The role of glycogen synthase kinase-3beta in normal haematopoiesis, angiogenesis and leukaemia.

Glycogen synthase kinase 3 beta (GSK-3beta) was one of the first kinases identified and studied, initially for its role in the regulation of glycogen synthesis. Over the past decade, interest in GSK-3beta has grown far beyond glycogen metabolism, and this is due in large measure to the critical role that GSK-3beta plays in the regulation of many other cellular processes, particularly cell proliferation and apoptosis. GSK-3beta has been shown to regulate the proteolysis and sub-cellular compartmentalization of a number of proteins directly involved in the regulation of cell cycling, proliferation, differentiation and apoptosis. GSK-3beta also regulates the degradation of proteins that regulate gene expression and thus affects a variety of important cell functions. Specifically, GSK-3beta controls the degradation of beta-catenin, the main effector of Wnt that regulates haematopoiesis and stem cell function. In this case GSK-3beta is a negative regulator of Wnt. In contrast, GSK-3beta positively regulates NF-kappaB, another important biochemical pathway also involved in the regulation of multiple aspects of normal and aberrant haematopoiesis. GSK-3beta regulates degradation of IkappaB, a central inhibitor of NF-kappaB. In this way, GSK-3beta acts to control the resistance of leukaemic cells to chemotherapy through the modulation of NF-kappaB, a critical factor in maintaining leukaemic cell growth. In addition, GSK-3beta regulates the pro-inflammatory activity of NF-kappaB. As GSK-3beta is a pleiotropic regulator, inhibitors may increase the range of novel anti-leukaemic and anti-inflammatory drugs that control immune response.

Gene-expressed RNA as a therapeutic: issues to consider, using ribozymes and small hairpin RNA as specific examples.

In recent years there has been a greater appreciation of both the role of RNA in intracellular gene regulation and the potential to use RNA in therapeutic modalities. In the latter case, RNA can be used as a therapeutic target or a drug. The chapters in this volume cover the varied and potent actions of RNA as antisense, ribozymes, aptamers, microRNA and small hairpin RNA in gene regulation, as well as their use as potential therapeutics for metabolic and infectious diseases. Our group has been involved in the development of anti-HIV gene expression constructs to treat HIV. In this chapter, we address the relevant scientific and some of the commercial issues in the use of RNA as a therapeutic. Specifically, the chapter discusses delivery, expression, potency, toxicity and commercial development using, as examples, hammerhead ribozymes and small hairpin RNA.

Cancer gene suppression strategies: issues and potential.

Oncogenes are ideal targets for therapies which down-regulate gene expression. However, effective modalities for altering gene expression in vivo have thus far proven to be elusive. Whilst there has been recent success with small molecule inhibitors of oncoprotein function, evolution of resistance to these agents has been observed in the clinical setting, indicating the need for combinations of therapies for cancer treatment. Strategies for in vivo gene down-regulation still hold promise for the treatment of cancer. The technologies relevant to such therapeutic strategies are discussed in terms of molecular action, delivery and choice of target gene. Consideration is given to the pre-clinical and clinical efficacy these agents have demonstrated to date.

Bone marrow reconstitution as a relevant model of genetically programmed leukemia.

Mouse model systems which allow bone marrow reconstitution can be used to analyse genetically programmed leukemia. The original and most widely used system is that of post 5-fluorouracil mouse hematopoietic stem cells (HSC) into lethally irradiated syngeneic mice. Another more recent system allows analysis of human HSCs in the NOD-SCID mouse. Both systems are discussed as models for analysis of gene induced leukemia.

Induced p21WAF1 expression acts to reverse myc myelomonocytic cell transformation.

Two murine myelomonocytic cells lines were used to examine p21WAF1 expression in myc-induced cell transformation. tEMmyc4 and FDLV are two v-myc-transformed immortalised myeloid cell lines exhibiting different transformed phenotypes. FDLV cells were derived from the transduction of v-myc into FDC-P1 cells and retain growth factor (IL-3) dependence, whereas tEMmyc4 cells were derived from the transduction of embryonal monocytes with v-myc and are growth factor-independent, constitutively express endogenous CSF-1, and are highly tumorigenic in syngeneic mice. Both cell lines were found to exhibit low p21WAF1 expression. When examined in tEMmyc4 cells, neither the p53-dependent pathway (mitomycin C or exogenous p53) nor p53-independent pathway (TPA or growth factor, CSF-1, stimulation) acted to increase p21WAF1 levels. Growth factor (IL-3) withdrawal, shown to reduce p21WAF1 levels in parental FDC-P1 cells, failed to do this in FDLV cells. The dependence of p21WAF1 expression on v-myc was further demonstrated by showing that a v-myc-targeted ribozyme, which acts to decrease v-myc RNA, increased p21WAF1 levels in tEMmyc4 cells. Enforced expression of exogenous p21WAF1 in tEMmyc4 cells with dysfunctional growth cycle (including growth arrest and increased susceptibility to apoptosis) was examined. p21WAF1 partially restored cell cycle regulation and apoptosis as well as inhibited the delayed cell cycle progression and apoptosis induced by mitomycin C or serum withdrawal. These results show p21WAF1 expression to be affected by v-myc and a restoration of p21WAF1 expression to partially reverse myc-mediated transformation.

