Feline leukemia virus integrase and capsid packaging functions do not change the insertion profile of standard Moloney retroviral vectors.

Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.

AAV5-mediated gene transfer to the parotid glands of non-human primates.

Salivary glands are potentially useful target sites for multiple clinical applications of gene transfer. Previously, we have shown that serotype 2 adeno-associated viral (AAV2) vectors lead to stable gene transfer in the parotid glands of rhesus macaques. As AAV5 vectors result in considerably greater transgene expression in murine salivary glands than do AAV2 vectors, herein we have examined the use of AAV5 vectors in macaques at two different doses (n = 3 per group; 10(10) or 3 x 10(11) particles per gland). AAV5 vector delivery, as with AAV2 vectors, led to no untoward clinical, hematological or serum chemistry responses in macaques. The extent of AAV5-mediated expression of rhesus erythropoietin (RhEpo) was dose-dependent and similar to that seen with an AAV2 vector. However, unlike results with the AAV2 vector, AAV5 vector-mediated RhEpo expression was transient. Maximal expression peaked at day 56, was reduced by approximately 80% on day 84 and thereafter remained near background levels until day 182 (end of experiment). Quantitative PCR studies of high-dose vector biodistribution at this last time point showed much lower AAV5 copy numbers in the targeted parotid gland (approximately 1.7%) than found with the same AAV2 vector dose. Molecular analysis of the conformation of vector DNA indicated a markedly lower level of concatamerization for the AAV5 vector compared with that of a similar AAV2 vector. In addition, cellular immunological studies suggest that host response differences may occur with AAV2 and AAV5 vector delivery at this mucosal site. The aggregate data indicate that results with AAV5 vectors in murine salivary glands apparently do not extend to macaque glands.

Improved engraftment of human hematopoietic cells in severe combined immunodeficient (SCID) mice carrying human cytokine transgenes.

We have generated immunodeficient scid-/scid- (SCID)-transgenic mice expressing the genes for human interleukin 3, granulocyte/macrophage-colony stimulating factor, and stem cell factor. We have compared engraftment and differentiation of human hematopoietic cells in transgenic SCID mice with two strains of nontransgenic SCID mice. Human bone marrow cells carrying the CD34 antigen or human umbilical cord blood were injected into sublethally irradiated recipients. Human DNA was detected by polymerase chain reaction in peripheral blood and bone marrow of 14 of 28 transgenic SCID mice after transplantation, but in only 2 of 15 nontransgenic SCID littermates at a 10-fold lower level. Bone marrow cultures 8 wk after transplantation of cord blood gave rise to human burst-forming unit erythroid, colony-forming unit granulocyte/macrophage, or granulocyte/erythroid/macrophage/megakaryocyte colonies. Engraftment was observed for up to 6 mo in transgenic SCID mice, twice as long as nontransgenic littermates or previous studies in which transplanted SCID mice were given daily injections of growth factors. We conclude that the level and duration of engraftment of human cells in SCID mice can be improved by expression of human cytokine transgenes and that transgenic SCID mice are an efficient model system for the study of human hematopoiesis.

Helper virus induced T cell lymphoma in nonhuman primates after retroviral mediated gene transfer.

Moloney Murine Leukemia Virus (MoMuLV) causes T cell neoplasms in rodents but is not known to be a pathogen in primates. The core protein and enzyme genes of the MoMuLV genome together with an amphotropic envelope gene are utilized to engineer the cell lines that generate retroviral vectors for use in current human gene therapy applications. We developed a producer clone that generates a very high concentration of retroviral vector particles to optimize conditions for gene insertion into pluripotent hematopoietic stem cells. This producer cell line also generates a much lower concentration of replication-competent virus that arose through recombination. Stem cells from rhesus monkeys were purified by immunoselection with an anti-CD34 antibody, incubated in vitro for 80-86 h in the presence of retroviral vector particles with accompanying replication-competent virus and used to reconstitute recipients whose bone marrow had been ablated by total body irradiation. The retroviral vector genome was detected in circulating cells of five of eight transplant recipients of CD34+ cells and in the circulating cells of two recipients of infected, unfractionated bone marrow mononuclear cells. Three recipients of CD34+ cells had a productive infection with replication-competent virus. Six or seven mo after transplantation, each of these animals developed a rapidly progressive T cell neoplasm involving the thymus, lymph nodes, liver, spleen, and bone marrow. Lymphoma cells contained 10-50 copies of the replication-competent virus, but lacked the retroviral vector genome. We conclude that replication-competent viruses arising from producer cells making retroviral vectors can be pathogenic in primates, which underscores the importance of carefully screening retroviral producer clones used in human trials to exclude contamination with replication-competent virus.

