PubMed Health⌕ Search

Biomedical subjects

Alan M Gewirtz

Publications and source records attributed to Alan M Gewirtz.

At least 19 recordsLinked to original sources
PubMed2007

RNA targeted therapeutics for hematologic malignancies.

In a variety of experimental systems, antisense nucleic acids (ASNA) of various composition, including antisense oligodeoxynucleotides (ODN) and siRNA, have been shown to have the ability to variably perturb gene expression in a sequence specific manner. Pilot clinical studies from our group, and others, have demonstrated that gene silencing is a therapeutic strategy that is starting to make a real contribution to the treatment of various diseases. The development of this field, with specific reference to hematologic malignancies, is reviewed very briefly below.

Gene Silencing↗
PubMed2006

Integrin inhibition through Lyn-dependent cross talk from CXCR4 chemokine receptors in normal human CD34+ marrow cells.

We studied the effects of Lyn ablation on CXCR4 receptor-mediated migration and adhesion of hematopoietic precursors. Down-regulation of Lyn expression with siRNA greatly reduced CXCR4-dependent hematopoietic cell movement, and increased cell adherence to stroma. This increase was associated with the up-regulated expression of activation-dependent epitopes of the beta(2) integrin LFA-1 and was prevented by antibodies that selectively block cell adhesion mediated by ICAM-1. Attachment to surfaces coated with ICAM-1 was also enhanced in Lyn-depleted hematopoietic cells, as compared with Lyn-expressing cells. Functional rescue experiments with Lyn siRNA targeting the 3' UTR indicated that the observed effects can be attributed directly to specific inhibition of Lyn. Our results show that in chemokine-stimulated hematopoietic cells Lyn kinase is a positive regulator of cell movement while negatively regulating adhesion to stromal cells by inhibiting the ICAM-1-binding activity of beta(2) integrins. These results provide a molecular mechanism for cross talk between the chemokine receptor CXCR4 and beta(2) integrins. This cross talk may allow chemokine receptors to modulate the arrest of rolling hematopoietic precursors on the surface of bone marrow stromal cells.

Antigens, CD34↗
PubMed2006

Nucleic acid therapeutics for hematologic malignancies--theoretical considerations.

Our work is motivated by the belief that RNA targeted gene silencing agents can be developed into effective drugs for treating hematologic malignancies. In many experimental systems, antisense nucleic acids of various composition, including antisense oligodeoxynucleotides (AS ODNs) and short interfering RNA (siRNA), have been shown to perturb gene expression in a sequence specific manner. Nevertheless, our clinical experience, and those of others, have led us to conclude that the antisense nucleic acids (ASNAs) we, and others, employ need to be optimized with regard to intracellular delivery, targeting, chemical composition, and efficiency of mRNA destruction. We have hypothesized that addressing these critical issues will lead to the development of practical and effective nucleic acid drugs. An overview of our recent work which seeks to addresses these core issues is contained within this review.

Hematologic Neoplasms↗
PubMed2005

Stemless self-quenching reporter molecules identify target sequences in mRNA.

The design of oligonucleotides for gene silencing requires a rational method for identifying hybridization-accessible sequences within the target RNA. To this end, we have developed stem-loop self-quenching reporter molecules (SQRMs) as probes for such sequence. SQRMs have a 5' fluorophore, a quenching moiety on the 3' end, an intervening sequence that forms an approximately 5-basepaired stem, and a loop sequence of approximately 20-30 bases. We have previously described a mapping strategy employing SQRMs to locate stem-loop structures in the target mRNA molecule. We now show that the original design constraint of a basepaired stem is not needed, either in vitro or in vivo. We propose that stemless probes possess sufficient signal-to-noise for use in vivo and that this ratio is an indication of hybridization of the probe to its target. Data showing that these SQRMs can specifically target and reduce c-Myb protein synthesis and can be used for real-time in vivo assays are presented.

Animals↗
PubMed2005

Multigene targeting with antisense oligodeoxynucleotides: an exploratory study using primary human leukemia cells.

