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Cynthia E Dunbar

Publications and source records attributed to Cynthia E Dunbar.

At least 19 recordsLinked to original sources
PubMed2006

Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy.

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent, life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs, therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment, and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific, large animal model using 2 clinically applicable conditioning regimens, and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD.

Animals↗
PubMed2006

Genotoxicity of retroviral integration in hematopoietic cells.

The experience of the past 3 years, since the first case of leukemia was reported in a child cured of X-linked severe combined immunodeficiency (X-SCID) by gene therapy, indicates that the potential genotoxicity of retroviral integration in hematopoietic cells will remain a consideration in evaluating the relative risks versus benefits of gene therapy for specific blood disorders. Although many unique variables may have contributed to an increased risk in X-SCID patients, clonal dominance or frank neoplasia in animal models, clonal dominance in humans with chronic granulomatous disease, and the ability of retroviral integration to immortalize normal bone marrow cells or convert factor-dependent cells to factor independence suggest that transduction of cells with an integrating retrovirus has the potential for altering their subsequent biologic behavior. The selective pressure imposed during in vitro culture or after engraftment may uncover a growth or survival advantage for cells in which an integration event has affected gene expression. Such cells then carry the risk that subsequent mutations may lead to neoplastic evolution of individual clones. Balancing that risk is that the vast majority of integration events seem to be neutral and that optimizing vector design may diminish the probability of altering gene expression by an integrated vector genome. Several cell culture systems and animal models designed to empirically evaluate the safety of vector systems are being developed and should provide useful data for weighing the relative risks and benefits for specific diseases and patient populations. Gene therapy interventions continue to have enormous potential for the treatment of disorders of the hematopoietic system. The future of such efforts seems bright as we continue to evolve and improve various strategies to make such interventions both effective and as safe as possible.

Adaptor Proteins, Signal Transducing↗
PubMed2006

In vitro culture during retroviral transduction improves thymic repopulation and output after total body irradiation and autologous peripheral blood progenitor cell transplantation in rhesus macaques.

Immunodeficiency after peripheral blood progenitor cell (PBPC) transplantation may be influenced by graft composition, underlying disease, and/or pre-treatment. These factors are difficult to study independently in humans. Ex vivo culture and genetic manipulation of PBPC grafts may also affect immune reconstitution, with relevance to gene therapy applications. We directly compared the effects of three clinically relevant autologous graft compositions on immune reconstitution after myeloblative total body irradiation in rhesus macaques, the first time these studies have been performed in a large animal model with direct clinical relevance. Animals received CD34(+) cell dose-matched grafts of either peripheral blood mononuclear cells, purified CD34(+) PBPCs, or purified CD34(+) PBPCs expanded in vitro and retrovirally transduced. We evaluated the reconstitution of T, B, natural killer, dendritic cells, and monocytes in blood and lymph nodes for up to 1 year post-transplantation. Animals receiving selected-transduced CD34(+) cells had the fastest recovery of T-cell numbers, along with the highest T-cell-receptor gene rearrangement excision circles levels, the fewest proliferating Ki-67(+) T-cells in the blood, and the best-preserved thymic architecture. Selected-transduced CD34(+) cells may therefore repopulate the thymus more efficiently and promote a higher output of naïve T-cells. These results have implications for the design of gene therapy trials, as well as for the use of expanded PBPCs for improved T-cell immune reconstitution after transplantation.

Animals↗
PubMed2006

AMD3100 mobilizes hematopoietic stem cells with long-term repopulating capacity in nonhuman primates.

