T Lymphocytes transduced with a lentiviral vector expressing F12-Vif are protected from HIV-1 infection in an APOBEC3G-independent manner.

The viral infectivity factor (Vif) is an essential component of the HIV-1 infectious cycle. Vif counteracts the action of the cytidine deaminase APOBEC3G (AP3G), which confers nonimmune antiviral defense against HIV-1 to T lymphocytes. Disabling or interfering with the function of Vif could represent an alternative therapeutic approach to AIDS. We have expressed a natural mutant of Vif, F12-Vif, in a VSV-G-pseudotyped lentiviral vector under the Tat-inducible control of the HIV-1 LTR. Conditional expression of F12-Vif prevents replication and spreading of both CXCR4 and CCR5 strains of HIV-1 in human primary T lymphocyte and T cell lines. T cells transduced with F12-Vif release few HIV-1 virions and with reduced infectivity. Several lines of evidence indicate that HIV-1 interference requires the presence of both wild-type and F12-Vif proteins, suggesting a dominant-negative feature of the F12-Vif mutant. Surprisingly, however, the F12-Vif-mediated inhibition does not depend on the reestablishment of the AP3G function.     

Multipotential marrow stromal cells transduced to produce L-DOPA: engraftment in a rat model of Parkinson disease.

Bone marrow stromal cells can be used as an alternative source of cells for neural transplantation and repair. Here, the efficacy of genetically modified marrow stromal cells was examined in a rat model of Parkinson disease. Rat marrow stromal cells (rMSCs) and human marrow stromal cells (hMSCs) were genetically engineered by transduction with retroviruses encoding tyrosine hydroxylase (TH) and GTP cyclohydrolase I, the enzyme necessary for production of the tetrahydrobiopterin cofactor for TH (BH4). Transduced cells synthesized 3,4-dihydroxyphenylalanine (L-DOPA) in vitro and maintained their multipotentiality after retroviral transduction. To examine the cells in vivo, transduced rMSCs were injected into the striatum of 6-hydroxydopamine-lesioned rats. L-DOPA and metabolites were detected by microdialysis in the denervated striatum of rats that received doubly transduced rMSCs. Also, there was a significant reduction in apomorphine-induced rotation when compared with controls. The cells engrafted and survived for at least 87 days. However, expression of the transgenes ceased at about 9 days, an observation consistent with reports from other laboratories in which similar retroviruses were used to express transgenes in the brain.     

Successful immunization with a single injection of non-integrating lentiviral vector.

We evaluated the ability of an integrase (IN)-defective self-inactivating lentiviral vector (sinLV) for the delivery of human immunodeficiency virus-1 (HIV-1) envelope sequences in mice to elicit specific immune responses. BALB/c mice were immunized with a single intramuscular injection of the IN-defective sinLV expressing the codon optimized HIV-1(JR-FL) gp120 sequence, and results were compared with those for the IN-competent counterpart. The IN-defective sinLV elicited specific and long-lasting immune responses, as evaluated up to 90 days from the immunization by enzyme-linked immunosorbent spot (ELISPOT) and intracellular staining (ICS) for interferon-gamma (IFN-gamma) assays in both splenocytes and bone marrow (BM) cells, chromium release assay in splenocytes, and antibody detection in sera, without integration of the vector into the host genome. These data provide evidence that a single administration of an IN-defective sinLV elicits a significant immune response in the absence of vector integration and may be a safe and useful strategy for vaccine development.     

Long-term engraftment of nonobese diabetic/severe combined immunodeficient mice with human CD34+ cells transduced by a self-inactivating human immunodeficiency virus type 1 vector

Human hematopoietic cells with in vivo repopulating potential hold much promise as a target for corrective gene transfer for numerous inherited or acquired hematopoietic disorders. Here we demonstrate long-term hematopoietic reconstitution of nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice with human CD34(+) cells transduced by an HIV-1-based self-inactivating (SIN) vector encoding the enhanced green fluorescent protein (EGFP). Human umbilical cord CD34(+) cells were transduced (up to 76%) at a low multiplicity of infection (MOI of 5) in the absence of cytokine prestimulation. Introduction of transduced hCD34(+) cells into irradiated recipients resulted in multilineage engraftment and stable transgene expression for 18 weeks posttransplantation. Bone marrow from transplanted mice contained up to 50% hCD45(+) cells and up to 63% hCD45(+)/EGFP(+) cells. Analysis of extramedullar splenic reconstitution showed up to 13% hCD45(+) cells and up to 41% hCD45(+)/EGFP(+) cells. Analysis of human progenitor cells isolated from bone marrow of recipient animals showed equivalent percentages of EGFP(+) colony-forming cells (CFCs) by fluorescence microscopy and by PCR analysis of provirus sequences, indicating minimal transgene silencing in vivo. These findings demonstrate the utility of lentivirus-based SIN vectors for hematopoietic stem cell gene transfer and provide strong support for their future clinical evaluation.     