Molecular characterisation of side population cells with cancer stem cell-like characteristics in small-cell lung cancer.

BACKGROUND: Side population (SP) fraction cells, identified by efflux of Hoechst dye, are present in virtually all normal and malignant tissues. The relationship between SP cells, drug resistance and cancer stem cells is poorly understood. Small-cell lung cancer (SCLC) is a highly aggressive human tumour with a 5-year survival rate of <10%. These features suggest enrichment in cancer stem cells. METHODS AND RESULTS: We examined several SCLC cell lines and found that they contain a consistent SP fraction that comprises <1% of the bulk population. Side population cells have higher proliferative capacity in vitro, efficient self-renewal and reduced cell surface expression of neuronal differentiation markers, CD56 and CD90, as compared with non-SP cells. Previous reports indicated that several thousand SP cells from non-small-cell lung cancer are required to form tumours in mice. In contrast, as few as 50 SP cells from H146 and H526 SCLC cell lines rapidly reconstituted tumours. Whereas non-SP cells formed fewer and slower-growing tumours, SP cells over-expressed many genes associated with cancer stem cell and drug resistance: ABCG2, FGF1, IGF1, MYC, SOX1/2, WNT1, as well as genes involved in angiogenesis, Notch and Hedgehog pathways. CONCLUSIONS: Side population cells from SCLC are highly enriched in tumourigenic cells and are characterised by a specific stem cell-associated gene expression signature. This gene signature may be used for development of targeted therapies for this rapidly fatal tumour.

Evaluation of a rapamycin-regulated serotype 2 adeno-associated viral vector in macaque parotid glands.

OBJECTIVES: Salivary glands are useful target organs for local and systemic gene therapeutics. For such applications, the regulation of transgene expression is important. Previous studies by us in murine submandibular glands showed that a rapamycin transcriptional regulation system in a single serotype 2, adeno-associated viral (AAV2) vector was effective for this purpose. This study evaluated if such a vector was similarly useful in rhesus macaque parotid glands. METHODS: A recombinant AAV2 vector (AAV-TF-RhEpo-2.3w), encoding rhesus erythropoietin (RhEpo) and a rapamycin-inducible promoter, was constructed. The vector was administered to macaques at either of two doses [1.5 x 10(11) (low dose) or 1.5 x 10(12) (high dose) vector genomes] via cannulation of Stensen's duct. Animals were followed up for 12-14 weeks and treated at intervals with rapamycin (0.1 or 0.5 mg kg(-1)) to induce gene expression. Serum chemistry, hematology, and RhEpo levels were measured at interval. RESULTS: AAV-TF-RhEpo-2.3w administration led to low levels of rapamycin-inducible RhEpo expression in the serum of most macaques. In five animals, no significant changes were seen in serum chemistry and hematology values over the study. One macaque, however, developed pneumonia, became anemic and subsequently required euthanasia. After the onset of anemia, a single administration of rapamycin led to significant RhEpo production in this animal. CONCLUSION: Administration of AAV-TF-RhEpo-2.3w to macaque parotid glands was generally safe, but led only to low levels of serum RhEpo in healthy animals following rapamycin treatment.

COOH-terminal-modified interleukin-3 is retained intracellularly and stimulates autocrine growth.

Autocrine growth due to dysregulated growth factor production may have a role in the development of neoplasia. Whether autocrine growth is stimulated by growth factor secretion in an autocrine loop or by intracellular binding of the growth factor to a receptor has been unclear. The carboxyl-terminus coding sequence for murine interleukin-3 (IL-3) was extended with an oligonucleotide encoding a four-amino acid endoplasmic reticulum retention signal. IL-3-dependent hematopoietic cells became growth factor-independent when the modified IL-3 gene was introduced by retroviral gene transfer, despite lack of secretion of the modified IL-3. Hence autocrine growth can occur as a result of the intracellular action of a growth factor and this mechanism may be important in neoplastic and normal cells.

