Lentiviral vectors incorporating a human elongation factor 1alpha 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.

LFA-1-deficient mice show normal CTL responses to virus but fail to reject immunogenic tumor.

The leukocyte integrin LFA-1 (CD11a/CD18) plays an important role in lymphocyte recirculation and homotypic interactions. Leukocytes from mice lacking CD11a displayed defects in in vitro homotypic aggregation, in proliferation in mixed lymphocyte reactions, and in response to mitogen. Mutant mice mounted normal cytotoxic T cell (CTL) responses against systemic LCMV and VSV infections and showed normal ex vivo CTL function. However, LFA-1-deficient mice did not reject immunogenic tumors grafted into footpads and did not demonstrate priming response against tumor-specific antigen. Thus CD11a deficiency causes a selective defect in induction of peripheral immune responses whereas responses to systemic infection are normal.

Potential genotoxicity from integration sites in CLAD dogs treated successfully with gammaretroviral vector-mediated gene therapy.

Integration site analysis was performed on six dogs with canine leukocyte adhesion deficiency (CLAD) that survived greater than 1 year after infusion of autologous CD34+ bone marrow cells transduced with a gammaretroviral vector expressing canine CD18. A total of 387 retroviral insertion sites (RIS) were identified in the peripheral blood leukocytes from the six dogs at 1 year postinfusion. A total of 129 RIS were identified in CD3+ T-lymphocytes and 102 RIS in neutrophils from two dogs at 3 years postinfusion. RIS occurred preferentially within 30 kb of transcription start sites, including 40 near oncogenes and 52 near genes active in hematopoietic stem cells. Integrations clustered around common insertion sites more frequently than random. Despite potential genotoxicity from RIS, to date there has been no progression to oligoclonal hematopoiesis and no evidence that vector integration sites influenced cell survival or proliferation. Continued follow-up in disease-specific animal models such as CLAD will be required to provide an accurate estimate of the genotoxicity using gammaretroviral vectors for hematopoietic stem cell gene therapy.

TLS-ERG leukemia fusion protein inhibits RNA splicing mediated by serine-arginine proteins.

The translocation liposarcoma (TLS) gene is fused to the ETS-related gene (ERG) in human myeloid leukemia, resulting in the generation of a TLS-ERG protein. We demonstrate that both TLS and the TLS-ERG leukemia fusion protein bind to RNA polymerase II through the TLS N-terminal domain, which is retained in the fusion protein; however, TLS recruits members of the serine-arginine (SR) family of splicing factors through its C-terminal domain, whereas the TLS-ERG fusion protein lacks the ability to recruit SR proteins due to replacement of the C-terminal domain by the fusion partner ERG. In transient-transfection assays, the TLS-ERG fusion protein inhibits E1A pre-mRNA splicing mediated by these TLS-associated SR proteins (TASR), and stable expression of the TLS-ERG fusion protein in K562 cells alters the splicing profile of CD44 mRNA. These results suggest that TLS fusion proteins may lead to cellular abnormalities by interfering with the splicing of important cellular regulators.

Conversion of the severe to the moderate disease phenotype with donor leukocyte microchimerism in canine leukocyte adhesion deficiency.

Leukocyte adhesion deficiency-1 (LAD-1), a genetic immunodeficiency disease characterized by life-threatening bacterial infections, results from the defective adherence and migration of leukocytes due to mutations in the leukocyte integrin CD18 molecule. Canine LAD (CLAD) represents the canine homologue of the severe phenotype of LAD-1 in children. In previous studies we demonstrated that non-myeloablative stem cell transplantation from matched littermates resulted in mixed donor-host chimerism and reversal of the disease phenotype in CLAD. In this study, we describe two CLAD dogs with less than 2% donor leukocyte chimerism following non-myeloablative transplant. Both dogs are alive more than 24 months after transplant with an attenuated CLAD phenotype resembling the moderate deficiency phenotype of LAD. The improvement in the CLAD phenotype with very low levels of donor CD18(+) leukocytes correlated with the preferential egress of the CD18(+) neutrophils into extravascular sites. The clinical response with very low levels of donor CD18(+) leukocytes in CLAD supports using this model for testing gene therapy strategies since the low levels of gene-corrected hematopoietic cells expected with hematopoietic gene therapy would likely have a therapeutic effect in CLAD.

