Lentiviral-transduced human mesenchymal stem cells persistently express therapeutic levels of enzyme in a xenotransplantation model of human disease.

Bone marrow-derived mesenchymal stem cells (MSCs) are a promising platform for cell- and gene-based treatment of inherited and acquired disorders. We recently showed that human MSCs distribute widely in a murine xenotransplantation model. In the current study, we have determined the distribution, persistence, and ability of lentivirally transduced human MSCs to express therapeutic levels of enzyme in a xenotransplantation model of human disease (nonobese diabetic severe combined immunodeficient mucopolysaccharidosis type VII [NOD-SCID MPSVII]). Primary human bone marrow-derived MSCs were transduced ex vivo with a lentiviral vector expressing either enhanced green fluorescent protein or the lysosomal enzyme beta-glucuronidase (MSCs-GUSB). Lentiviral transduction did not affect any in vitro parameters of MSC function or potency. One million cells from each population were transplanted intraperitoneally into separate groups of neonatal NOD-SCID MPSVII mice. Transduced MSCs persisted in the animals that underwent transplantation, and comparable numbers of donor MSCs were detected at 2 and 4 months after transplantation in multiple organs. MSCs-GUSB expressed therapeutic levels of protein in the recipients, raising circulating serum levels of GUSB to nearly 40% of normal. This level of circulating enzyme was sufficient to normalize the secondary elevation of other lysosomal enzymes and reduce lysosomal distention in several tissues. In addition, at least one physiologic marker of disease, retinal function, was normalized following transplantation of MSCs-GUSB. These data provide evidence that transduced human MSCs retain their normal trafficking ability in vivo and persist for at least 4 months, delivering therapeutic levels of protein in an authentic xenotransplantation model of human disease.

In vivo biosafety model to assess the risk of adverse events from retroviral and lentiviral vectors.

Serious adverse events in some human gene therapy clinical trials have raised safety concerns when retroviral or lentiviral vectors are used for gene transfer. We evaluated the potential for generating replication-competent retrovirus (RCR) and assessed the risk of occurrence of adverse events in an in vivo system. Human hematopoietic stem and progenitor cells (HSCs) and mesenchymal stem cells (MSCs) transduced with two different Moloney murine leukemia virus (MoMuLV)-based vectors were cotransplanted into a total of 481 immune-deficient mice (that are unable to reject cells that become transformed), and the animals were monitored for 18 months. Animals with any signs of illness were immediately killed, autopsied, and subjected to a range of biosafety studies. There was no detectable evidence of insertional mutagenesis leading to human leukemias or solid tumors in the 18 months during which the animals were studied. In 117 serum samples analyzed by vector rescue assay there was no detectable RCR. An additional 149 mice received HSCs transduced with lentiviral vectors, and were followed for 2-6 months. No vector-associated adverse events were observed, and none of the mice had detectable human immunodeficiency virus (HIV) p24 antigen in their sera. Our in vivo system, therefore, helps to provide an assessment of the risks involved when retroviral or lentiviral vectors are considered for use in clinical gene therapy applications.

19F magnetic resonance imaging for stem/progenitor cell tracking with multiple unique perfluorocarbon nanobeacons.

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninvasively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle-labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro-15-crown-5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+ CD133+ CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC-labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.

Long-term persistence of donor nuclei in a Duchenne muscular dystrophy patient receiving bone marrow transplantation.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle-wasting disorder caused by mutations in the dystrophin gene. Studies have shown that bone marrow cells transplanted into lethally irradiated mdx mice, the mouse model of DMD, can become part of skeletal muscle myofibers. Whether human marrow cells also have this ability is unknown. Here we report the analysis of muscle biopsies from a DMD patient (DMD-BMT1) who received bone marrow transplantation at age 1 year for X-linked severe combined immune deficiency and who was diagnosed with DMD at age 12 years. Analysis of muscle biopsies from DMD-BMT1 revealed the presence of donor nuclei within a small number of muscle myofibers (0.5-0.9%). The majority of the myofibers produce a truncated, in-frame isoform of dystrophin lacking exons 44 and 45 (not wild-type). The presence of bone marrow-derived donor nuclei in the muscle of this patient documents the ability of exogenous human bone marrow cells to fuse into skeletal muscle and persist up to 13 years after transplantation.

Leaky ribosomal scanning in mammalian genomes: significance of histone H4 alternative translation in vivo.