Dysregulated interleukin 6 expression produces a syndrome resembling Castleman's disease in mice.

Interleukin 6 (IL-6) is an important regulator of the acute phase response, T cell function, and terminal B cell differentiation. Excessive or inappropriate production of this cytokine may be involved in a variety of autoimmune and neoplastic disorders. To investigate the consequences of dysregulated synthesis of IL-6 in vivo, a high-titer recombinant retroviral vector produced in psi-2 packaging cells was used to introduce the coding sequences of murine IL-6 into mouse hematopoietic cells. Congenitally anemic W/Wv mice reconstituted with bone marrow cells transduced with the retroviral vector developed a syndrome characterized by anemia, transient granulocytosis, hypoalbuminemia, and polyclonal hypergammaglobulinemia, with marked splenomegaly and peripheral lymphadenopathy. Extensive plasma cell infiltration of lymph nodes, spleen, liver, and lung was noted. The similarity of these findings to those of multicentric Castleman's disease, taken together with the observation that lymph nodes from these patients elaborate large amounts of this cytokine, suggest that the inappropriate synthesis of IL-6 has a primary role in the pathogenesis of this systemic lymphoproliferative disorder.

Avoidance of stimulation improves engraftment of cultured and retrovirally transduced hematopoietic cells in primates.

Recent reports suggest that cells in active cell cycle have an engraftment defect compared with quiescent cells. We used nonhuman primates to investigate this finding, which has direct implications for clinical transplantation and gene therapy applications. Transfer of rhesus CD34(+) cells to culture in stem cell factor (SCF) on the CH-296 fibronectin fragment (FN) after 4 days of culture in stimulatory cytokines maintained cell viability but decreased cycling. Using retroviral marking with two different gene transfer vectors, we compared the engraftment potential of cytokine-stimulated cells versus those transferred to nonstimulatory conditions (SCF on FN alone) before reinfusion. In vivo competitive repopulation studies showed that the level of marking originating from the cells continued in culture for 2 days with SCF on FN following a 4-day stimulatory transduction was significantly higher than the level of marking coming from cells transduced for 4 days and reinfused without the 2-day culture under nonstimulatory conditions. We observed stable in vivo overall gene marking levels of up to 29%. This approach may allow more efficient engraftment of transduced or ex vivo expanded cells by avoiding active cell cycling at the time of reinfusion.

Retrovirus-mediated transfer and expression of the interleukin-3 gene in mouse hematopoietic cells result in a myeloproliferative disorder.

A high-titer, recombinant retroviral vector produced in psi 2 packaging cells has been used to introduce the murine interleukin-3 (IL-3) gene into mouse hematopoietic cells. Integration and expression of the IL-3 gene was observed in spleen foci from which could be derived factor-independent, continuously proliferating cell lines. Irradiated or genetically anemic W/Wv recipients of infected hematopoietic cells developed a myeloproliferative syndrome characterized by a marked elevation in leukocyte count, bone marrow hyperplasia, and enlargement of the liver and spleen. The syndrome reflected proliferation of one or more stem cell clones, the progeny of which were capable of repopulating secondary recipients. One animal developed the syndrome primarily by a paracrine mechanism. Endogenous IL-3 production caused amplification of hematopoietic cells but did not appear to alter the maturational or self-renewal potential of these cells.

A pilot study of nonmyeloablative allogeneic hematopoietic stem cell transplant for advanced systemic mastocytosis.

Systemic mastocytosis (SM) is a disease characterized by tissue infiltration of neoplastic mast cells originating from hematopoietic stem cells. Patients with advanced SM have a poor prognosis, and there is no mast cell ablative therapy available for most patients who carry an activating point mutation in the c-kit gene. We report results of a prospective study evaluating the safety, engraftment, and possibility of inducing a graft-versus-mast cell (GvMC) effect after allogeneic nonmyeloablative hematopoietic cell transplantation (HCT) from an HLA-identical sibling. Three patients with advanced SM were transplanted. All achieved complete donor T cell chimerism followed by clinical evidence for GvMC effect. However, all patients experienced disease progression with the longest response duration of 39 months. The GvMC effect can be observed after nonmyeloablative HCT with limited efficacy. Effective cytoreductive therapy prior to HCT may be required for long-term disease control and cure.