AMD3100, a bicyclam antagonist of the chemokine receptor CXCR4, has been shown to induce rapid mobilization of CD34(+) hematopoietic cells in mice, dogs, and humans, offering an alternative to G-CSF mobilization of peripheral-blood hematopoietic stem cells. In this study, AMD3100-mobilized CD34(+) cells were phenotypically analyzed, marked with Neo(R)-containing retroviral vectors, and subsequently transplanted into myeloablated rhesus macaques. We show engraftment of transduced AMD3100-mobilized CD34(+) cells with Neo(R) gene marked myeloid and lymphoid cells up to 32 months after transplantation, demonstrating the ability of AMD3100 to mobilize true long-term repopulating hematopoietic stem cells. More AMD3100-mobilized CD34(+) cells are in the G(1) phase of the cell cycle and more cells express CXCR4 and VLA-4 compared with G-CSF-mobilized CD34(+) cells. In vivo gene marking levels obtained with AMD3100-mobilized CD34(+) cells were better than those obtained using CD34(+) cells mobilized with G-CSF alone. Overall, these results indicate that AMD3100 mobilizes a population of hematopoietic stem cells with intrinsic characteristics different from those of hematopoietic stem cells mobilized with G-CSF, suggesting fundamental differences in the mechanism of AMD3100-mediated and G-CSF-mediated hematopoietic stem cell mobilization. Thus, AMD3100-mobilized CD34(+) cells represent an alternative source of hematopoietic stem cells for clinical stem cell transplantation and genetic manipulation with integrating retroviral vectors.

Animals↗
PubMed2006

Mobilization as a preparative regimen for hematopoietic stem cell transplantation.

Current myeloablative conditioning regimens for hematopoietic stem cell (HSC) transplantation are associated with significant morbidity and mortality. Thus, alternative strategies to promote engraftment of infused HSCs with increased safety warrant investigation. Using parabiotic mice, we determined that, after mobilization with AMD3100 (a CXCR4 antagonist), HSCs exited from marrow, transited blood, and engrafted in open niches in partner marrow. We then hypothesized that mobilization before transplantation might vacate niches and improve HSC engraftment. When PeP3(b) mice were treated with AMD3100 at 2 hours before the transplantation of 4 x 10(7) marrow cells, donor cell engraftment was higher (4.6% +/- 1.1%) than in control animals (no AMD3100; 1.0% +/- 0.24%, P < .001). When mice received weekly injections of AMD3100 on 3 consecutive weeks and marrow cells were transplanted 2 hours after each mobilization, donor cell engraftment further increased (9.1% +/- 1.7%, P = .001). In contrast, in similar experiments with Balb/cByJ mice that mobilize poorly, there was no difference between the donor cell engraftment of AMD3100-treated and control recipients. These results indicate that the number of available niches regulates the number of HSCs. In addition, mobilization with AMD3100 may provide a safer preparative approach for HSC transplantation in genetic and other nonmalignant disorders.

Animals↗
PubMed2006

Acute myeloid leukemia is associated with retroviral gene transfer to hematopoietic progenitor cells in a rhesus macaque.

We report, for the first time, a replication-defective retroviral vector-associated neoplasia in a nonhuman primate. Five years after transplantation with CD34+ cells transduced with a retroviral vector expressing enhanced green fluorescent protein (eGFP) and a drug-resistant variant of the dihydrofolate reductase gene (L22Y), a rhesus macaque developed a fatal myeloid sarcoma, a type of acute myeloid leukemia. Tumor cells contained 2 clonal vector insertions. One insertion was found in BCL2-A1, an antiapoptotic gene. This event suggests that currently available retroviral vectors may have long-term side effects, particularly in hematopoietic stem and progenitor cells.

Animals↗
PubMed2006

Anti-interleukin-2 receptor antibody (daclizumab) treatment of corticosteroid-refractory autoimmune thrombocytopenic purpura.

We administered daclizumab, a humanized monoclonal anti-interleukin-2 receptor (IL-2R) antibody, to 11 patients with corticosteroid-refractory autoimmune thrombocytopenic purpura (AITP) every 2 weeks for five treatments. Of nine evaluable patients, one individual experienced a partial response. Lymphocyte phenotyping by flow cytometry indicated post-treatment binding of IL-2Ra by daclizumab in all patients. Mid-study serum soluble IL-2R levels in all patients increased 4-15 -fold over baseline values (p=0.004). Despite these measurable immunologic effects, blockade of the IL-2/IL-2R axis did not effectively abrogate the autoimmune response in this group of patients with corticosteroid-refractory AITP.

Adolescent↗
PubMed2005

Outcomes and risks of granulocyte colony-stimulating factor in patients with coronary artery disease.