Autologous transplantation of SM/C-2.6(+) satellite cells transduced with micro-dystrophin CS1 cDNA by lentiviral vector into mdx mice.

Duchenne muscular dystrophy (DMD) is a lethal muscle disorder caused by mutations in the dystrophin gene. Transplantation of autologous myogenic cells genetically corrected ex vivo is a possible treatment for this disorder. In order to test the regenerative efficiency of freshly isolated satellite cells, we purified quiescent satellite cells from limb muscles of 8-12-week-old green fluorescent protein-transgenic (GFP-Tg) mice using SM/C-2.6 (a recently developed monoclonal antibody) and flow cytometry. Freshly isolated satellite cells were shown to participate in muscle regeneration more efficiently than satellite cell-derived myoblasts passaged in vitro do, when transplanted into tibialis anterior (TA) muscles of 8-12-week-old cardiotoxin-injected C57BL/6 mice and 5-week-old dystrophin-deficient mdx mice, and analyzed at 4 weeks after injection. Importantly, expansion of freshly isolated satellite cells in vitro without passaging had no detrimental effects on their regenerative capacity. Therefore we directly isolated satellite cells from 5-week-old mdx mice using SM/C-2.6 antibody and cultured them with lentiviral vectors expressing micro-dystrophin CS1. The transduced cells were injected into TA muscles of 5-week-old mdx mice. At 4 weeks after transplantation, the grafted cells efficiently contributed to regeneration of mdx dystrophic muscles and expressed micro-dystrophin at the sarcolemma. These results suggest that there is potential for lentiviral vector-mediated ex vivo gene therapy for DMD.     

Corticospinal tract transduction: a comparison of seven adeno-associated viral vector serotypes and a non-integrating lentiviral vector

The corticospinal tract (CST) is extensively used as a model system for assessing potential therapies to enhance neuronal regeneration and functional recovery following spinal cord injury (SCI). However, efficient transduction of the CST is challenging and remains to be optimised. Recombinant adeno-associated viral (AAV) vectors and integration-deficient lentiviral vectors are promising therapeutic delivery systems for gene therapy to the central nervous system (CNS). In the present study the cellular tropism and transduction efficiency of seven AAV vector serotypes (AAV1, 2, 3, 4, 5, 6, 8) and an integration-deficient lentiviral vector were assessed for their ability to transduce corticospinal neurons (CSNs) following intracortical injection. AAV1 was identified as the optimal serotype for transducing cortical and CSNs with green fluorescent protein (GFP) expression detectable in fibres projecting through the dorsal CST (dCST) of the cervical spinal cord. In contrast, AAV3 and AAV4 demonstrated a low efficacy for transducing CNS cells and AAV8 presented a potential tropism for oligodendrocytes. Furthermore, it was shown that neither AAV nor lentiviral vectors generate a significant microglial response. The identification of AAV1 as the optimal serotype for transducing CSNs should facilitate the design of future gene therapy strategies targeting the CST for the treatment of SCI.     

Development of an enhanced B-specific lentiviral vector expressing BTK: a tool for gene therapy of XLA

Further development of haematopoietic stem cell (HSC) gene therapy will depend on enhancement of gene transfer safety: ad hoc improvement of vector design relating to each particular disease is thus a crucial issue for HSC gene therapy. We modified a previously described lentiviral vector by adding the Emumar B-specific enhancer to a human CD19 promoter-derived sequence (Mol Ther 2004;10:45-56). We thus significantly improved the level of expression of the green fluorescent protein (GFP) reporter gene while retaining the specificity of expression in B-cell progeny of transduced human CD34+ progenitor cells obtained from cord blood or adult bone marrow. Indeed, GFP was strongly expressed from early medullary pro-B cells to splenic mature B cells whereas transgene expression remained low in transduced immature progenitors as in myeloid and T-lymphoid progeny retrieved from xenografted NOD/SCID/gammac(null) mice. Using this lentiviral vector, we further demonstrated the possibility to express a functional human BTK protein in long-term human CD34+ cell B-lymphoid progeny. This newly designed lentiviral vector fulfils one of the pre-requisites for the development of efficient and safe gene therapy for X-linked agammaglobulinaemia, the most common primary humoral immunodeficiency disorder.     