PURPOSE: We previously reported that the c-myb and Vav proto-oncogenes are amenable to silencing with antisense oligodeoxynucleotides and that inhibition of either impairs leukemic cell growth. Because the expression of these genes is not known to be linked, we sought to determine the therapeutic value of silencing both genes simultaneously in K562 and primary patient (n = 9) chronic myelogenous leukemia cells. EXPERIMENTAL DESIGN: K562 and primary chronic myelogenous leukemia cells were exposed to antisense oligodeoxynucleotides (alone or in combination) for 24 or 72 hours and then cloned in methylcellulose cultures. Effects on K562 cluster, and blast-forming unit-erythroid colonies and granulocyte-macrophage colony-forming units were determined and correlated with the ability to down-regulate the targeted mRNA. RESULTS: After 24-hour exposure, K562 cell growth was inhibited in a sequence specific, dose-responsive manner with either c-myb or Vav antisense oligodeoxynucleotides. Exposure to both oligodeoxynucleotides simultaneously considerably enhanced growth inhibition and accelerated apoptosis. Primary cell results were more complex. After 24- and 72-hour exposures to either anti-vav or anti-myb antisense oligodeoxynucleotides, equivalent colony-forming unit inhibition was observed. Exposing cells to both antisense oligodeoxynucleotides simultaneously for 24 hours did not result in additional inhibition of colony formation. However, after 72-hour incubation with both oligodeoxynucleotides, colony formation was diminished significantly when compared with either oligodeoxynucleotides alone (from approximately 30% to approximately 78% for granulocyte-macrophage colony-forming unit; approximately 50% to approximately 80% for blast-forming unit-erythroid). CONCLUSIONS: We hypothesize that exposing primary leukemic cells to antisense oligodeoxynucleotides targeted to two, or possibly more, genes might significantly augment the therapeutic utility of these molecules.

Apoptosis↗
PubMed2005

Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.

We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

Animals↗
PubMed2005

RNA interference for treating haematological malignancies.

RNA interference (RNAi) is a method for silencing gene expression. It is relatively gene-specific, potent, and minimally toxic. For these reasons, RNAi holds great promise for the treatment of haematological malignancies. Much has already been learned about RNAi in the laboratory, although many fundamental questions about its mechanisms remain to be elucidated. For human trials, major hurdles to be overcome include the induction of a nonspecific immune response to RNAi, the selection of the most appropriate targets, the design of more specific molecules, and the assurance of efficient delivery and safety in patients. Translational research efforts are currently well on their way to solving these problems, and will be reviewed here.

Animals↗
PubMed2005

Nucleic acid modulation of gene expression: approaches for nucleic acid therapeutics against cancer.

Most cancers are characterized by abnormal gene expression, which is thought to contribute to the pathogenesis and maintenance of the malignant phenotype; abnormal proliferation, maturation, and apoptosis. Silencing such genes would appear to be a rational approach to the therapy of cancer, and some preliminary clinical studies support this concept. Of the strategies available, the anti-mRNA gene silencing approach has attracted much attention and is the focus of this review. This strategy includes three types of agents: (1) single-stranded antisense oligonucleotides; (2) catalytically active oligonucleotides, such as ribozymes, and DNAzymes that possess inherent RNA cleaving activity; and (3) small interfering RNA (siRNA) molecules that induce RNA interference (RNAi). Among these agents, antisense oligonucleotides, especially phosphorothioate (PS) oligonucleotides, have been the most frequently used in clinical trials. In this article, we provide an overview of anti-mRNA gene silencing agents and their development for use as cancer therapeutics.

Animals↗
PubMed2004

Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells.

We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80-95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.

Apoptosis↗
PubMed2004

Neuromedin U: a Myb-regulated autocrine growth factor for human myeloid leukemias.