OBJECTIVES: Cytokine mobilization of progenitor cells from bone marrow may promote myocardial neovascularization with relief of ischemia. BACKGROUND: Patients with coronary artery disease (CAD) have low numbers of endothelial progenitor cells compared with healthy subjects. METHODS: Granulocyte colony-stimulating factor (G-CSF), 10 microg/kg/day for five days, was administered to 16 CAD patients. Progenitor cells were measured by flow cytometry; ischemia was assessed by exercise stress testing and by dobutamine stress cardiac magnetic resonance imaging. RESULTS: Granulocyte colony-stimulating factor increased CD34+/CD133+ cells in the circulation from 1.5 +/- 0.2 microl to 52.4 +/- 10.4 microl (p < 0.001), similar to the response observed in 15 healthy subjects (75.1 +/- 12.6 microl, p = 0.173). Indices of platelet and coagulation activation were not changed by treatment, but C-reactive protein increased from 4.5 +/- 1.3 mg/l to 8.6 +/- 1.3 mg/l (p = 0.017). Two patients experienced serious adverse events: 1) non-ST-segment elevation myocardial infarction (MI) 8 h after the fifth G-CSF dose, and 2) MI and death 17 days after treatment. At 1 month after treatment, there was no improvement from baseline values (i.e., reduction) in wall motion score (from 25.7 +/- 2.1 to 28.3 +/- 1.9, p = 0.196) or segments with abnormal perfusion (7.6 +/- 1.1 to 7.7 +/- 1.1, p = 0.916) and a trend towards a greater number of ischemic segments (from 4.5 +/- 0.6 to 6.1 +/- 1.0, p = 0.068). There was no improvement in exercise duration at 1 month (p = 0.37) or at 3 months (p = 0.98) versus baseline. CONCLUSIONS: Granulocyte colony-stimulating factor administration to CAD patients mobilizes cells with endothelial progenitor potential from bone marrow, but without objective evidence of cardiac benefit and with the potential for adverse outcomes in some patients.

Aged↗
PubMed2005

Hematopoietic stem cell gene therapy: dead or alive?

Despite some reports of toxicity in recent clinical trials, many scientists believe that the use of gene therapy in the treatment of congenital genetic defects and acquired disorders has too much potential to abandon. Hematopoietic stem cells (HSCs) have been primary targets for gene therapy owing to their capacity for differentiation and self-renewal, whereby multiple cell lineages can potentially be corrected for the lifetime of an individual. These efforts represent a long-term investment towards broadening physicians' treatment options for patients whose diseases, in particular certain immunodeficiencies, are fatal and where no other therapy is available. We review the recent progress and clinical triumphs as well as the reported toxicity related to insertional mutagenesis. We also discuss the current risk-to-benefit estimates and future strategies to reduce the risks and allow full realization of clinical potential. Scientists are continually revising protocols: going both from "bench to bedside" and, as strikingly demonstrated by HSC gene therapy, from "bedside to bench."

Animals↗
PubMed2005

Recurrent retroviral vector integration at the Mds1/Evi1 locus in nonhuman primate hematopoietic cells.

Recent reports linking insertional activation of LMO2 following gene therapy for X-linked severe combined immunodeficiency (X-SCID) have led to a re-evaluation of risks following gene therapy with retroviral vectors. In our analysis of 702 integration sites in rhesus macaques that underwent transplantation up to 7 years earlier with autologous CD34+ cells transduced with amphotropic murine leukemia virus (MLV)-derived retroviral vectors containing marker genes, we detected insertion into one locus, the Mds1/Evi1 region, a total of 14 times in 9 animals. Mds1/Evi1 integrations were observed stably long term, primarily in myeloid cells. We hypothesize that this over-representation likely results from an impact on the self-renewal and engraftment potential of CD34+ progenitor cells via insertional mutagenesis at this specific locus. There is no evidence of ongoing in vivo clonal expansion of the Mds1/Evi1 populations, and all animals are hematologically normal without evidence for leukemia. Characterization of integration sites in this relevant preclinical model provides critical information for gene therapy risk assessment as well as identification of genes controlling hematopoiesis.