Paclitaxel chemotherapy after autologous stem-cell transplantation and engraftment of hematopoietic cells transduced with a retrovirus containing the multidrug resistance complementary DNA (MDR1) in metastatic breast cancer patients.

The MDR1 multidrug resistance gene confers resistance to natural-product anticancer drugs including paclitaxel. We conducted a clinical gene therapy study to determine whether retroviral-mediated transfer of MDR1 in human hematopoietic cells would result in stable engraftment, and possibly expansion, of cells containing this gene after treatment with myelosuppressive doses of paclitaxel. Patients with metastatic breast cancer who achieved a complete or partial remission after standard chemotherapy were eligible for the study. Hematopoietic stem cells (HSCs) were collected by both peripheral blood apheresis and bone marrow harvest after mobilization with a single dose of cyclophosphamide (4 g/m2) and daily filgrastim therapy (10 microg/kg/day). After enrichment for CD34+ cells, one-third of each collection was incubated ex vivo for 72 h with a replication-incompetent retrovirus containing the MDR1 gene (G1MD) in the presence of stem-cell factor, interleukin 3, and interleukin 6. The remaining CD34+ cells were stored without further manipulation. All of the CD34+ cells were reinfused for hematopoietic rescue after conditioning chemotherapy with ifosfamide, carboplatin, and etoposide regimen. After hematopoietic recovery, patients received six cycles of paclitaxel (175 mg/m2 every 3 weeks). Bone marrow and serial peripheral blood samples were obtained and tested for the presence of the MDR1 transgene using a PCR assay. Six patients were enrolled in the study and four patients received infusion of genetically altered cells. The ex vivo transduction efficiency, estimated by the PCR assay, ranged from 0.1 to 0.5%. Three of the four patients demonstrated engraftment of cells containing the MDR1 transgene. The estimated percentage of granulocytes containing the MDR1 transgene ranged from a maximum of 9% of circulating nucleated cells down to the limit of detection of 0.01%. One patient remained positive for the MDR1 transgene throughout all six cycles of paclitaxel therapy, whereas the other 2 patients showed a decrease in the number of cells containing the transgene to undetectable levels. Despite the low level of engraftment of MDR1-marked cells, a correlation was observed between the relative number of granulocytes containing the MDR1 transgene and the granulocyte nadir after paclitaxel therapy. No adverse reactions to the genetic manipulation procedures were detected. Therefore, engraftment of human HSCs transduced with the MDR1 gene can be achieved. However, the overall transduction efficiency and stable engraftment of gene-modified HSCs must be improved before MDR1 gene therapy and in vivo selection with anticancer drugs can be reliably used to protect cancer patients from drug-related myelosuppression.

Isolation and identification of antifungal and antialgal alkaloids from Haplophyllum sieversii.

Bioassay-guided fractionation of the hexane/ethyl acetate/water (H/EtOAc/H2O) crude extract of the aerial parts of Haplophyllum sieversii was performed because of preliminary screening data that indicated the presence of growth inhibitory components against Colletotrichum fragariae, Colletotrichum gloeosporioides, and Colletotrichum acutatum. Fractionation was directed using bioautographical methods resulting in the isolation of the bioactive alkaloids flindersine, anhydroevoxine, haplamine, and a lignan eudesmin. These four compounds were evaluated for activity against C. fragariae, C. gloeosporioides, C. acutatum, Botrytis cinerea, Fusarium oxysporum, and Phomopsis obscurans in a dose-response growth-inhibitory bioassay at 50.0, 100.0, and 150.0 microM. Of the four compounds tested, flindersine demonstrated the highest level of antifungal activity. Additionally, flindersine, eudesmin, and haplamine were screened against the freshwater phytoplanktons Oscillatoria perornata, Oscillatoria agardhii, Selenastrum capricornutum, and Pseudanabaena sp. (strain LW397). Haplamine demonstrated selective inhibition against the odor-producing cyanobacterium O. perornata compared to the activity against the green alga S. capricornutum, with lowest observed effect concentration values of 1.0 and 10.0 microM, respectively.

