Delivery and mechanistic considerations for the production of knock-in mice by single-stranded oligonucleotide gene targeting.

Single-stranded oligodeoxynucleotide (ssODN) gene targeting may facilitate animal model creation and gene repair therapy. Lipofection of ssODN can introduce point mutations into target genes. However, typical efficiencies in mouse embryonic stem cells (ESC) are <10(-4), leaving corrections too rare to effectively identify. We developed ESC lines with an integrated mutant neomycin resistance gene (Tyr22Ter). After targeting with ssODN, repaired cells survive selection in G418. Correction efficiencies varied with different lipofection procedures, clonal lines, and ssODN designs, ranging from 1 to 100 corrections per million cells plated. Uptake studies using cell sorting of Cy5-labelled ssODN showed 40% of the corrections concentrated in the best transfected 22% of cells. Four different basepair mismatches were tested and results show that the base-specificity of the mismatch is critical. Dual mismatch ssODN also showed mismatch preferences. These ESC lines may facilitate development of improved ssODN targeting technologies for either animal production or ex vivo gene therapy.

Progress in the development of nucleic acid therapeutics.

Abnormal gene expression is a hallmark of many diseases. Gene-specific downregulation of aberrant genes could be useful therapeutically and potentially less toxic than conventional therapies due its specificity. Over the years, many strategies have been proposed for silencing gene expression in a gene-specific manner. Three major approaches are antisense oligonucleotides (AS-ONs), ribozymes/DNAzymes, and RNA interference (RNAi). In this brief review, we will discuss the successes and shortcomings of these three gene-silencing methods, and the approaches being taken to improve the effectiveness of antisense molecules. We will also provide an overview of some of the clinical applications of antisense therapy.

2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) modified oligonucleotides (ON) effect highly efficient, and persistent, gene silencing.

To be effective in vivo, antisense oligonucleotides (AS ON) should be nuclease resistant, form stable ON/RNA duplexes and support ribonuclease H mediated heteroduplex cleavage, all with negligible non-specific effects on cell function. We report herein that AS ONs containing a 2'-deoxy-2'-fluoro-beta-D-arabinonucleic acid (2'F-ANA) sugar modification not only meet these criteria, but have the added advantage of maintaining high intracellular concentrations for prolonged periods of time which appears to promote longer term gene silencing. To demonstrate this, we targeted the c-MYB protooncogene's mRNA in human leukemia cells with fully phosphorothioated 2'F-ANA-DNA chimeras (PS-2'FANA-DNA) and compared their gene silencing efficiency with AS ON containing unmodified nucleosides (PS-DNA). When delivered by nucleofection, chemically modified ON of both types effected a >90% knockdown of c-MYB mRNA and protein expression, but the PS-2'F-ANA-DNA were able to accomplish this at 20% of the dose of the PS-DNA, and in contrast to the PS-AS DNA, their silencing effect was still present after 4 days after a single administration. Therefore, our data demonstrate that PS-2'F-ANA-DNA chimeras are efficient gene silencing molecules, and suggest that they could have significant therapeutic potential.

Rationally targeted, conformationally constrained, oxetane-modified oligonucleotides demonstrate efficient gene-silencing activity in a cellular system.

Antisense oligodeoxynucleotides (AS ODN) have been employed as gene-silencing agents in the laboratory and, in the clinic. The in vivo use of these molecules has been facilitated by chemical modifications to the DNA backbone which augment their nuclease stability. Attempts to further improve the efficacy of AS ODN have largely focused on 2' alterations of the ribose sugar that make the molecules more RNA like in structure. This increases the T(m) of formed DNA/RNA hybrids but simultaneously prevents binding of RNaseH which is important for ODN effectiveness. Herein, we demonstrate the use of AS ODN containing nucleosides with a novel oxetane (OXE) modification [oxetane, 1-(1', 3'-O-anhydro-beta-D-psicofuranosyl nucleosides)] which augments Tm, enhances nuclease stability, and is permissive of RNaseH activation. We also illustrate herein the value of rational targeting of OXE modified, and by analogy, AS ODN of any chemical modification.

Oxetane modified, conformationally constrained, antisense oligodeoxyribonucleotides function efficiently as gene silencing molecules.

