopoietic stem cells poses one of the most significant challenges to the success of gene therapy. If the transferred gene is not completely silenced, a progressive decline in gene expression as the mice age often is encountered. These phenomena were observed to various degrees in mouse transplant experiments using retroviral vectors containing a human β-globin gene, even when cis-linked to locus control region derivatives. Here, we have investigated whether ex vivo preselection of retrovirally transduced stem cells on the basis of expression of the green fluorescent protein driven by the CpG island phosphoglycerate kinase promoter can ensure subsequent long-term expression of a cis-linked β-globin gene in the erythroid lineage of transplanted mice. We observed that 100% of mice (n = 7) engrafted with preselected cells concurrently expressed human β-globin and the green fluorescent protein in 20–95% of their RBC for up to 9.5 mo posttransplantation, the longest time point assessed. This expression pattern was successfully transferred to secondary transplant recipients. In the presence of β-locus control region hypersensitive site 2 alone, human β-globin mRNA expression levels ranged from 0.15% to 20% with human β-globin chains detected by HPLC. Neither the proportion of positive blood cells nor the average expression levels declined with time in transplanted recipients. Although suboptimal expression levels and heterocellular position effects persisted, in vivo stem cell gene silencing and age-dependent extinction of expression were avoided. These findings support the further investigation of this type of vector for the gene therapy of human hemoglobinopathies.

Chromatin structure and gene expression.

It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes. This is most obvious in the neighborhood of promoters and enhancers, where hypersensitivity to nucleases marks sites that no longer carry canonical nucleosomes, and to which transcription factors bind. To study the relationship between transcription factor binding and the generation of these hypersensitive regions, we mutated individual cis-acting regulatory elements within the enhancer that lies between the chicken beta- and epsilon-globin genes. Constructions carrying the mutant enhancer were introduced by stable transformation into an avian erythroid cell line. We observed that weakening the enhancer resulted in creation of two classes of site: those still completely accessible to nuclease attack and those that were completely blocked. This all-or-none behavior suggests a mechanism by which chromatin structure can act to sharpen the response of developmental systems to changing concentrations of regulatory factors. Another problem raised by chromatin structure concerns the establishment of boundaries between active and inactive chromatin domains. We have identified a DNA element at the 5' end of the chicken beta-globin locus, near such a boundary, that has the properties of an insulator; in test constructions, it blocks the action of an enhancer on a promoter when it is placed between them. We describe the properties and partial dissection of this sequence. A third problem is posed by the continued presence of nucleosomes on transcribed genes, which might prevent the passage of RNA polymerase. We show, however, that a prokaryotic polymerase can transcribe through a histone octamer on a simple chromatin template. The analysis of this process reveals that an octamer is capable of transferring from a position in front of the polymerase to one behind, without ever losing its attachment to the DNA.

Complexity of the erythroid transcription factor NF-E2 as revealed by gene targeting of the mouse p18 NF-E2 locus.

High-level globin expression in erythroid precursor cells depends on the integrity of NF-E2 recognition sites, transcription factor AP-1-like protein-binding motifs, located in the upstream regulatory regions of the alpha- and beta-globin loci. The NF-E2 transcription factor, which recognizes these sites, is a heterodimer consisting of (i) p45 NF-E2 (the larger subunit), a hematopoietic-restricted basic leucine zipper protein, and (ii) a widely expressed basic leucine zipper factor, p18 NF-E2, the smaller subunit. p18 NF-E2 protein shares extensive homology with the maf protooncogene family. To determine an in vivo role for p18 NF-E2 protein we disrupted the p18 NF-E2-encoding gene by homologous recombination in murine embryonic stem cells and generated p18 NF-E2-/- mice. These mice are indistinguishable from littermates throughout all phases of development and remain healthy in adulthood. Despite the absence of expressed p18 NF-E2, DNA-binding activity with the properties of the NF-E2 heterodimer is present in fetal liver erythroid cells of p18 NF-E2-/- mice. We speculate that another member of the maf basic leucine zipper family substitutes for the p18 subunit in a complex with p45 NF-E2. Thus, p18 NF-E2 per se appears to be dispensable in vivo.

Transfer of beta-amyloid precursor protein gene using adenovirus vector causes mitochondrial abnormalities in cultured normal human muscle.