Like alternative splicing, leaky ribosomal scanning (LRS), which occurs at suboptimal translational initiation codons, increases the physiological flexibility of the genome by allowing alternative translation. Comprehensive analysis of 22 208 human mRNAs indicates that, although the most important positions relative to the first nucleotide of the initiation codon, -3 and +4, are usually such that support initiation (A-3 = 42%, G-3 = 36% and G+4 = 47%), only 37.4% of the genes adhere to the purine (R)-3/G+4 rule at both positions simultaneously, suggesting that LRS may occur in some of the remaining (62.6%) genes. Moreover, 12.5% of the genes lack both R-3 and G+4, potentially leading to sLRS. Compared with 11 genes known to undergo LRS, 10 genes with experimental evidence for high fidelity A+1T+2G+3 initiation codons adhered much more strongly to the R-3/G+4 rule. Among the intron-less histone genes, only the H3 genes adhere to the R-3/G+4 rule, while the H1, H2A, H2B and H4 genes usually lack either R-3 or G+4. To address in vivo the significance of the previously described LRS of H4 mRNAs, which results in alternative translation of the osteogenic growth peptide, transgenic mice were engineered that ubiquitously and constitutively express a mutant H4 mRNA with an A+1T+1 mutation. These transgenic mice, in particular the females, have a high bone mass phenotype, attributable to increased bone formation. These data suggest that many genes may fulfill cryptic functions by LRS.

Immune-deficient mouse models for analysis of human stem cells.

The field of murine models of xenotransplantation has grown immensely over the past two decades. The explosive growth in this field is in part due to the fact that good in vitro methods do not exist yet to allow examination of human stem cell homing into the bone marrow compartment versus other tissues, long-term survival of human stem cells, or differentiation into tissues outside of the hematopoietic system. Since these important aspects of human stem cell biology can be examined in vivo using immune-deficient mice, the number of different strains and models is constantly increasing. The current review discusses the merits and drawbacks of each immune-deficient mouse xenograft system as it stands to date and reviews how each immune-deficient mouse model has been used to further our knowledge of human hematopoietic stem cell biology.

Mesenchymal stem cells for the sustained in vivo delivery of bioactive factors.

Mesenchymal stem cells (MSC) are a promising tool for cell therapy, either through direct contribution to the repair of bone, tendon and cartilage or as an adjunct therapy through protein production and immune mediation. They are an attractive vehicle for cellular therapies due to a variety of cell intrinsic and environmentally responsive properties. Following transplantation, MSC are capable of systemic migration, are not prone to tumor formation, and appear to tolerize the immune response across donor mismatch. These attributes combine to allow MSC to reside in many different tissue types without disrupting the local microenvironment and, in some cases, responding to the local environment with appropriate protein secretion. We describe work done by our group and others in using human MSC for the sustained in vivo production of supraphysiological levels of cytokines for the support of cotransplanted hematopoietic stem cells and enzymes that are deficient in animal models of lysosomal storage disorders such as MPSVII. In addition, the use of MSC engineered to secrete protein products has been reviewed in several fields of tissue injury repair, including but not limited to revascularization after myocardial infarction, regeneration of intervertebral disc defects and spine therapy, repair of stroke, therapy for epilepsy, skeletal tissue repair, chondrogenesis/knee and joint repair, and neurodegenerative diseases. Genetically engineered MSC have thus proven safe and efficacious in numerous animal models of disease modification and tissue repair and are poised to be tested in human clinical trials. The potential for these interesting cells to secrete endogenous or transgene products in a sustained and long-term manner is highly promising and is discussed in the current review.

In vivo distribution of human adipose-derived mesenchymal stem cells in novel xenotransplantation models.

The potential for human adipose-derived mesenchymal stem cells (AMSC) to traffic into various tissue compartments was examined using three murine xenotransplantation models: nonobese diabetic/severe combined immunodeficient (NOD/SCID), nude/NOD/SCID, and NOD/SCID/MPSVII mice. Enhanced green fluorescent protein was introduced into purified AMSC via retroviral vectors to assist in identification of cells after transplantation. Transduced cells were administered to sublethally irradiated immune-deficient mice through i.v., intraperitoneal, or subcutaneous injection. Up to 75 days after transplantation, tissues were harvested and DNA polymerase chain reaction (PCR) was performed for specific vector sequences as well as for human Alu repeat sequences. Duplex quantitative PCR using human beta-globin and murine rapsyn primers assessed the contribution of human cells to each tissue. The use of the novel NOD/SCID/MPSVII mouse as a recipient allowed rapid identification of human cells in the murine tissues, using an enzyme reaction that was independent of surface protein expression or transduction with an exogenous transgene. For up to 75 days after transplantation, donor-derived cells were observed in multiple tissues, consistently across the various administration routes and independent of transduction parameters. Tissue localization studies showed that the primary MSC did not proliferate extensively at the sites of lodgement. We conclude that human AMSC represent a population of stem cells with a ubiquitous pattern of tissue distribution after administration. AMSC are easily obtained and highly amenable to current transduction protocols for retroviral transduction, making them an excellent avenue for cell-based therapies that involve a wide range of end tissue targets.