Incorporation of nucleosides with novel base-constraining oxetane (OXE) modifications [oxetane, 1-(1',3'-O-anhydro-beta-d-psicofuranosyl nucleosides)] into antisense (AS) oligodeoxyribonucleotides (ODNs) should greatly improve the gene silencing efficiency of these molecules. This is because OXE modified bases provide nuclease protection to the natural backbone ODNs, can impart T(m) values similar to those predicted for RNA-RNA hybrids, and not only permit but also accelerate RNase H mediated catalytic activity. We tested this assumption in living cells by directly comparing the ability of OXE and phosphorothioate (PS) ODNs to target c-myb gene expression. The ODNs were targeted to two different sites within the c-myb mRNA. One site was chosen arbitrarily. The other was a 'rational' choice based on predicted hybridization accessibility after physical mapping with self-quenching reporter molecules (SQRM). The Myb mRNA and protein levels were equally diminished by OXE and PS ODNs, but the latter were delivered to cells with approximately six times greater efficiency, suggesting that OXE modified ODNs were more potent on a molar basis. The rationally targeted molecules demonstrated greater silencing efficiency than those directed to an arbitrarily chosen mRNA sequence. We conclude that rationally targeted, OXE modified ODNs, can function efficiently as gene silencing agents, and hypothesize that they will prove useful for therapeutic purposes.

Chimeric RNA-DNA molecular beacon assay for ribonuclease H activity.

Current methods to detect and assay ribonuclease H (RNase H) activity are indirect and time-consuming. Here we introduce a direct and sensitive method, based on the fluorescence quenching mechanism of molecular beacons, to assay RNA cleavage in RNA:DNA hybrids. An RNA-DNA chimeric beacon assay for RNase H enzymatic activity was developed. The substrate is a single-stranded RNA-DNA chimeric oligonucleotide labeled with a 5'-fluorescein and a 3'-DABCYL. The fluorophore (fluorescein) of the probe is held in close proximity to the quencher (DABCYL) by the RNA:DNA stem-loop structure. When the RNA sequence of the RNA:DNA hybrid stem is cleaved, the fluorophore is separated from the quencher and fluorescence can be detected as a function of time. Chimeric beacons with different stem lengths and sequences have been surveyed for this assay with E. coli RNase H. We found that the beacon kinetic parameters are in qualitative agreement with previously reported values using more cumbersome assays. This method permits real-time detection of RNase H activity and a convenient approach to RNase H kinetic and mechanistic study.

Gemtuzumab ozogamicin (Mylotarg) monotherapy for relapsed AML after hematopoietic stem cell transplant: efficacy and incidence of hepatic veno-occlusive disease.

Gemtuzumab ozogamicin (GO) (Mylotarg, CMA-676) is a novel chemotherapeutic agent consisting of an anti-CD33 monoclonal antibody linked to calicheamicin, and is associated with a 30% response rate in patients with CD33-positive acute myeloid leukemia (AML) in first relapse. GO therapy has a 20% incidence of grade 3 or 4 hepatotoxicity, and has recently been associated with hepatic veno-occlusive disease (VOD). The efficacy and toxicity of GO in patients with AML who have relapsed after hematopoietic stem cell transplant (HSCT) is unknown, as this population was largely excluded from phase II studies. We reviewed the outcomes of eight consecutive patients with AML who received GO following relapse after HSCT. Two (25%) had responses to GO. One patient, who had had two previous HSCT and prior hyperbilirubinemia, developed severe VOD and died 14 days after GO therapy. The other seven patients did not meet diagnostic criteria for VOD. We conclude that GO can be safe and effective in patients who relapse following HSCT, but that caution is warranted in patients with multiple risk factors for VOD.

Numerous growth factors, cytokines, and chemokines are secreted by human CD34(+) cells, myeloblasts, erythroblasts, and megakaryoblasts and regulate normal hematopoiesis in an autocrine/paracrine manner.

