Comparison of mesenchymal stem cells from different tissues to suppress T-cell activation.

Graft-versus-host disease (GvHD) and graft rejection have remained significant complications of allogeneic stem cell transplantation. Mesenchymal stem cells (MSCs) from the bone marrow have been shown to suppress T-cell activation in vitro and in vivo, and may be used to reduce GvHD in the recipient or to facilitate engraftment across MHC barriers. MSCs can be derived from a variety of tissues.

Hematopoietic stem cell engraftment: a direct comparison between intramarrow and intravenous injection in nonhuman primates.

Objective: Recent studies in the mouse model have shown improved engraftment of repopulating cells when cells were administered by intramarrow (IM) vs intravenous (IV) injection. Here we wished to determine if IM injection was feasible and would result in improved engraftment in a clinically relevant large animal model.

Materials And Methods: We used a competitive repopulation assay to directly compare IM vs IV injection in four baboons.

In vivo selection and chemoprotection after drug resistance gene therapy in a nonmyeloablative allogeneic transplantation setting in dogs.

We have previously demonstrated successful in vivo selection, chemoprotection, and modulation of donor chimerism in dogs that received myeloablative allogeneic stem cell transplantation with cells expressing the P140K mutant of the DNA repair enzyme methylguanine methyltransferase (MGMTP140K). Here, we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after nonmyeloablative transplantation, which could allow for less toxic transplantation regimens for patients with malignant and genetic diseases. Three dogs received a nonmyeloablative conditioning regimen and infusion of allogeneic stem cells transduced with MGMTP140K.

Hematopoietic stem-cell behavior in nonhuman primates.

Little is known about the behavior of hematopoietic stem cells (HSCs) in primates because direct observations and competitive-repopulation assays are not feasible. Therefore, we used 2 different and independent experimental strategies, the tracking of transgene expression after retroviral-mediated gene transfer (N = 11 baboons; N = 7 rhesus macaques) and quantitation of the average telomere length of granulocytes (N = 132 baboons; N = 14 macaques), together with stochastic methods, to study HSC kinetics in vivo. The average replication rate for baboon HSCs is once per 36 weeks according to gene-marking analyses and once per 23 weeks according to telomere-shortening analyses.

Unique integration profiles in a canine model of long-term repopulating cells transduced with gammaretrovirus, lentivirus, or foamy virus.

Recent advances have allowed for improved retrovirus-mediated gene transfer, and therapeutic benefits have been described in patients. These successes have shown the potential of hematopoietic stem cell (HSC) gene therapy, but treatment-related leukemia and benign expansion of gene-modified clones have shifted the attention toward safety. The delayed onset of adverse events in gene therapy clinical trials emphasizes the importance of long-term integration site studies in large animal models.

Effects of HOXB4 overexpression on ex vivo expansion and immortalization of hematopoietic cells from different species.

Overexpression of the human HOXB4 has been shown to induce the expansion and self-renewal of murine hematopoietic stem cells. In preparation for clinical studies, we wished to investigate the effects of HOXB4 on cells from other species, in particular preclinical large animals such as dogs and nonhuman primates. Thus, we transduced CD34(+) cells from nonhuman primates, dogs, and humans with a HOXB4-expressing gammaretroviral vector and a yellow fluorescent protein-expressing control vector.

Differential effects of bexarotene on intrinsic and extrinsic pathways in TRAIL-induced apoptosis in two myeloid leukemia cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces programmed cell death (apoptosis) preferentially in tumor cells. However, not all cancer cells are sensitive to TRAIL. We determined whether ligation of the retinoid receptor, RXR, would sensitize cells to TRAIL-mediated apoptosis.

Comparison of HIV-derived lentiviral and MLV-based gammaretroviral vector integration sites in primate repopulating cells.

The potential for leukemia caused by retroviral vector integration has become a significant concern for hematopoietic stem cell gene therapy. We analyzed the distribution of vector integrants in pigtailed macaque and baboon repopulating cells for the two most commonly used retroviral vector systems, human immunodeficiency virus (HIV)-based lentiviral vectors and murine leukemia virus (MLV)-based gammaretroviral vectors, to help define their relative genotoxicity. All animals had polyclonal engraftment with no apparent adverse effects from transplantation with gene-modified cells.