Retroviral-mediated gene transfer into CD34-enriched human peripheral blood stem cells.

Retroviral-mediated gene transfer has been shown to be a feasible method for the introduction of new genes into bone marrow hematopoietic stem cells. We have investigated the application of this technology to primitive CD34-enriched human peripheral blood cells as a potential alternative stem cell source. Bone marrow (BM) and peripheral blood (PB) CD34-enriched cells from normal volunteers and patients with multiple myeloma were exposed to retroviral vectors containing the neomycin-resistance gene and gene transfer efficiency into colony-forming unit colonies (CFU-C) and CD34+ cells was assessed by polymerase chain reaction (PCR). Peripheral blood was a target equally efficient to BM, and PB cells mobilized with chemotherapy and growth factors were also shown to take up retroviral vectors readily. Conditions favoring gene transfer were investigated, and exposure of cells to interleukin-3 (IL-3), interleukin-6 (IL-6), and stem cell factor (SCF) during a 72-hour transduction was found to be most effective. The use of PB stem cells as targets for gene transfer could allow repeated collections and transductions, with obvious advantages over a single BM collection.

Positive selection of CD34+ hematopoietic cells using an immunoaffinity column results in T cell-depletion equivalent to elutriation.

Acute graft-vs.-host disease (GVHD) continues to present a barrier to successful allogeneic marrow transplantation. T cell-depletion may prevent severe GVHD but carries an increased risk of graft rejection and relapse posttransplant. Clinical trials have defined the number of lymphocytes associated with sustained engraftment but low risk of significant GVHD (greater than grade I or II skin only) as < or = 10(5)/kg. We examined T cell-depletion resulting from positive selection of CD34+ hematopoietic cells with a biotinylated monoclonal anti-CD34 antibody and an immunoaffinity column. Eleven patients (six myeloma and five breast cancer) underwent both peripheral blood stem cell (PBSC) collection and marrow harvest prior to autologous transplantation. One PBSC collection and one-third of each marrow underwent column separation. PBSCs were enriched for CD34+ cells from an initial mean of 1.5 to 53.3%, while marrow went from an initial mean of 2.8 to 65.4%. PBSC were depleted of CD3+ cells from an initial mean of 9.6 x 10(9) to 8.6 x 10(6). Marrow CD3+ lymphocyte content was reduced from an initial mean of 5.6 x 10(9) to 8 x 10(5). Since the column permits quantification and salvage of depleted T cells, its use should allow re-addition of T cell-aliquots associated with minimal risk for GVHD and rejection. In addition, since PBSCs were as readily depleted as marrow, allogeneic PBSC transplant may be feasible using this method.

Expression of interferon-gamma by stromal cells inhibits murine long-term repopulating hematopoietic stem cell activity.

Several lines of evidence suggest that overexpression of interferon gamma (IFN-gamma) in the marrow microenvironment may play a role in the pathogenesis of marrow suppression in aplastic anemia. We previously showed that overexpression of IFN-gamma by marrow stromal cells inhibits human long-term culture initiating cell activity assayed in vitro to a much greater degree than the addition of soluble IFN-gamma. The effect of IFN-gamma on true repopulating stem cells assayed in vivo has not been studied previously. We compared the effect of co-culture of murine marrow cells in the presence of stromal cells transduced with a retroviral vector expressing murine IFN-gamma vs stromal cells transduced with a control neo vector. Using a murine congenic competitive repopulation assay, there was significantly less long-term repopulating stem cell activity remaining after culture on mIFN-gamma-expressing stroma as compared to control stroma. We also investigated the effect of directly transducing murine bone marrow cells with the mIFN-gamma or control vector. Marrow cells transduced with either vector were transplanted into W/Wv recipient mice. The percentage of vector-containing cells in the mIFN-gamma mice was significantly lower than in the control mice, suggesting that mIFN-gamma-transduced primitive cells may not have survived culture, or that mIFN-gamma directly decreases gene transfer into repopulating cells. Despite no significant differences in white or red blood cells in the mice transplanted with the mIFN-gamma-transduced cells, the number of bone marrow colony-forming unit-C 16 weeks after transplantation was significantly lower in the IFN-gamma group. These data indicate that ectopic or overexpression of mIFN-gamma, especially by marrow microenvironmental elements, may have a marked effect on primitive hematopoiesis as assayed in vivo.