The aim of this study was to explore further the hypothesis that early stages of normal human hematopoiesis might be coregulated by autocrine/paracrine regulatory loops and by cross-talk among early hematopoietic cells. Highly purified normal human CD34(+) cells and ex vivo expanded early colony-forming unit-granulocyte-macrophage (CFU-GM)-derived, burst forming unit-erythroid (BFU-E)-derived, and CFU-megakaryocyte (CFU-Meg)-derived cells were phenotyped for messenger RNA expression and protein secretion of various growth factors, cytokines, and chemokines to determine the biological significance of this secretion. Transcripts were found for numerous growth factors (kit ligand [KL], FLT3 ligand, fibroblast growth factor-2 [FGF-2], vascular endothelial growth factor [VEGF], hepatocyte growth factor [HGF], insulinlike growth factor-1 [IGF-1], and thrombopoietin [TPO]); cytokines (tumor necrosis factor-alpha, Fas ligand, interferon alpha, interleukin 1 [IL-1], and IL-16); and chemokines (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, regulated upon activation, normal T cell expressed and secreted [RANTES], monocyte chemotactic protein-3 [MCP-3], MCP-4, IL-8, interferon-inducible protein-10, macrophage-derived chemokine [MDC], and platelet factor-4 [PF-4]) to be expressed by CD34(+) cells. More importantly, the regulatory proteins VEGF, HGF, FGF-2, KL, FLT3 ligand, TPO, IL-16, IGF-1, transforming growth factor-beta1 (TGF-beta1), TGF-beta2, RANTES, MIP-1alpha, MIP-1beta, IL-8, and PF-4 were identified in media conditioned by these cells. Moreover, media conditioned by CD34(+) cells were found to inhibit apoptosis and slightly stimulate the proliferation of other freshly isolated CD34(+) cells; chemo-attract CFU-GM- and CFU-Meg-derived cells as well as other CD34(+) cells; and, finally, stimulate the proliferation of human endothelial cells. It was also demonstrated that these various hematopoietic growth factors, cytokines, and chemokines are expressed and secreted by CFU-GM-, CFU-Meg-, and BFU-E-derived cells. It is concluded that normal human CD34(+) cells and hematopoietic precursors secrete numerous regulatory molecules that form the basis of intercellular cross-talk networks and regulate in an autocrine and/or a paracrine manner the various stages of normal human hematopoiesis.

Hybrid HIV/MSCV LTR enhances transgene expression of lentiviral vectors in human CD34(+) hematopoietic cells.

HIV-based lentiviral vectors can transduce nondividing cells, an important advantage over murine leukemia virus (MLV)-based vectors when transducing slowly dividing hematopoietic stem cells. However, we find that in human CD34(+) hematopoietic cells, the HIV-based vectors with an internal cytomegalovirus (CMV) promoter express transgenes 100- to 1,000-fold less than the MLV-based retroviral vector murine stem cell virus (MSCV). To increase the expression of the integrated lentivirus, we replaced CMV promoter with that of the Rous sarcoma virus or MSCV and obtained a modest augmentation in expression. A more dramatic effect was seen when the CMV enhancer/promoter was removed and the HIV long-terminal repeat (LTR) was replaced by a novel HIV/MSCV hybrid LTR. This vector retains the ability to transduce nondividing cells but now expresses its transgene (enhanced green fluorescent protein) 10- to 100-fold greater than the original HIV-based vector. When compared under identical conditions, the HIV vector with the hybrid LTR transduced a higher percentage of CD34(+) cells than the MSCV-based retroviral vector (19.4% versus 2.4%). The number of transduced cells and level of transgene expression remain constant over 5-8 weeks as determined by long-term culture-initiating cells, fluoresence-activated cell sorting, and nonobese diabetic/severe combined immunodeficiency repopulation assay.

Investigating the platelet-sparing mechanism of paclitaxel/carboplatin combination chemotherapy.

Paclitaxel and carboplatin chemotherapy is reported to be a platelet-sparing drug combination. This study investigated potential mechanisms for this observation by studying the effects of paclitaxel and carboplatin on (1) normal donor and chemotherapy patient-derived erythroid (burst-forming units-erythroid [BFU-E]), myeloid (colony-forming units-granulocyte/macrophage [CFU-GM]), and megakaryocyte (CFU-Meg) progenitor cell growth; (2) P-glycoprotein (P-gp) protein and glutathione S-transferase (GST) messenger RNA (mRNA) expression; (3) serum thrombopoietin (Tpo), stem cell factor (SCF), interleukin-6 (IL-6), IL-11, IL-1beta, IL-8, and tumor necrosis factor-alpha levels in patients treated with paclitaxel and carboplatin; and (4) stromal cell production of Tpo and SCF after paclitaxel and carboplatin exposure. CFU-Meg were more resistant to paclitaxel alone, or in combination with carboplatin, than CFU-GM and BFU-E. Although all progenitors expressed P-gp protein and GST mRNA, verapamil treatment significantly, and selectively, increased the toxicity of paclitaxel and carboplatin to CFU-Meg, suggesting an important role for P-gp in megakaryocyte drug resistance. Compared to normal controls, serum Tpo levels in patients receiving paclitaxel and carboplatin were significantly elevated 5 hours after infusion and remained elevated at day 7 (287% +/- 63% increase, P <.001). Marrow stroma was shown to be the likely source of this Tpo. It is concluded here that P-gp-mediated efflux of paclitaxel, and perhaps GST-mediated detoxification of carboplatin, results in relative sparing of CFU-Meg, which may then respond to locally high levels of stromal cell-derived Tpo. The confluence of these events might lead to the platelet-sparing phenomenon observed in patients treated with paclitaxel and carboplatin chemotherapy.