Animals↗
PubMed2005

Large granular lymphocytic proliferation-associated cyclic thrombocytopenia.

Cyclic thrombocytopenia is a rare condition characterized by regular fluctuations in the platelet count, resulting in bleeding at the time of platelet count nadir. We evaluated a male patient whose platelet count cycled between <10x10(9)/L and a maximum of >1300x10(9)/L over a median of every 42 days (range, 28-57 days). Antiplatelet antibodies were present at highest titer just prior to platelet nadirs. A pathologic expansion of CD3+CD57+ large granular lymphocytes (LGLs) along with a clonal rearrangement of the T-cell receptor (TCR) gamma gene in circulating T cells was detected. LGLs decreased in response to treatment with cyclosporine-A (CsA), but the cycling of the platelet count continued. This is the first report of cyclic thrombocytopenia associated with a T-LGL lymphoproliferative disorder.

Adult↗
PubMed2005

A new direction for gene therapy: intrathymic T cell-specific lentiviral gene transfer.

Reports of neoplasia related to insertional activation of protooncogenes by retroviral vectors have raised serious safety concerns in the field of gene therapy. Modification of current approaches is urgently required to minimize the deleterious consequences of insertional mutagenesis. In this issue of the JCI, Adjali and colleagues report on their treatment of SCID mice lacking the 70-kDa protein tyrosine kinase, ZAP-70, with direct intrathymic injection of a ZAP-70-expressing T cell-specific lentiviral vector, which resulted in T cell reconstitution. Using lentiviral vectors and in situ gene transfer may represent a safer approach than using retroviral vectors for ex vivo gene transfer into HSCs, avoiding 3 factors potentially linked to leukemogenesis, namely HSC targets, ex vivo transduction and expansion, and standard Moloney leukemia virus-based retroviral vectors.

Animals↗
PubMed2005

Stem cell gene transfer: insights into integration and hematopoiesis from primate genetic marking studies.

Genetic marking strategies in the nonhuman primate model have elucidated a number of principles with relevance to implementation of clinical stem cell therapies, including the lineage potential, number, and life span of hematopoietic stem and progenitor cells. The recent occurrence of leukemias likely related to insertional activation of a proto-oncogene in two patients with X-severe combined immunodeficiency (SCID) syndromes treated with CD34(+) cells transduced with retroviral vectors expressing the corrective common gamma cytokine receptor gene has refocused attention on the issue of retroviral integration. We have analyzed >1500 independent insertions from rhesus macaques transplanted with CD34(+) cells transduced with either MLV or SIV vectors. Of these, 46 rhesus macaques followed long term have not had progression to leukemia, abnormal hematopoiesis, or clonal hematopoiesis. However, the pattern of both MLV and SIV integrants in cells of these animals was found to be highly nonrandom, with a propensity for insertions of both vectors within genes: for MLV particularly near the transcription start site, and for SIV particularly in gene-dense regions.

Animals↗
PubMed2004

Imatinib inhibits T-cell receptor-mediated T-cell proliferation and activation in a dose-dependent manner.

The tyrosine kinase inhibitor imatinib (imatinib, STI571, Glivec, and Gleevec) is increasingly used in patients undergoing allogeneic transplantation for leukemia. However, little is known regarding its potential immunoregulatory effects. Here, we investigate the effect of imatinib on T-cell receptor (TCR)-mediated activation of human T cells. Following stimulation with the anti-CD3 antibody 12F6, proliferation of activated T cells was almost completely inhibited by 10 microM imatinib. Furthermore, antigen-triggered expansion of CD8+ T cells in response to immunodominant cytomegalovirus (CMV) and Epstein-Barr virus (EBV) peptides was significantly reduced. Up-regulation of the activation markers CD25 and CD69 in response to TCR cross-linking was suppressed by imatinib at a mean inhibitory concentration 50% (IC50) of 5.4 microM and 7.3 microM, respectively; interleukin 2 (IL-2) production was also impaired. Analysis of the TCR-induced signaling cascade showed that imatinib substantially reduced tyrosine phosphorylation of ZAP70 and LAT in response to activation through the TCR. Sequence comparisons of all 90 tyrosine kinase genes in the human genome for homology in the adenosine triphosphate (ATP) binding pocket identified LCK, which is required for ZAP70 activation, as a likely target for imatinib. The IC50 for LCK inhibition by imatinib was 0.6 microM to 0.8 microM in an in vitro tyrosine kinase assay. In summary, imatinib can interfere with T-cell activation in vitro, and its impact on the frequency of opportunistic infections and graft-versus-host or graft-versus-leukemia reactions after transplantation should be investigated in clinical trials.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Distinct genomic integration of MLV and SIV vectors in primate hematopoietic stem and progenitor cells.