Improved short-term engraftment of lentivirally versus gammaretrovirally transduced allogeneic canine repopulating cells.

Gammaretroviral vectors require cell division for efficient transduction. Thus, extended cell culture times are necessary for efficient transduction with gammaretroviral vectors, which in turn can lead to stem cell loss and impaired engraftment. Lentiviral vectors transduce nondividing cells and are therefore able to transduce stem cells in short transduction protocols.

Extended core sequences from the cHS4 insulator are necessary for protecting retroviral vectors from silencing position effects.

The prototypic chromatin insulator cHS4 has proven effective at reducing repressive chromosomal position effects on retroviral vector expression. We report here studies designed to identify the minimal chicken hypersensitive site-4 (cHS4) sequences necessary for this activity. Using a gammaretroviral reporter vector and expression analysis in cell lines and primary mouse hematopoietic progenitor colonies, we found that a 250-bp core fragment reported to contain most of the cHS4 insulating activity failed to prevent silencing when used alone, although some barrier activity was observed when this fragment was combined with a 790-bp, but not 596-bp, spacer.

Foamy-virus-mediated gene transfer to canine repopulating cells.

Foamy virus (FV) vectors are particularly attractive gene-transfer vectors for stem-cell gene therapy because they form a stable transduction intermediate in quiescent cells and can efficiently transduce hematopoietic stem cells. Here, we studied the use of FV vectors to transduce long-term hematopoietic repopulating cells in the dog, a clinically relevant large animal model. Mobilized canine peripheral blood (PB) CD34+ cells were transduced with an enhanced green fluorescent protein (EGFP)-expressing FV vector in an 18-hour transduction protocol.

Continuous electrical oscillations emerge from a coupled network: a study of the inferior olive using lentiviral knockdown of connexin36.

Do continuous subthreshold oscillations in membrane potential within an electrically coupled network depend on gap junctional coupling? For the inferior olive (IO), modeling and developmental studies suggested that the answer is yes, although physiological studies of connexin36 knock-out mice lacking electrical coupling suggested that the answer is no. Here we addressed the question differently by using a lentivirus-based vector to express, in the IO of adult rats, a single amino acid mutation of connexin36 that disrupts the intracellular trafficking of wild-type connexin36 and blocks gap junctional coupling. Confocal microscopy of green fluorescence protein-labeled dendrites revealed that the mutant connexin36 prevented wild-type connexin36 from being expressed in dendritic spines of IO neurons.

Complications of a temozolomide overdose: a case report.

Purpose And Background: This is a report of a 53 year-old man with a glioblastoma multiforme (GBM) treated with an excessive dose of temozolomide (TMZ).

Methods: This is a single case review of all clinically relevant records. O6-methylguanine-DNA methyltransferase activity was determined by a biochemical assay.

Differential effects of HOXB4 on nonhuman primate short- and long-term repopulating cells.

Background: Hematopoietic stem cells (HSCs) or repopulating cells are able to self-renew and differentiate into cells of all hematopoietic lineages, and they can be enriched using the CD34 cell surface marker. Because of this unique property, HSCs have been used for HSC transplantation and gene therapy applications. However, the inability to expand HSCs has been a significant limitation for clinical applications.

Reduced incidence of acute and chronic graft-versus-host disease with the addition of thymoglobulin to a targeted busulfan/cyclophosphamide regimen.

To reduce the incidence of graft-versus-host disease (GVHD), we added Thymoglobulin (THY) to dose-adjusted oral busulfan plus cyclophosphamide (targeted BUCY). The starting dose of THY was 4.5 mg/kg given over days -3, -2, and -1, escalated in steps of 1.

Efficient transduction and engraftment of G-CSF-mobilized peripheral blood CD34+ cells in nonhuman primates using GALV-pseudotyped gammaretroviral vectors.