Suppression of gene expression by targeted disruption of messenger RNA: available options and current strategies.

At least three different approaches may be used for gene targeting including: A) gene knockout by homologous recombination; B) employment of synthetic oligonucleotides capable of hybridizing with DNA or RNA, and C) use of polyamides and other natural DNA-bonding molecules called lexitropsins. Targeting mRNA is attractive because mRNA is more accessible than the corresponding gene. Three basic strategies have emerged for this purpose, the most familiar being to introduce antisense nucleic acids into a cell in the hopes that they will form Watson-Crick base pairs with the targeted gene's mRNA. Duplexed mRNA cannot be translated, and almost certainly initiates processes which lead to its destruction. The antisense nucleic acid can take the form of RNA expressed from a vector which has been transfected into the cell, or take the form of a DNA or RNA oligonucleotide which can be introduced into cells through a variety of means. DNA and RNA oligonucleotides can be modified for stability as well as engineered to contain inherent cleaving activity. It has also been proven that because RNA and DNA are very similar chemical compounds, DNA molecules with enzymatic activity could also be developed. This assumption proved correct and led to the development of a "general-purpose" RNA-cleaving DNA enzyme. The attraction of DNAzymes over ribozymes is that they are very inexpensive to make and that because they are composed of DNA and not RNA, they are inherently more stable than ribozymes. Although mRNA targeting is impeccable in theory, many additional considerations must be taken into account in applying these strategies in living cells including mRNA site selection, drug delivery and intracellular localization of the antisense agent. Nevertheless, the ongoing revolution in cell and molecular biology, combined with advances in the emerging disciplines of genomics and informatics, has made the concept of nontoxic, cancer-specific therapies more viable then ever and continues to drive interest in this field.

Expression, regulation and function of AC133, a putative cell surface marker of primitive human haematopoietic cells.

To explore the physiological significance of AC133 expression on human haematopoietic cells, we phenotyped normal and malignant human haematopoietic cells for AC133 expression, evaluated the utility of AC133 for isolating human stem/progenitor cells in comparison to other known early haematopoietic cell markers, investigated the role of AC133 in regulating hematopoiesis, and evaluated the possibility that MYB might regulate AC133. We found that while human CD34+ progenitor cells expressed AC133, expression was rapidly downregulated during differentiation. In apparent contrast, AC133 mRNA was detectable in cells isolated from CFU-Mix, BFU-E, CFU-GM and CFU-Meg colonies. Human cord blood CD34+ cells expressed AC133 at higher levels than their normal bone marrow counterparts. In apparent contrast to normal primitive haematopoietic cells, the AC133 protein was undetectable on cells from 24 different human haematopoietic cells lines, even though the majority of these cells expressed AC133 mRNA. Since CD34, AC133 and the c-kit (KIT) receptor are all co-expressed on human stem/progenitor cells, we compared the ability of monoclonal antibodies directed against each of these proteins to isolate early progenitor cells. Using these antibodies and magnetized particles in a standard immunoaffinity isolation protocol, we found that anti-CD34 and anti-KIT MoAbs could isolate > 80-90% of the clonogeneic cell population present in a given marrow sample. Anti-AC133 MoAbs recovered approximately 75-80% of CFU-GM and CFU-Meg, but only about 30% of CFU-Mix and BFU-E. Perturbation of AC133 expression with antisense oligodeoxynucleotides (AS ODN) resulted in transient downregulation of AC133 protein on human CD34+ cells but no apparent effect on cell survival or cloning efficiency ex vivo. Finally, downregulation of MYB expression with AS ODN had no effect on the AC133 expression at either the mRNA or protein level. Based on these results, we conclude that AC133 offers no distinct advantage over CD34 or c-kit as a target for immunoaffinity based isolation of primitive hematopoietic cells, that AC133 expression is not required for normal hematopoietic progenitor cell development in vitro, and finally that AC133 expression may not be MYB-dependent.