The beta-galactosidase gene as a marker for hematopoietic reconstitution: individual cell analysis in a murine bone marrow transplantation model.

We have used a simple, single-gene retrovirus carrying the Escherichia coli beta-galactosidase reporter gene (lacZ), termed LlacZ. This virus was found to infect immortalized myeloid and lymphoid precursor/leukemic cell lines efficiently as well as primary murine bone marrow clonogenic progenitors, without apparent modulation of growth or phenotype. Following infection of bone marrow cells, a significant proportion of progenitors--36% of lineage-negative cells with low levels of c-kit expression (lin-/c-kit(lo)) known to be enriched with pluripotent hemopoietic stem cells, and 19% of Sca1-positive cells known to be enriched with transplantable cells with lymphomyeloid-reconstituting ability--were shown to express lacZ. Use of an LlacZ-infected population of post 5-fluorouracil bone marrow cells to reconstitute lethally irradiated mice demonstrated the presence of lacZ-expressing cells in the spleen at day 12 post-transplantation with provirus detected in individual spleen colonies (CFU-S). In the long term (3-6 months following transplantation), lacZ expression was detected in hematopoietic tissues of all recipient mice. The use of two-color in situ and flow cytometry analysis combined with lineage-specific antibodies showed lacZ expression in both myeloid and lymphoid cells in spleen and bone marrow. In addition, lacZ-expressing cells were detected in secondary recipient mice injected with bone marrow cells derived from primary LlacZ recipients. Overall, these data show the efficacy of a single gene vector for stem cell transduction, the utility of beta-galactosidase as a single cell marker for stem cell transduction and reconstitution ability, and the need for protocol optimization to see high-level multilineage gene expression.

The use of ribozyme gene therapy for the inhibition of HIV replication and its pathogenic sequelae.

Human immunodeficiency virus (HIV) is a lentivirus, a separate genus of the Retroviridae which are RNA viruses that integrate as DNA copies into the genomes of host cells and replicate intracellularly through various RNA intermediates. Several of these RNA molecules can be targeted by ribozymes and a number of investigators, including our group, have demonstrated the ability of ribozymes to suppress HIV replication in cultured cells. It is argued that the use of this ribozyme gene therapy approach for the treatment of HIV infection may act as an adjunct to chemotherapeutic drugs and may affect not just viral suppression, but also immune restoration. This approach can be tested in Clinical Trials, several of which are currently under way.

Artificial capillary culture: expansion and retroviral transduction of CD4+ T-lymphocytes for clinical application.

An artificial capillary culture/transduction technique has been developed for application in a phase I gene therapy clinical trial for HIV. The trial protocol involves isolation of CD4+ T-lymphocytes from a genetically matched HIV negative twin, retroviral transduction of equal numbers of cells with the ribozyme therapeutic and control genes, and expansion in Cellmax artificial capillary modules. Preclinical studies showed transduction efficiencies in the range of 3-30%, with preferential expansion of CD4+ lymphocytes over a culture period of 10-14 days. Over this time period, an average yield of 1.7 x 10(9) lymphocytes was readily attainable from 5 x 10(7) CD8-depleted lymphocytes. In addition, a sensitive and reliable quantitative competitive PCR method was developed to assess the levels of transduction before infusion into the recipient. The transduction data suggest that the efficiency of retroviral transduction was affected by the presence of inhibitory factors present in the virus preparations or generated as a result of the transduction process itself. It is hypothesised that the method of transduction could significantly affect the extent of this inhibition, and thus impact on clinical efficacy of retrovirus mediated gene therapy.

Ribozymes in gene therapy of HIV-1.

Human immunodeficiency virus type 1 (HIV-1) is the primary etiologic agent for Aquired Immune Deficiency Syndrome (AIDS). HIV-1 is a lentivirus, a separate genus of the Retroviridae, which are complex RNA viruses that integrate into the genome of host cells and replicate intracellularly. Ribozymes are catalytic RNA molecules with enzyme-like cleavage properties, that can be designed to target specific RNA sequences within the HIV-1 genome. In addition to the genomic RNA, several RNA intermediates, including splice variants, can be targeted by a single ribozyme. We and others have demonstrated the ability of ribozymes to suppress HIV-1 replication in a variety of cultured cells. Ribozyme gene therapy for HIV-1 infection is a therapeutic approach that offers several potential advantages over conventional therapies in that it can potentially impact on both viral load and restoration of the immune system. Ribozyme gene therapy may be used as an adjunct to chemotherapeutic drugs, effecting viral suppression, and facilitating immune restoration without problems of patient compliance. Currently, an anti-HIV-1 ribozyme is being tested in two separate Phase I Clinical Trials.