Oncogenic TLS/ERG and EWS/Fli-1 fusion proteins inhibit RNA splicing mediated by YB-1 protein.

The translocation liposarcoma protein TLS has recently been shown to function as an adapter molecule coupling gene transcription to RNA splicing. Here we demonstrate that YB-1, a protein known to play important roles in transcription and translation, interacts with the COOH-terminal domains of TLS and the structurally related Ewing's sarcoma protein EWS. Through this interaction, YB-1 is recruited to RNA polymerase II and promotes splicing of E1A pre-mRNA to the 13S isoform. This splicing function of YB-1 is inhibited by exogenous TLS/ERG or EWS/Fli-1 fusion proteins, which bind to RNA polymerase II but fail to recruit the YB-1 protein. In Ewing's sarcoma cells that express endogenous EWS/Fli-1, this linkage between YB-1 and RNA Pol II via EWS (or TLS) was found to be defective. Together, these results suggest that TLS and EWS fusion proteins may contribute to malignant transformation through disruption of RNA splicing mediated by TLS- and EWS-binding proteins such as YB-1.

The Tel-PDGFRbeta fusion gene produces a chronic myeloproliferative syndrome in transgenic mice.

Chronic myelomonocytic leukemia (CMML) is a pre-leukemic syndrome that displays both myelodysplastic and myeloproliferative features. The t(5;12) chromosomal translocation, present in a subset of CMML patients with myeloproliferation fuses the amino terminal portion of the ets family member, Tel, with the transmembrane and tyrosine kinase domains of platelet-derived growth factor receptor beta (PDGFRbeta) gene. To investigate the role of this fusion protein in the pathogenesis of CMML, we expressed the Tel-PDGFRbeta fusion cDNA in hematopoietic cells of transgenic mice under the control of the human CD11a promoter. Transgenic founders and their offspring express the transgene specifically in hematopoietic tissues and develop a myeloproliferative syndrome characterized by: overproduction of mature neutrophils and megakaryocytes in the bone marrow; splenomegaly with effacement of splenic architecture by extramedullary hematopoiesis; an abnormal population of leukocytes co-expressing lymphoid and myeloid markers; and increased numbers of colonies in in vitro bone marrow CFU assays. All mice expressing the transgene exhibited at least one of these features of dysregulated myelopoiesis, and 20% progressed to a myeloid or lymphoid malignancy. This murine model of CMML parallels a myeloproliferative syndrome in humans and implicates the Tel-PDGFRbeta fusion protein in its pathogenesis.

Transduction of human hematopoietic cells and cell lines using a retroviral vector containing a modified murine CD4 reporter gene.

To investigate conditions for improving transduction efficiencies of human hematopoietic stem or progenitor cells using retroviral vectors, we constructed a retroviral vector containing a modified murine CD4 cDNA reporter gene with a truncated cytoplasmic domain to prevent signaling. The advantages of using this truncated murine CD4 reporter gene include: (i) CD4 is well characterized with well-known cell signaling pathways, (ii) truncation of the cytoplasmic domain of CD4 has been demonstrated to abrogate signaling, (iii) the truncated murine CD4 is easily detectable on the cell surface with no cross-reaction to human CD4, (iv) a variety of monoclonal antibodies directed against the murine CD4 molecule are available commercially, and (v) expression of a truncated CD4 molecule in a transgenic mouse in vivo does not interfere with hematopoiesis. We cloned the truncated murine CD4 reporter gene into the retroviral vector LXSN, packaged this vector using PG13 retrovirus packaging cells, and transduced hematopoietic cell lines representing erythroid, myeloid, megakaryocyte, and lymphoid lineages using vector-containing medium harvested from the murine CD4 producer line. After seven daily exposures to vector-containing medium, all cell lines expressed murine CD4 on the cell surface, and 5-7% of human CD34+ cells expressed murine CD4 on the cell surface after 3 days of exposure to murine CD4 vector-containing medium. Colony-forming cell assays assessing progenitor cells demonstrated the presence of transduced cells in the CD34+ population. These results demonstrate the utility of using a modified murine CD4 gene in a retroviral vector to allow optimization of in vitro transduction conditions of human hematopoietic cells and to facilitate identification of the lineages that have been transduced using different growth factors, prior to clinical trials using retroviral vectors.