The c-myb proto-oncogene has been implicated in leukemogenesis, but possible mechanisms remain ill defined. To gain further insight to this process, we used transcript profiling in K562 cells expressing a dominant-negative Myb (MERT) protein. A total of 105 potential Myb gene targets were identified. Neuromedin U (NmU), a peptide affecting calcium transport, underwent the greatest expression change ( approximately 5-fold decrease). To verify a linkage between c-myb and NmU, their mRNA levels were quantitated using real-time polymerase chain reaction in primary acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL), as well as normal hematopoietic cells. We found that c-myb was elevated in AML and ALL samples, but NmU expression was increased only in AML cells. Significantly, only AML cells expressed the cognate receptor of NmU, NMU1R, suggesting the presence of a novel autocrine loop. We examined this possibility in detail. Exogenous NmU "rescued" growth suppression in K562-MERT cells and stimulated the growth of primary AML cells. Short interfering RNA "knockdown" of NmU in K562 cells arrested cell growth. Exposing Indo-1-labeled K562 cells to NmU induced an intracellular Ca(++) flux consistent with engagement of the NMU1R. Combined, these results suggest that NmU expression is related to Myb and that the NmU/NMU1R axis constitutes a previously unknown growth-promoting autocrine loop in myeloid leukemia cells.

Autocrine Communication↗
PubMed2004

Progress in the development of nucleic acid therapeutics for cancer.

Many cancers are characterized by abnormal gene expression. Silencing these aberrantly expressed genes could therefore have therapeutic utility and by virtue of specific targeting, prove less toxic than conventional cancer therapies. A number of strategies for inhibiting gene expression have been developed. Some, such as triple helix forming, or decoy transcription factor binding, oligodeoxynucleotides seek to disrupt gene expression at the level of transcription. Others, such as antisense oligonucleotides (ODN) and short interfering RNA (siRNA) molecules attempt to disrupt expression at the level of mRNA translation. In this review, we provide an overview of gene silencing agents and their development for use as cancer therapeutics. We will focus on mRNA targeting methodologies and discuss issues core to the clinical success of these molecules including cellular delivery, and successful targeting. The potential utility of nucleic acid based therapeutics in the clinic will also be addressed.

Antineoplastic Agents↗
PubMed2003

Therapeutic potential of antisense nucleic acid molecules.

Elucidation of many disease-related signal transduction and gene expression pathways has provided unparalleled opportunities for the development of targeted therapeutics. The types of molecules in development are increasingly varied and include small-molecule enzyme inhibitors, humanized antibodies to cell surface receptors, and antisense nucleic acids for silencing the expression of specific genes. This Perspective reviews the basis for various antisense strategies for modulating gene expression, including RNA interference, and discusses the prospects for their clinical use.

Animals↗
PubMed2003

28. Embryonic and adult stem cell therapy.

Stem cells are characterized by the ability to remain undifferentiated and to self-renew. Embryonic stem cells derived from blastocysts are pluripotent (able to differentiate into many cell types). Adult stem cells, which were traditionally thought to be monopotent multipotent, or tissue restricted, have recently also been shown to have pluripotent properties. Adult bone marrow stem cells have been shown to be capable of differentiating into skeletal muscle, brain microglia and astroglia, and hepatocytes. Stem cell lines derived from both embryonic stem and embryonic germ cells (from the embryonic gonadal ridge) are pluripotent and capable of self-renewal for long periods. Therefore embryonic stem and germ cells have been widely investigated for their potential to cure diseases by repairing or replacing damaged cells and tissues. Studies in animal models have shown that transplantation of fetal, embryonic stem, or embryonic germ cells may be able to treat some chronic diseases. In this review, we highlight recent developments in the use of stem cells as therapeutic agents for three such diseases: Diabetes, Parkinson disease, and congestive heart failure. We also discuss the potential use of stem cells as gene therapy delivery cells and the scientific and ethical issues that arise with the use of human stem cells.

Diabetes Mellitus↗
PubMed2002

Crosstalk between BCR/ABL oncoprotein and CXCR4 signaling through a Src family kinase in human leukemia cells.