In vivo transduction of hematopoietic stem cells after neonatal intravenous injection of an amphotropic retroviral vector in mice.

Hematopoietic stem cells (HSC) are important targets for gene therapy. Most protocols involve ex vivo modification, in which HSC are transduced in vitro and injected into the recipient. An in vivo delivery method might simplify HSC gene therapy. We previously demonstrated that iv injection of an amphotropic retroviral vector (RV) into newborn mice resulted in long-term expression from hepatocytes. The goal of this study was to determine if HSC were also transduced. After neonatal administration of 1 x 10(10) transducing units/kg of RV, peripheral blood cells had approximately 0.1 copy of RV per cell for up to 22 months. At 18 months, RV sequences were detected in T, B, and myeloid cells from bone marrow (BM). Unfractionated BM was transplanted into naive recipients after total body irradiation. Recipients maintained similar levels of the RV in their blood cells for 10 months, at which time RV sequences were present at the same integration site in all lineages of cells from BM. We conclude that neonatal iv injection of RV results in transduction of HSC in mice, which might be used for BM-directed gene therapy. Transduction of blood cells after liver-directed neonatal gene therapy might have adverse effects in patients, although no leukemias developed here.

Transduction and selection of human T cells with novel CD34/thymidine kinase chimeric suicide genes for the treatment of graft-versus-host disease.

Clinical trials evaluating the herpes simplex virus thymidine kinase (HSV-tk)/ganciclovir (GCV) suicide gene therapy system for the control of graft-versus-host disease (GVHD) have been limited by low transduction efficiencies and inefficient selection procedures. In this study, we designed and evaluated a novel chimeric suicide gene consisting of the extracellular and transmembrane domains of human CD34 and full-length HSV-tk (DeltaCD34-tk). High-efficiency transfer of DeltaCD34-tk to primary human T cells was accomplished after a single exposure to VSV-G-pseudotyped, Moloney murine leukemia virus-based retrovirus 48 h after activation of human PBMCs with anti-CD3 and anti-CD28 antibodies immobilized on magnetic beads. Using an optimized 5-day transduction and selection procedure, transduction efficiencies averaged 71%, with isolation purities greater than 95% and yields exceeding 90%. The immunoselected T cells were selectively eliminated by GCV (IC(50) approximately 3 nM), maintained a normal subset composition, exhibited a polyclonal TCR Vbeta family repertoire, and contained 5 or 6 vector copies per transduced cell when optimally transduced. No increase in GCV sensitivity was observed upon incorporation of highly active mutant HSV-tk enzymes into the DeltaCD34-tk suicide gene. T cells modified with the DeltaCD34-tk gene using the optimized protocol should improve the overall efficacy of the HSV-tk/GCV suicide gene therapy method of GVHD control.

Functional characterization of highly purified human hematopoietic repopulating cells isolated according to aldehyde dehydrogenase activity.

Human hematopoietic stem cells (HSCs) are commonly purified by the expression of cell surface markers such as CD34. Because cell phenotype can be altered by cell cycle progression or ex vivo culture, purification on the basis of conserved stem cell function may represent a more reliable way to isolate various stem cell populations. We have purified primitive HSCs from human umbilical cord blood (UCB) by lineage depletion (Lin(-)) followed by selection of cells with high aldehyde dehydrogenase (ALDH) activity. ALDH(hi)Lin(-) cells contained 22.6% +/- 3.0% of the Lin(-) population and highly coexpressed primitive HSC phenotypes (CD34(+) CD38(-) and CD34(+)CD133(+)). In vitro hematopoietic progenitor function was enriched in the ALDH(hi)Lin(-) population, compared with ALDH(lo)Lin(-) cells. Multilineage human hematopoietic repopulation was observed exclusively after transplantation of ALDH(hi)Lin(-) cells. Direct comparison of repopulation with use of the nonobese diabetic/severe combined immunodeficient (NOD/SCID) and NOD/SCID beta2 microglobulin (beta2M) null models demonstrated that 10-fold greater numbers of ALDH(hi)-Lin(-) cells were needed to engraft the NOD/SCID mouse as compared with the more permissive NOD/SCID beta2M null mouse, suggesting that the ALDH(hi)Lin(-) population contained committed progenitors as well as primitive repopulating cells. Cell fractionation according to lineage depletion and ALDH activity provides a viable and prospective purification of HSCs on the basis of cell function rather than cell surface phenotype.