Expression from leukocyte integrin promoters in retroviral vectors.

Human gene therapy for diseases involving leukocytes would be facilitated by the identification of specific promoter/enhancer sequences capable of directing high levels of tissue and stage-specific expression of the requisite cDNA when used in a retroviral vector. We tested the promoter sequences from the leukocyte integrin CD11a (LFA-1), CD11b (Mac-1), and CD18 subunits in retroviral vectors to express a reporter gene, adenosine deaminase, in the human leukocyte cell lines K562 and HL-60. The leukocyte integrins are expressed in leukocytes, and they are inducible in HL-60 cells, a model system for myeloid differentiation. Although the leukocyte integrin promoter/enhancer sequences direct the expression of reporter genes in myeloid lineage cell lines in transient transfection assays, in these studies, the leukocyte integrin promoters direct low levels of reporter gene expression following retroviral-mediated transduction in K562 and HL-60 cells and selection of stable integrants. Treatment of HL-60 cells transduced with retroviral vectors containing the leukocyte integrin promoters with retinoic acid or phorbol myristate acetate results in less than a two-fold increase in reporter gene expression. These studies indicate that: (i) expression from the leukocyte integrin promoters from stable integrants in retroviral vectors does not parallel the results observed in transient transfection assays, and (ii) additional promoter/enhancer sequences will likely be required for these promoters to direct high levels of tissue and stage-specific expression in retroviral vectors.

Gene therapy for leukocyte adhesion deficiency.

Leukocyte adhesion deficiency (LAD) is an autosomal recessive immunodeficiency disease characterized by severe, recurrent bacterial infections. In patients with LAD, the leukocytes, particularly the neutrophils, fail to adhere to the endothelial cell wall and migrate to the site of infection. LAD results from heterogeneous molecular defects in the leukocyte integrin CD18, which prevent CD11/CD18 heterodimer formation and surface expression. To date, hematopoietic stem cell transplantation remains the only curative treatment for LAD, however, this approach is limited by transplant-related toxicities and graft-versus-host disease. During the course of the preceding decade we have conducted extensive experimental studies demonstrating that gene transfer of the CD18 subunit corrects the structural and functional defect in LAD leukocytes. These studies provided the support for the initiation of a clinical trial of retroviral-mediated gene transfer of CD18 in two patients with the severe deficiency phenotype or LAD. This review will present an overview of LAD, preclinical CD18 gene transfer studies and the initial results from the current clinical trial.

EWS.Fli-1 fusion protein interacts with hyperphosphorylated RNA polymerase II and interferes with serine-arginine protein-mediated RNA splicing.