Mammography in the 1990s: the United States and Canada.

PURPOSE: To evaluate trends in mammography quality before and after the implementation of the Mammography Quality Standards Act (MQSA) of 1992 and to compare technical data collected in the United States with corresponding data obtained from the first survey of mammography facilities conducted in 1994-1995 in Canada. MATERIALS AND METHODS: Data from MQSA inspections conducted in 1995-1997 were analyzed and compared with survey data on U.S. mammography facilities acquired before the MQSA. Technical indicators of mammography quality such as radiation dose phantom image score, film processing, and darkroom fog were analyzed. RESULTS: In the United States, phantom image scores, along with other technical measures of performance such as film processing, darkroom fog, and x-ray beam quality, have improved continuously since 1985. The U.S. mean glandular dose has increased to 1.6 mGy compared with the Canadian dose of 1.1 mGy. The mean total phantom image score with artifact subtraction was 11.1 in Canada in 1994-1995 and 11.8 in the U.S. in 1997. CONCLUSION: Mammography quality is better today than it has been at any other time in the United States. With the exception of radiation dose. Canadian technical measures of performance are comparable to measures before MQSA in the United States.

Mutant N-ras induces myeloproliferative disorders and apoptosis in bone marrow repopulated mice.

Mutations that activate the N-ras oncogene are among the most frequently detected genetic alterations in human acute myeloid leukemias (AMLs), Philadelphia chromosome-negative myeloproliferative disorders (MPDs), and myelodysplastic syndromes (MDSs). However, because N-ras has not been shown to induce these disorders in an in vivo model, the role of N-ras in the evolution of myeloid leukemia is unclear. To investigate the potential of N-ras to induce myeloid leukemia, lethally irradiated mice were reconstituted with bone marrow (BM) cells infected with a retroviral vector carrying activated N-ras. Approximately 60% of these mice developed hematopoietic disorders, including severe MPDs resembling human chronic myelogenous leukemia (CML) or AML with differentiation (French-American-British [FAB] classification M2). Other reconstituted mice succumbed to hematopoietic defects that were pathologically similar to human MDSs. The latter disorders appeared to be due to a myeloid impairment that was demonstrated by enumeration of day-12 colony-forming units-spleen (CFU-S) and by in vitro colony assays. A high level of apoptosis associated with thymic atrophy and peripheral blood (PB) lymphopenia was also evident in N-ras reconstituted mice. Our results are consistent with a model in which antiproliferative effects are a primary consequence of N-ras mutations and secondary transforming events are necessary for the development of myeloid leukemia. This is the first report of an in vivo model for N-ras induced MPD and leukemia.

Preclinical characterization of an anti-tat ribozyme for therapeutic application.

A hammerhead ribozyme retroviral construct, denoted RRz2, targeting the coding region of the human immunodeficiency virus type 1 (HIV-1) tat gene, has shown itself to be effective in a range of test systems. Inhibition of the replication of HIV-1 IIIB and primary drug-resistant strains in pooled transduced CEMT4 cells was consistently found to be more than 80% compared with the control-vector transduced cells, whereas a mutant RRz2 gave approximately 45% inhibition. A multiple HIV-1 passage assay showed the absence of emergence of mutations within the specific viral RNA ribozyme target sequences. This lack of generation of ribozyme "escape mutants" occurred despite the almost complete disappearance of a HIV-1 quasi-species in the testing virus. When RRz2 was tested in peripheral blood lymphocytes (PBLs) from HIV-1-infected patients, paired analysis showed that cell viability in the ribozyme-transduced HIV-1-infected PBLs was significantly higher than that in the vector-transduced cells. This difference in viability (vector versus RRz2) was not observed in PBLs from non-HIV-1-infected donors. Taken together, these results indicate that the transfer of an anti-HIV-1 ribozyme gene into human T lymphocytes could have major impact on viral replication and T cell viability in the HIV-1-infected individual.

V-myc in a simple, single gene retroviral vector causes rapid induction of leukemia and concomitant apoptosis following bone marrow transplantation.