The optimal stem cell source for stem cell gene therapy has yet to be determined. Most large-animal studies have utilized peripheral blood or marrow-derived cells collected after administration of granulocyte colony-stimulating factor (G-SCF) and stem cell factor (SCF); however, SCF is unavailable for clinical use in the United States and the European Union. A recent study in a competitive repopulation assay in the rhesus macaque showed very inefficient marking of G-CSF-mobilized (G/only) peripheral blood (G-PBSC) CD34(+) cells relative to G-CSF and SCF-mobilized cells using vectors with an amphotropic pseudotype.

Foamy virus vector integration sites in normal human cells.

Foamy viruses (FVs) or spumaviruses are retroviruses that have been developed as vectors, but their integration patterns have not been described. We have performed a large-scale analysis of FV integration sites in unselected human fibroblasts (n = 1,008) and human CD34(+) hematopoietic cells (n = 1,821) by using a bacterial shuttle vector and a comparable analysis of lentiviral vector integration sites in CD34(+) cells (n = 1,331). FV vectors had a distinct integration profile relative to other types of retroviruses.

Hematopoietic stem cell transduction and amplification in large animal models.

Progress in retroviral gene transfer to large animal hematopoietic stem cells (HSCs) has led to efficient, reproducible long-term marking in both canine and nonhuman primate models. Successes for HSC gene therapy have occurred in the severe combined immunodeficiency setting, in which transduced cells have a selective advantage. However, for most diseases, the therapeutic transgene does not confer a sufficient survival advantage, and increasing the percentage of gene-marked cells in vivo will be necessary to observe a therapeutic effect.

Survival of the fittest: in vivo selection and stem cell gene therapy.

Stem cell gene therapy has long been limited by low gene transfer efficiency to hematopoietic stem cells. Recent years have witnessed clinical success in select diseases such as X-linked severe combined immunodeficiency (SCID) and ADA deficiency. Arguably, the single most important factor responsible for the increased efficacy of these recent protocols is the fact that the genetic correction provided a selective in vivo survival advantage.

Rapid 1-hour transduction of whole bone marrow leads to long-term repopulation of murine recipients with lentivirus-modified hematopoietic stem cells.

Efficient gene transfer to hematopoietic stem cells by Moloney murine leukemia virus-derived retroviral vectors benefits from ex vivo culture and cytokine support. Both also increase the risks of apoptosis and differentiation among cells targeted for transduction. In an effort to maximize the retention of stem cell properties in target cells, we developed a transduction protocol with a focus on minimizing graft manipulation, cytokine stimulation, and ex vivo exposure duration.

Genetically modified CD34+ cells do not contribute to the mesenchymal compartment after autologous transplantation in the baboon.

Background: There is ongoing controversy about the transdifferentiation of hematopoietic stem cells (HSC) into different tissues such as mesenchymal cells. This transdifferentiation or 'plasticity' would be an appealing concept for many therapeutic strategies. While studies in the murine model show encouraging results, reports from clinical allogeneic stem cell transplantations do not support the concept of HSC plasticity.

A phase I/II study of mycophenolate mofetil in combination with cyclosporine for prophylaxis of acute graft-versus-host disease after myeloablative conditioning and allogeneic hematopoietic cell transplantation.

Abstract In a phase I/II study, the combination of cyclosporine (CSP) and mycophenolate mofetil (MMF) was investigated as graft-versus-host disease (GVHD) prophylaxis after myeloablative conditioning and hematopoietic cell transplantation from an HLA-matched sibling donor. In phase I, 3 groups, each with 10 or 11 patients, received MMF (15 mg/kg) from day 0 to day 27 at decreasing dose intervals of every 12, 8, and 6 hours to determine a safe and effective total daily dose. At the 45 mg/kg/d dosage level, 4 of 11 patients developed only grade II GVHD, and a concentration at steady state of mycophenolic acid (the active moiety of MMF) consistent with a therapeutic range described for solid-organ transplantation was achieved.

Low-dose cyclophosphamide conditioning for haematopoietic cell transplantation from HLA-matched related donors in patients with Fanconi anaemia.

Allogeneic haematopoietic cell transplantation (HCT) is effective therapy for Fanconi anaemia (FA). FA patients do not tolerate conditioning with 200 mg/kg of cyclophosphamide (Cy), typically used in aplastic anaemia. We previously published results of studies in which Cy doses were gradually reduced from 200 to 100 mg/kg.