Ewing's sarcoma displays a characteristic chromosomal translocation that results in fusion of the N-terminal domain of the Ewing's sarcoma protein (EWS) to the C-terminal DNA-binding domain of the ETS family transcription factor Fli-1 (Friend leukemia integration-1). EWS possesses structural motifs suggesting a role in transactivation as well as RNA binding. We demonstrate that wild-type EWS protein functions as an adapter molecule coupling transcription to RNA splicing by binding to hyperphosphorylated RNA polymerase II through the N-terminal domain of EWS and recruiting serine-arginine (SR) splicing factors through the C-terminal domain of EWS. The oncogenic EWS.Fli-1 fusion protein retains the ability to bind to hyperphosphorylated RNA polymerase II but lacks the ability to recruit SR proteins because of replacement of the C-terminal domain of EWS by Fli-1. In an in vivo splicing assay, the EWS.Fli-1 fusion protein inhibits SR protein-mediated E1A pre-mRNA splicing in a dominant-negative manner. These results indicate that EWS.Fli-1 interferes with the normal function of EWS and implicate uncoupling of gene transcription from RNA splicing in the pathogenesis of Ewing's sarcoma.

Description of the leukocyte function-associated antigen 1 (LFA-1 or CD11a) promoter.

The CD11a/CD18 (leukocyte function-associated antigen 1 or LFA-1) leukocyte integrin is expressed at high levels on the cell surface of T lymphocytes and macrophages, where it mediates homotypic and heterotypic adherence between leukocytes and other cell types by binding to intracellular adhesion molecules 1 and 2 on the conjugate cell. To initiate studies of the molecular regulation of expression of the CD11a molecule, we isolated genomic clones corresponding to the 5'-flanking region of CD11a, identified the transcriptional start sites for CD11a, and characterized the CD11a promoter sequence in transient expression assays. The CD11a promoter (1.7 kb) directed functional activity of a heterologous reporter gene in the T-lymphocyte cell line Jurkat and the myeloid cell line HL-60 but did not direct functional activity in three different nonleukocyte cell lines. Deletional analysis of the CD11a promoter sequence indicated the presence of distinct, cell-type-specific regulatory sequences with the region from -40 to -17 relative to the transcription start sites responsible for most of the in vitro activity of the CD11a promoter in the Jurkat T-cell line, and the promoter sequence located within the first 17 bp relative to the transcription start sites responsible for CD11a promoter activity in the HL-60 cell line. Identification of the CD11a promoter provides the opportunity to identify unique cis-acting elements and trans-acting factors responsible for the cell-type-specific expression of CD11a in human leukocytes. Further, the CD11a promoter may be useful in transgenic constructs and in retroviral vectors to direct expression of heterologous genes selectively in leukocytes.

The spectrum of pituitary adenoma hemorrhage.

In 34 cases of pituitary adenoma hemorrhage at one institution, the clinical manifestations of adenoma hemorrhage depended upon the size of the adenoma, the presence of suprasellar extension, the amount of hemorrhage and the extent of pituitary glandular destruction. Recognition of the spectrum of acute, subacute and chronic pituitary adenoma hemorrhage should expedite diagnosis and treatment.

Identification of two molecular defects in a child with leukocyte adherence deficiency.

Children with leukocyte adherence deficiency (LAD), or leukocyte cell adhesion molecule deficiency, experience recurrent, life-threatening bacterial infections related to severe deficiency in surface expression of the leukocyte integrin molecules. The leukocyte integrins consist of a common CD18 (beta) subunit and individual, noncovalently associated alpha subunits designated CD11a, CD11b, and CD11c. Defects in the CD18 subunit prevent surface expression of the CD11/CD18 complexes in children with this disease. We investigated the molecular basis of the disease in a child with the severe deficiency form of LAD and identified two molecular defects in the CD18 subunit. The first defect is a single-base pair C----T transposition resulting in an amino acid substitution of a leucine for a proline at amino acid 178. This amino acid substitution is located in a region that is highly conserved among the integrin beta subunits and where two previous defects have been located in LAD. The second mutation involves a deletion of 220 base pairs in the cDNA coding for a portion of the extracellular domain and results in a frameshift into a premature stop codon. The deleted region corresponds to a single exon in the CD18 gene. Identification of these two molecular defects in a single child with this disease indicates the compound heterozygous nature of the disorder in this child and identifies regions of the CD18 subunit that may be important for CD11/CD18 heterodimer formation and surface expression.