Brain attack: a new approach to stroke

'Brain' attack' is a new term to describe the acute presentation of stroke which emphasises the need for urgent action. The article describes the basis for this new approach to acute stroke treatment. Rational treatment requires individual causes of stroke to be identified early and treatment targeted at the mechanism. Acute stroke treatment aims to preserve the ischaemic penumbra, protect neurons against further ischaemia and enhance brain plasticity to maximise recovery. There is a strong evidence base supporting the routine use of aspirin, but not heparin, in acute ischaemic stroke. There is also convincing evidence supporting intravenous thrombolysis using recombinant tissue plasminogen activator in selected patients within 3 hours of stroke onset. Surprisingly, as many as 33% of suspected-stroke patients arrive in Accident & Emergency departments in the UK within 3 hours of onset. New techniques in MR imaging, particularly diffusion weighted imaging, are transforming the approach to diagnosis of acute stroke. Although neuroprotective drugs have proved disappointing, active neuroprotection in acute stroke should include control of blood pressure within certain limits, antipyretic therapy, maintenance of blood glucose, and early feeding and fluid replacement. Surgical hemicraniectomy should be considered in patients with malignant cerebral oedema. There is good evidence that the best way to enhance recovery from stroke is to admit the patient to a stroke unit. To enable patients to benefit from the early active approach outlined in the article, the following are needed: the development of acute stroke units; imaging protocols; and education of patients, general practitioners and the ambulance services. Stroke care has become a specialised field, requiring input from stroke physicians, as well as the multidisciplinary rehabilitation team. The British Association of Stroke Physicians (BASP) has therefore developed a curriculum which is designed to lead to the development of a new sub-specialty of stroke medicine.     

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector

Gene transfer using viral vectors offers the potential for the sustained expression of proteins in specific target tissues. However, in the case of calcified tissues, in vivo delivery remains problematic because of limited accessibility. The aim of this study was to test the efficiency of lentiviral vectors (LVs) on osteogenic cells in vitro, and determine the feasibility of directly transducing resident bone cells in vivo. LVs encoding for green fluorescent protein (GFP) and ameloblastin (AMBN), a protein associated with mineralization not reported in bone, were generated. The transduction efficiency of the LVs was evaluated using the MC3T3 cell line and primary calvaria-derived osteogenic cells. For in vivo delivery, the LVs were infused using osmotic minipumps through holes created in the bone of the rat hemimandible and tibia. The production of GFP and AMBN in vitro and in vivo was monitored using fluorescence microscopy. Both transgenes were expressed in MC3T3 and primary osteogenic cells. In vivo, GFP was detected at the infusion site and fibroblast-like cells, osteoblasts, osteocytes and osteoclasts expressed AMBN. Our data demonstrate, for the first time, that primary osteogenic cells are efficiently transduced with LVs and that their infusion is advantageous for locally delivering DNA to bone cells.     

Low-dose lenograstim to enhance engraftment after autologous stem cell transplantation: a prospective randomized evaluation of two different fixed doses

BACKGROUND: G-CSF is used to enhance hematopoietic recovery after autologous stem cell transplantation (ASCT), but the optimal dose of G-CSF during engraftment has not been established. The medical cost of ASCT is a serious financial burden in developing countries, and G-CSF is the most costly drug used in this procedure. We evaluated whether a lower, vial-size fitted dose of lenograstim is clinically equivalent to a higher fixed dose. STUDY DESIGN AND METHODS: A prospective randomized study was performed on 33 patients (11 non-Hodgkin's lymphoma, 8 multiple myeloma, 14 breast cancer) undergoing ASCT. Patients were randomly administered 100 micro g or 250 micro g lenograstim daily starting on the next day of ASCT, with a minimum infusion of 3 x 10(6) CD34+ cells per kg. RESULTS: For both lenograstim doses, median time to neutrophil engraftment was 9 days and median time to PLT engraftment was 11 days. Episodes of clinically documented infections were 10 per 379 patient-days in the 100 microg per day group and 10 per 320 patient-days in the 250 microg per day group. There were no between-group differences in requirements for transfusion of RBCs or PLTs. Duration of hospitalization was 16 days for the 100 microg per day group and 17 days for the 250 microg per day group. Daily lenograstim dose per patient's body weight and total amount of lenograstim used during ASCT were both significantly lower in the 100 microg per day group. CONCLUSION: Administration of 100 microg per day of lenograstim showed comparable clinical efficacy to 250 microg per day lenograstim for immediate hematopoietic recovery after ASCT. Use of the lower dose was associated with lower overall lenograstim usage and lower cost.     