We have previously developed an in vivo model of leukemogenesis utilizing mice reconstituted with genetically modified bone marrow cells. Based on those studies, a new single gene retroviral vector has been engineered which efficiently transfers v-myc into immature murine bone marrow cells. All reconstituted mice developed leukemia with a short latency period (5-11 weeks). In addition to hyperproliferation associated with elevated levels of PCNA, extensive apoptosis was also observed in all leukemic animals with p53 accumulating in the apoptotic cells. Whereas bax encoded protein, an effector of p53 apoptotic activity was detected in apoptotic cells, p21Waf1 protein, a potential mediator of p53 growth suppression was not detected in these cells suggesting that v-myc-induced apoptosis was independent of the ability of p53 to induce p21Waf1. These results indicate that apoptosis, a part of the cellular response to v-myc expression, does not prevent leukemia development and that hyperproliferation rather than abrogation of oncogene-induced apoptosis appears to be a critical event in v-myc-induced leukemia.

Effects of megakaryocyte growth and development factor on survival and retroviral transduction of T lymphoid progenitor cells.

Murine retroviral vectors have the potential to mediate stable gene transfer into hematopoietic progenitor cells. A known drawback to the use of these vectors is that transduction can only take place in cells actively progressing through the cell cycle. Thrombopoietin, the c-mpl ligand, is known to support division of hematopoietic precursors of primitive origin. Polyethylene glycol (PEG)-conjugated recombinant human megakaryocyte growth and development factor (MGDF) is a polypeptide related to thrombopoietin that stimulates megakaryocyte production. To investigate whether MGDF would also induce stem cell division and support retroviral transduction of CD34+ cells, we compared the effects of MGDF, stem cell factor (SCF), interleukin-3 (IL-3), and IL-6, alone or in combination, using amphotropic and vesicular stomatitis virus (VSV-G) pseudotyped murine retroviral vectors. Similar transduction efficiency was observed when CD34+ cells were transduced in the presence of SCF and MGDF as compared to SCF, IL-3, and IL-6. Using the SCID-hu mouse model of thymopoiesis, we investigated whether CD34+ cells transduced in the presence of these cytokines could reconstitute irradiated thymic implants, and whether vector sequences were present in mature thymocytes. At early timepoints, no significant differences were observed on engraftment of donor progenitors incubated with each cytokine combination. However, a significant difference in the percentage of donor derived CD4+/CD8+ immature thymocytes was observed 9 weeks after implantation of CD34+ cells exposed to the combination of SCF and MGDF as compared to SCF, IL-3, and IL-6 (p = 0.04), indicating that MGDF/SCF better supported the survival of thymocyte precursor cells. Approximately 4% of thymocytes in both cytokine groups harbored vector sequences. These studies provide evidence that MGDF and SCF in combination can mediate transduction of hematopoietic progenitors capable of contributing to long-term thymopoiesis. These results may have important applications for the implementation of gene therapy strategies in disorders affecting the T lymphoid system.

Myeloproliferative disease and myelodysplastic syndrome induced by transplantation of bone marrow cells expressing mutant p53.

p53 mutations are the most common genetic alterations observed in human cancers including lymphomas and leukemias. We have previously shown that transduction of normal murine hematopoietic cells with mutant p53 alone in vitro results in an enhanced proliferative capacity and modified differentiation potential of transduced cells. In order to investigate further the role of mutant p53 in hematopoietic cell transformation, mutant p53-transduced bone marrow cells were used to reconstitute the hematopoietic system of lethally irradiated mice. The results show that overexpression of mutant p53 can initiate the transformation of immature murine hematopoietic cells in vivo and induce two types of hematopoietic disorders, myeloproliferative disease and myelodysplastic syndrome.

Anti-HIV ribozymes.

HIV is an RNA virus that replicates intracellularly through various RNA intermediates. Several of these can be targeted by ribozymes (catalytic RNA molecules), and a number of investigators, including this group, have demonstrated the ability of ribozymes to suppress HIV replication in this way. It is argued that this gene therapy approach may be viewed as an adjunct to chemotherapeutic drugs, which may allow not just viral suppression, but also immune restoration. This can only finally be tested in clinical trials, and several are planned. The basic ribozyme unit, the potential of which was described less than 10 years ago, is about to be tested in an amunable disease state.

Inhibition of Moloney murine leukaemia virus by a retroviral vector, LNL6, carrying ribozymes targeted to the 5' non-coding sequence.

The packaging region psi is the RNA sequence which directs the inclusion of genomic viral RNA into virions. As such it represents an apparently accessible site for ribozyme action. Ribozymes directed against the psi site of Moloney murine leukaemia virus (MoMLV) were delivered to target cells using a related retroviral vector, LNL6. LNL6 is a vector predominantly derived from MoMLV and, like all retroviruses, requires the psi region to be packaged. By exploiting the heterogeneity between nucleotide sequences of the MoMLV target and LNL6 vector in the psi packaging region, two ribozymes were designed and shown to selectively cleave the target but not the vector sequence. Clonally derived cell lines expressing ribozymes showed inhibition of virus replication. These results show the utility of catalytic RNA as specific antiviral agents.