Regulation of expression of the leukocyte integrin CD11a (LFA-1) molecule during differentiation of HL-60 cells along the monocyte/macrophage pathway.

The CD11a/CD18 (LFA-1) leukocyte integrin receptor mediates homotypic and heterotypic leukocyte adhesion by binding to one of two defined ligands, ICAM-1 or 2, on the conjugate cell. In this study we investigated the molecular regulation of expression of the CD11a subunit during myeloid differentiation of HL-60 cells. Induction of monocyte/macrophage differentiation of HL-60 promyelocytic leukemia cells with PMA results in an increase in CD11a surface Ag expression and the acquisition of CD11a/CD18-mediated homotypic adherence. These changes are accompanied by a 40-fold increase in CD11a mRNA levels. Nuclear run-on transcription assays indicate that the increase in CD11a mRNA in PMA-induced HL-60 cells is not caused by an increase in CD11a RNA transcription. We assessed the posttranscriptional regulation of CD11a using two methods. By using actinomycin D to block RNA transcription, we demonstrate that the CD11a mRNA half-life in HL-60 cells is prolonged after PMA treatment. Inhibition of protein synthesis with cycloheximide also results in enhanced expression of CD11a mRNA in HL-60 cells without increasing CD11a transcription. These findings indicate that, in HL-60 cells induced with PMA to differentiate along the monocyte/macrophage pathway, CD11a expression is regulated primarily at the posttranscriptional level by a labile protein. Identification of the specific CD11a RNA sequences, and the proteins that bind to these sequences may provide insight into lineage commitment during human monocyte/macrophage differentiation.

Transcriptional regulation of the leukocyte adherence protein beta subunit during human myeloid cell differentiation.

Adherence reactions involving human leukocytes are mediated by a family of glycoprotein surface antigens composed of three different alpha subunits designated alpha L, alpha M, and alpha X, each of which is associated with a single beta subunit in an alpha 1 beta 1 heterodimer structure. We cloned the cDNA for the common beta subunit and investigated beta subunit mRNA expression in HL-60 promyelocytic leukemia cells and human granulocytic cells. Leukocyte adherence receptor beta subunit mRNA transcripts were present in low levels in HL-60 myeloblasts and promyelocytes and increased 10-fold or greater with chemically induced differentiation to more mature granulocytes (using retinoic acid and dimethylformamide) or monocyte/macrophages (using phorbol myristate acetate). Levels of beta subunit mRNA expression were also increased both in normal human peripheral blood granulocytes and in granulocytes from patients with chronic myelogenous leukemia. Nuclear run-off assays indicated that the increased steady state level of the beta subunit mRNA in retinoic acid-differentiated HL-60 cells was secondary to enhanced beta subunit gene transcription. We conclude that mRNA levels for the beta subunit of the receptor on human leukocytes that mediates cellular adherence are increased in more mature granulocytic cells compared to immature myeloid precursors and that this enhanced mRNA expression is transcriptionally regulated.

Regulation of expression of the CD11b and CD18 subunits of the neutrophil adherence receptor during human myeloid differentiation.

The receptor on human myeloid cells that mediates cellular adherence consists of a heterodimer complex (designated CD11b/CD18). This receptor complex plays a critical role in leukocyte chemotaxis, adherence to vascular endothelium, and phagocytosis. We investigated expression of the CD11b subunit of this adherence receptor complex in various leukocyte populations. As previously described for the CD18 subunit, enhanced CD11b surface antigen and mRNA expression are present in peripheral blood granulocytes, as well as in chemically induced, differentiating HL-60 cells. However, in contrast to CD18 mRNA expression, which is transcriptionally regulated in differentiating HL-60 cells, the steady state levels of CD11b mRNA appear to be post-transcriptionally regulated in these cells. Thus, although the steady state levels of mRNA for the individual subunits of the CD11b/CD18 adherence receptor complex generally parallel each other during human myeloid differentiation, the mechanisms responsible for regulating these levels are distinctly different.