Induction of immune tolerance to a transplantation carbohydrate antigen by gene therapy with autologous lymphocytes transduced with adenovirus containing the corresponding glycosyltransferase gene

Induction of tolerance to transplantation carbohydrate antigens is of clinical significance in recipients of ABO-incompatible allografts, or of xenografts. The experimental animal model used for studying such tolerance was that of alpha1,3galactosyltransferase (alpha1,3GT) knockout (KO) mice, which lacks the alpha-gal epitope (Galalpha1-3Galbeta1-4GlcNAc-R) and which can produce the anti-Gal antibody against it. In contrast, wild-type (WT) mice synthesize the alpha-gal epitope and are immunotolerant to it. KO lymphocytes transduced in vitro with adenovirus containing the alpha1,3GT gene (AdalphaGT) express alpha-gal epitopes. Administration of such lymphocytes into KO mice resulted in tolerization of na?ve and memory anti-Gal B cells. Mice tolerized by AdalphaGT transduced lymphocytes failed to produce anti-Gal following immunizations with pig kidney membranes (PKM) expressing multiple alpha-gal epitopes. This tolerance was perpetuated by transplanted syngeneic WT mouse hearts expressing alpha-gal epitopes. Transplanted WT hearts survived in the tolerized KO mice for at least 100 days, despite repeated PKM immunizations. Control mice receiving lymphocytes transduced with adenovirus lacking the alpha1,3GT gene were not tolerized, but produced anti-Gal and rejected transplanted WT hearts. This study suggests that autologous lymphocytes transduced with adenovirus containing A or B transferase genes may induce a similar tolerance to blood group antigens in humans.     

Activation of the complement system by different autologous transfusion devices: an in vitro study

BACKGROUND: The aim of the present investigation was to study whether autologous transfusion devices activate the complement system and whether complement-activated blood is more vulnerable to further activation during processing. STUDY DESIGN AND METHODS: Forty-eight blood units were randomized to be processed by one of three different salvage systems: Group 1 underwent whole blood filtration (hemofiltration) (n=16); Group 2 underwent continuous processing, saline washing, and centrifugation (CATS, Fresenius AG ) (n=16); and Group 3 underwent saline washing and centrifugation (Cell-Saver, Haemonetics Corp.) (n=16). Eight blood units for each system were activated with cobra venom factor (CVF) at a concentration of 0.2 U per mL whole blood before processing. C activation was studied by determinations of C4d, Bb, C3a, and SC5b-9. Samples were drawn from whole blood, processed blood, and the waste bags. RESULTS: The concentrations of Bb, C3a, and SC5b-9 in whole blood after activation with CVF were significantly elevated compared to blood that was not activated (p < 0.01). Processed blood from hemofiltration contained significantly higher levels of complement-split products than techniques that use washing and centrifugation. The concentrations of SC5b-9 in blood processed by hemofiltration were higher in the experiments with CVF activation (p < 0.05). CONCLUSION: The tested autologous transfusion systems did not themselves activate the complement system, and complement-activated blood was not more vulnerable to further activation during processing. A blood-salvaging technique that used washing and centrifugation reduced elevated concentrations of complement-split products, whereas hemofiltration did not.     

Mindboggle: a scatterbrained approach to automate brain labeling

Mindboggle (http://www.binarybottle.com/mindboggle.html) is a fully automated, feature matching approach to label cortical structures and activity anatomically in human brain MRI data. This approach does not assume that the existence of component structures and their relative spatial relationship is preserved from brain to brain, but instead disassembles a labeled atlas and reassembles its pieces to match corresponding pieces in an unlabeled subject brain before labeling. Mindboggle: (1) converts linearly coregistered subject and atlas MRI data into sulcus pieces, (2) matches each atlas piece with a combination of subject pieces by minimizing a cost function, (3) transforms atlas label boundaries to the matching subject pieces, (4) warps atlas labels to their transformed boundaries, and (5) propagates labels to fill remaining gaps in a mask derived from the subject brain. We compared Mindboggle with four registration methods: linear registration, and nonlinear registration using SPM2, AIR, and ANIMAL. Automated labeling by all of the nonlinear methods was found to be at least comparable with linear registration. Mindboggle outperformed every other method, as measured by the agreement between overlapping atlas labels and manually assigned subject labels, with respect to the union or the intersection of voxels. After applying the same procedure that Mindboggle uses to fill a subject's segmented gray matter mask with labels (step 5), the results of the other methods improved. However, after performing a one-way ANOVA (and Tukey's honestly significant difference criterion) in a multiple comparison between the results obtained by the different methods, Mindboggle was still found to be the only nonlinear method whose labeling performance was significantly better than that of linear registration or SPM2. Further advantages to Mindboggle include a high degree of robustness against image artifacts, poor image quality, and incomplete brain data. We tested the latter hypothesis by conducting all of the tests again, this time registering the atlas to an artificially lesioned version of itself, and found that Mindboggle was the only method whose performance did not degrade significantly as the lesion size increased.     

Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice.

Gene therapy by use of integrating vectors carrying therapeutic transgene sequences offers the potential for a permanent cure of genetic diseases by stable vector insertion into the patients' chromosomes. However, three cases of T cell lymphoproliferative disease have been identified almost 3 years after retrovirus gene therapy for X-linked severe combined immune deficiency. In two of these cases vector insertion into the LMO2 locus was implicated in leukemogenesis, demonstrating that a more profound understanding is required of the genetic and molecular effects imposed on the host by vector integration or transgene expression. In vivo models to test for retro- and lentiviral vector safety prior to clinical application are therefore needed. Here we present a high incidence of lentiviral vector-associated tumorigenesis following in utero and neonatal gene transfer in mice. This system may provide a highly sensitive model to investigate integrating vector safety prior to clinical application.     

Predicting nurses' intention to quit with a support vector machine: a new approach to set up an early warning mechanism in human resource management

This project developed a Support Vector Machine for predicting nurses' intention to quit, using working motivation, job satisfaction, and stress levels as predictors. This study was conducted in three hospitals located in southern Taiwan. The target population was all nurses (389 valid cases). For cross-validation, we randomly split cases into four groups of approximately equal sizes, and performed four training runs. After the training, the average percentage of misclassification on the training data was 0.86, while that on the testing data was 10.8, resulting in predictions with 89.2% accuracy. This Support Vector Machine can predict nurses' intention to quit, without asking these nurses whether they have an intention to quit.     

In vivo methotrexate selection of murine hemopoietic cells transduced with a retroviral vector for Gaucher disease.

The studies described were performed to investigate whether in vivo selection of retrovirus-transduced hemopoietic cells is feasible starting from a low percentage of transduced hemopoietic stem cells (PHSCs). The vector used is an amphotropic bicistronic retroviral vector carrying a cDNA for human lysosomal glucocerebrosidase (hGC) for treatment of Gaucher disease and a methotrexate (MTX) resistant mutant cDNA encoding human dihydrofolate reductase (DHFR). We tested the effect of MTX selection in mice that were either myeloablated or not before infusion of transduced cells. In addition, we determined whether repeated administration of transduced bone marrow cells has an additional effect on the percentage of hGC expressing cells. The results obtained have shown that, in myeloablated mice transplanted once with transduced bone marrow and treated twice weekly with 10 mg/kg of MTX for a total of 6 months, a two- to three-fold increased numbers of hGC expressing cells could be detected in both peripheral blood and bone marrow as compared with non-MTX treated mice. In mice transplanted with transduced bone marrow once every 2 weeks for a total of four times, percentages of hGC expressing cells were not significantly increased as compared with mice transplanted once. In non-ablated mice neither MTX selection nor multiple infusions of transduced bone marrow resulted in detection of hGC expressing cells 6 months after transplantation, indicating that the success of in vivo selection using MTX is highly dependent on the ratio of transduced hemopoietic stem cells transplanted versus residing and untransduced stem cells.     

Regulatory considerations for novel gene therapy products: a review of the process leading to the first clinical lentiviral vector

This review is intended to exemplify the roles and responsibilities of the two agencies under the Department of Health and Human Services, the National Institutes of Health and the Food and Drug Administration, that have oversight for human gene transfer clinical protocols, as seen through our experience of bringing a first-in-its-class lentiviral vector to clinical trials. In response to the changing circumstances in gene therapy research between 1999 and 2002, the concerns of these agencies regarding gene therapy have been evolving. This review provides an overview of the major safety concerns regarding insertional oncogenesis, the generation of a replication- competent lentivirus (RCL), and vector mobilization thought to be related to lentiviral vectors, which had to be addressed during the regulatory review process before initiating the clinical trial. Specific monitoring assays to address these concerns were established to test for RCL generation, vector mobilization, persistence of vector-modified cells, and abnormal clonal expansion of modified cells. We hope to provide a basic understanding and appreciation of the regulatory process and major safety concerns, toward providing useful insight to those presently embarking on the development of clinical application of lentiviral vectors.