Murine leukemia virus (MLV)-derived vectors are widely used for hematopoietic stem cell (HSC) gene transfer, but lentiviral vectors such as the simian immunodeficiency virus (SIV) may allow higher efficiency transfer and better expression. Recent studies in cell lines have challenged the notion that retroviruses and retroviral vectors integrate randomly into their host genome. Medical applications using these vectors are aimed at HSCs, and thus large-scale comprehensive analysis of MLV and SIV integration in long-term repopulating HSCs is crucial to help develop improved integrating vectors. We studied integration sites in HSCs of rhesus monkeys that had been transplanted 6 mo to 6 y prior with MLV- or SIV-transduced CD34(+)cells. Unique MLV (491) and SIV (501) insertions were compared to a set of in silico-generated random integration sites. While MLV integrants were located predominantly around transcription start sites, SIV integrants strongly favored transcription units and gene-dense regions of the genome. These integration patterns suggest different mechanisms for integration as well as distinct safety implications for MLV versus SIV vectors.

Animals↗
PubMed2004

Low-dose total body irradiation causes clonal fluctuation of primate hematopoietic stem and progenitor cells.

Due to high frequency of side effects caused by high-dose total body irradiation (TBI) the nonmyeloablative regimen together with cytotoxic agents is currently used especially for elderly patients. However, immediate and long-term effects of low-dose irradiation used in allogeneic transplantation on stem cells is less well known. We have studied the effect of low-dose 3 Gy TBI on the number of hematopoietic stem cell (HSC) clones contributing simultaneously to granulocyte production in rhesus macaque. The number of clones after 3 Gy TBI decreased markedly by 2 to 3 weeks after 3 Gy TBI, followed by a period of clonal instability, and recovery to almost pre-3 Gy TBI clonal diversity. The clones accounting for this recovery contributed before 3 Gy TBI, suggesting the profound initial impact of TBI was on a pool of progenitor cells, whereas most of the more primitive HSCs remained unaffected and were able to again contribute to hematopoiesis after recovery. Clonal fluctuation may indirectly suggest the presence of short-term/long-term HSC populations in rhesus macaque bone marrow as reported in a mouse model. The results indicate that even low-dose irradiation affects hematopoietic clonal dynamics and have implications for design of conditioning regimens for transplantation purposes.

Animals↗
PubMed2004

MRI detection of single particles for cellular imaging.

There is rapid growth in the use of MRI for molecular and cellular imaging. Much of this work relies on the high relaxivity of nanometer-sized, ultrasmall dextran-coated iron oxide particles. Typically, millions of dextran-coated ultrasmall iron oxide particles must be loaded into cells for efficient detection. Here we show that single, micrometer-sized iron oxide particles (MPIOs) can be detected by MRI in vitro in agarose samples, in cultured cells, and in mouse embryos. Experiments studying effects of MRI resolution and particle size from 0.76 to 1.63 microm indicated that T(2)* effects can be readily detected from single MPIOs at 50-microm resolution and significant signal effects could be detected at resolutions as low as 200 microm. Cultured cells were labeled with fluorescent MPIOs such that single particles were present in individual cells. These single particles in single cells could be detected both by MRI and fluorescence microscopy. Finally, single particles injected into single-cell-stage mouse embryos could be detected at embryonic day 11.5, demonstrating that even after many cell divisions, daughter cells still carry individual particles. These results demonstrate that MRI can detect single particles and indicate that single-particle detection will be useful for cellular imaging.

Animals↗