Stromal-derived factor (SDF)-1 and its G protein-coupled receptor, CXCR4, regulate stem/progenitor cell migration and retention in the marrow and are required for hematopoiesis. We show here an interaction between CXCR4 and the Src-related kinase, Lyn, in normal progenitors. We demonstrate that CXCR4-dependent stimulation of Lyn is associated with the activation of phosphatidylinositol 3-kinase (PI3-kinase). This chemokine signaling, which involves a Src-related kinase and PI3-kinase, appears to be a target for BCR/ABL, a fusion oncoprotein expressed only in leukemia cells. We show that the binding of phosphorylated BCR/ABL to Lyn results in the constitutive activation of Lyn and PI3-kinase, along with a total loss of responsiveness of these kinases to SDF-1 stimulation. Inhibition of BCR/ABL tyrosine kinase with STI571 restores Lyn responsiveness to SDF-1 signaling. Thus, BCR/ABL perturbs Lyn function through a tyrosine kinase-dependent mechanism. Accordingly, the blockade of Lyn tyrosine kinase inhibits both BCR/ABL-dependent and CXCR4-dependent cell movements. Our results demonstrate, for the first time, that Lyn-mediated pathological crosstalk exists between BCR/ABL and the CXCR4 pathway in leukemia cells, which disrupts chemokine signaling and chemotaxis, and increases the ability of immature cells to escape from the marrow. These results define a Src tyrosine kinases-dependent mechanism whereby BCR/ABL (and potentially other oncoproteins) dysregulates G protein-coupled receptor signaling and function of mammalian precursors.

Animals↗
PubMed2002

Oligodeoxynucleotide-mediated inhibition of c-myb gene expression in autografted bone marrow: a pilot study.

Antisense oligodeoxynucleotide (ODN) drugs might be more effective if their delivery was optimized and they were targeted to short-lived proteins encoded by messenger RNA (mRNA) species with equally short half-lives. To test this hypothesis, an ODN targeted to the c-myb proto-oncogene was developed and used to purge marrow autografts administered to allograft-ineligible chronic myelogenous leukemia patients. CD34(+) marrow cells were purged with ODN for either 24 (n = 19) or 72 (n = 5) hours. After purging, Myb mRNA levels declined substantially in approximately 50% of patients. Analysis of bcr/abl expression in long-term culture-initiating cells suggested that purging had been accomplished at a primitive cell level in more than 50% of patients and was ODN dependent. Day-100 cytogenetics were evaluated in surviving patients who engrafted without infusion of unmanipulated "backup" marrow (n = 14). Whereas all patients were approximately 100% Philadelphia chromosome-positive (Ph(+)) before transplantation, 2 patients had complete cytogenetic remissions; 3 patients had fewer than 33% Ph(+) metaphases; and 8 remained 100% Ph(+). One patient's marrow yielded no metaphases, but fluorescent in situ hybridization evaluation approximately 18 months after transplantation revealed approximately 45% bcr/abl(+) cells, suggesting that 6 of 14 patients had originally obtained a major cytogenetic response. Conclusions regarding clinical efficacy of ODN marrow purging cannot be drawn from this small pilot study. Nevertheless, these results lead to the speculation that enhanced delivery of ODN, targeted to critical proteins of short half-life, might lead to the development of more effective nucleic acid drugs and the enhanced clinical utility of these compounds in the future.

Adult↗
PubMed2002

Nucleic-acid therapeutics: basic principles and recent applications.

The sequencing of the human genome and the elucidation of many molecular pathways that are important in disease have provided unprecedented opportunities for the development of new therapeutics. The types of molecule in development are increasingly varied, and include antisense oligonucleotides and ribozymes. Antisense technology and catalytic nucleic-acid enzymes are important tools for blocking the expression of abnormal genes. One FDA-approved antisense drug is already in the clinic for the treatment of cytomegalovirus retinitis, and other nucleic-acid therapies are undergoing clinical trials. This article reviews different strategies for modulating gene expression, and discusses the successes and problems that are associated with this type of therapy.

Apoptosis↗