The role of HIV-related chemokine receptors and chemokines in human erythropoiesis in vitro.

In order to better define the role of HIV-related chemokines in human erythropoiesis we studied: A) the expression of chemokine receptors, both on human CD34(+) cells which include erythroid progenitors and on more mature erythroid cells; B) the functionality of these receptors by calcium flux, chemotaxis assay and phosphorylation of mitogen-activated protein kinases (MAPK) p42/44 (ERK1/ERK2) and AKT, and finally C) the influence of chemokines on BFU-E formation. We found that HIV-related chemokine receptor CXCR4, but not CCR5, is detectable on human CD34(+) BFU-E cells. CXCR4 surface expression decreased during erythroid maturation, although CXCR4 mRNA was still present in cells isolated from differentiated erythroid colonies. SDF-1, a CXCR4 ligand, induced calcium flux and phosphorylation of MAPK (p42/44) and AKT in CD34(+)KIT(+) bone marrow mononuclear cells which contain BFU-E, as well as chemotactic activity of both human CD34(+) BFU-E progenitors and erythroid cells isolated from day 2-6 BFU-E colonies. Responsiveness to SDF-1 decreased when the cells differentiated to the point of surface expression of the erythroid-specific marker Glycophorin-A. In contrast, the CCR5 ligands (macrophage inflammatory protein-1alpha [MIP-1alpha], MIP-1beta, and RANTES) did not activate calcium flux, MAPK and AKT phosphorylation or chemotaxis of CD34(+)KIT(+) cells or cells isolated from the BFU-E colonies. Interestingly, none of the chemokines tested in this study had any effect on BFU-E colony formation. In conclusion, only CXCR4 is functional, and its specific ligand SDF-1 may therefore play an important role in the homing and/or retention of early erythroid precursors in the bone marrow environment.

Crosstalk between G protein-coupled receptors and tyrosine kinase signaling: Src take centre stage.

Although the role of G protein-coupled receptors in the regulation of metabolic, secretory and contractile responses is well established, they have only recently been recognized as important mediators of cellular growth and differentiation. G protein-coupled signaling pathways had been previously thought to be totally independent of the tyrosine kinase receptor pathway. It was previously believed that molecular switches responsible for growth factor tyrosine kinase receptor signaling and G protein-coupled signaling were divided into a distinct sets of protein families. Recent evidence has demonstrated, however, that G protein-coupled receptors can crosstalk to tyrosine kinase signaling. In the past few years several groups have found that G protein-coupled receptors utilize non-receptor tyrosine kinases, mostly that of Src family, and some adapter proteins, to regulate tyrosine kinase cascades in cells.

Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Myb targeted therapeutics for the treatment of human malignancies.

For the past several years, we have been engaged in developing a therapeutically effective strategy for disrupting gene function with reverse complementary, or so called 'antisense', oligodeoxynucleotides (ODN). This pursuit has focused on finding appropriate diseases in which to apply this approach, and suitable gene targets. Of the genes that we have targeted for disruption using the antisense ODN strategy (Clevenger et al., 1995; Gewirtz and Calabretta, 1988; Ratajczak et al., 1992c; Small et al., 1994) one that has been of particular interest, and one where therapeutically motivated disruptions are now in clinical trial, is the myb gene (reviewed in Lyon et al., 1994). These trials involve treatment of human leukemias. These diseases are a logical choice for developing oncogene targeted therapies because of easy access to tissues, and the abundance of knowledge about the cell and molecular biology of these diseases. Nevertheless, as will be touched on below, other malignancies have also been examined as models for Myb targeted therapy with some surprisingly encouraging results. Finally, while we have focused our efforts on the ODN strategy, I will allude briefly to other strategies for disrupting Myb function with therapeutic intent.

Relationship between megakaryocyte mass and serum thrombopoietin levels as revealed by a case of cyclic amegakaryocytic thrombocytopenic purpura.

Cyclic amegakaryocytic thrombocytopenic purpura is a rare syndrome characterized by periodic failure of megakaryocytopoiesis. In this report we describe a patient with cyclic amegakaryocytic thrombocytopenic purpura associated with a megakaryocyte specific serum IgG antibody, who responded to cyclosporin A therapy. Serial serum thrombopoietin assays during an episode of platelet cycling demonstrated a reciprocal relationship between serum thrombopoietin level and megakaryocyte mass, suggesting that megakaryocytes have an important role in the regulation of thrombopoietin metabolism.