As in Alzheimer-disease (AD) brain, vacuolated muscle fibers of inclusion-body myositis (IBM) contain abnormally accumulated beta-amyloid precursor protein (beta APP), including its beta-amyloid protein epitope, and increased beta APP-751 mRNA. Other similarities between IBM muscle and AD brain phenotypes include paired helical filaments, hyperphosphorylated tau protein, apolipoprotein E, and mitochondrial abnormalities, including decreased cytochrome-c oxidase (COX) activity. The pathogenesis of these abnormalities in IBM muscle and AD brain is not known. We now report that direct transfer of the beta APP gene, using adenovirus vector, into cultured normal human muscle fibers causes structural abnormalities of mitochondria and decreased COX activity. In this adenovirus-mediated beta APP gene transfer, we demonstrated that beta APP overproduction can induce mitochondrial abnormalities. The data suggest that excessive beta APP may be responsible for mitochondrial and COX abnormalities in IBM muscle and perhaps AD brain.

Repair of thalassemic human β-globin mRNA in mammalian cells by antisense oligonucleotides

In one form of β-thalassemia, a genetic blood disorder, a mutation in intron 2 of the β-globin gene (IVS2-654) causes aberrant splicing of β-globin pre-mRNA and, consequently, β-globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β-globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β-globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

Long-range disruption of gene expression by a selectable marker cassette

Recent studies have suggested that the retention of selectable marker cassettes (like PGK–Neo, in which a hybrid gene consisting of the phosphoglycerate kinase I promoter drives the neomycin phosphotransferase gene) in targeted loci can cause unexpected phenotypes in “knockout” mice due to disruption of expression of neighboring genes within a locus. We have studied targeted mutations in two multigene clusters, the granzyme B locus and the β-like globin gene cluster. The insertion of PGK–Neo into the granzyme B gene, the most 5′ gene in the granzyme B gene cluster, severely reduced the normal expression of multiple genes within the locus, even at distances greater than 100 kb from the mutation. Similarly, the insertion of a PGK–Neo cassette into the β-globin locus control region (LCR) abrogates the expression of multiple globin genes downstream from the cassette. In contrast, a targeted mutation of the promyelocyte-specific cathepsin G gene (which lies just 3′ to the granzyme genes in the same cluster) had minimal effects on upstream granzyme gene expression. Although the mechanism of these long distance effects are unknown, the expression of PGK–Neo can be “captured” by the regulatory domain into which it is inserted. These results suggest that the PGK–Neo cassette can interact productively with locus control regions and thereby disrupt normal interactions between local and long-distance regulatory regions within a tissue-specific domain.

Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb βs-globin yeast artificial chromosome: A mouse model of sickle cell anemia

Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide. Current approaches to the development of murine models of SCA involve the elimination of functional murine α- and β-globin genes and substitution with human α and βs transgenes. Recently, two groups have produced mice that exclusively express human HbS. The transgenic lines used in these studies were produced by coinjection of human α-, γ-, and β-globin constructs. Thus, all of the transgenes are integrated at a single chromosomal site. Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human β-globin gene family are required for appropriate developmental and stage-restricted expression of the genes. As the cis-acting sequences that participate in activation and silencing of the γ- and β-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA. To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb βs yeast artificial chromosome. These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

Essential role for complex N-glycans in forming an organized layer of bronchial epithelium.

Mice lacking the complex subset of N-glycans due to inactivation of the Mgat1 gene die at mid-gestation, making it difficult to identify specific biological functions for this class of cell surface carbohydrates. To circumvent this embryonic lethality and to uncover tissue-specific functions for complex N-glycans, WW6 embryonic stem cells with inactivated Mgat1 alleles were tracked in chimeric embryos. The Mgat1 gene encodes N-acetylglucosaminyltransferase I (Glc-NAc-TI; EC 2.4.1.101), the transferase that initiates the synthesis of complex N-glycans. WW6 cells carry an inert beta-globin transgene that allows their identification in chimeras by DNA-DNA in situ hybridization. Independent Mgat1-/- and Mgat1+/- mutant WW6 isolates contributed like parent WW6 cells to the tissues of embryonic day (E) 10.5 to E16.5 chimeras. However, a cell type-specific difference was observed in lung. Homozygous null Mgat1-/- WW6 cells did not contribute to the epithelial layer in more than 99% bronchi. This deficiency was corrected by transfection of a Mgat1 transgene. Interestingly, heterozygous Mgat1+/- WW6 cells were also deficient in populating the layer of bronchial epithelium. Furthermore, examination of lung bud in E9.5 Mgat1-/- mutant embryos showed complete absence of an organized epithelial cell layer in the bronchus. Thus, complex N-glycans are required to form a morphologically recognizable bronchial epithelium, revealing an in vivo, cell type-specific function for this class of N-glycans.