Retroviral-mediated transfer of the murine interleukin-3 gene engineered for intracellular retention results in a myeloproliferative syndrome but is associated with circulating interleukin-3 levels.

Myeloid leukemias have been shown to secrete as well as respond to cytokines such as interleukin-3 (IL-3) with an increased growth rate and may therefore become self-stimulatory through an external autocrine mechanism. In vitro evidence that IL-3 is functional within the intracellular compartment has been obtained through modification of the murine IL-3 gene to encode for the amino acids SEKDEL on the carboxyl terminus of the protein, resulting in preferential intracellular retention. The ability of bone marrow-derived hematopoietic progenitor cells to increase their proliferative capacity through intracellular mechanisms was investigated in vivo using retroviruses containing the wild-type or SEKDEL-modified IL-3 gene, transcriptionally regulated by the retroviral long terminal repeat (LTR) or by the SV40 early promoter, in lethally irradiated, bone marrow-reconstituted mice. Bone marrow cells exposed to the N2KDEL virus containing the SEKDEL-modified IL-3 gene were shown by bioassay to retain large amounts of IL-3 intracellularly, and the presence of an integrated provirus containing the SEKDEL sequences was demonstrated by polymerase chain reaction (PCR) in the spleen and bone marrow of these animals. Transduction with all four types of IL-3 viruses resulted in dramatic increases in the circulating white blood cell (WBC) count; this myeloproliferative state occurred within several weeks following bone marrow transplantation (BMT), when viruses expressing the IL-3 or modified gene were under transcriptional regulation of the viral LTR, and approximately 2 months post-BMT, when they were under control of the SV40 internal promoter. Serum levels of IL-3 were measured in transplanted animals and found to be markedly increased in each case in which WBC elevation was observed, including mice receiving marrow transduced with constructs containing the IL-3 gene modified for intracellular retention. No animals were observed in which myeloproliferation occurred without secretion. From these experiments, it seems unlikely that exclusively intracellular mechanisms are a major contributor to the development of the myeloproliferative syndrome observed in these animals.

Targeting protein tyrosine phosphatase σ after myocardial infarction restores cardiac sympathetic innervation and prevents arrhythmias.

Millions of people suffer a myocardial infarction (MI) every year, and those who survive have increased risk of arrhythmias and sudden cardiac death. Recent clinical studies have identified sympathetic denervation as a predictor of increased arrhythmia susceptibility. Chondroitin sulfate proteoglycans present in the cardiac scar after MI prevent sympathetic reinnervation by binding the neuronal protein tyrosine phosphatase receptor σ (PTPσ). Here we show that the absence of PTPσ, or pharmacologic modulation of PTPσ by the novel intracellular sigma peptide (ISP) beginning 3 days after injury, restores sympathetic innervation to the scar and markedly reduces arrhythmia susceptibility. Using optical mapping we observe increased dispersion of action potential duration, supersensitivity to ß-adrenergic receptor stimulation and Ca(2+) mishandling following MI. Sympathetic reinnervation prevents these changes and renders hearts remarkably resistant to induced arrhythmias.

Retroviral mediated transfer of the cDNA for human glucocerebrosidase into hematopoietic stem cells of patients with Gaucher disease. A phase I study.

Patients with Gaucher disease suffer from a lack of functional glucocerebrosidase enzyme (Gc). Disease symptoms are a result of macrophage engorgement secondary to this enzyme deficiency. This study is designed to determine if cDNA encoding normal Gc can be introduced into macrophage precursors using a retroviral vector. CD34+ cells obtained from G-CSF mobilized peripheral blood stem cells or from bone marrow will be transduced ex vivo using one of the following three methods of transduction: 1) GlGc retroviral supernatant in the presence of autologous stroma over a period of 72 hours, 2) GlGc retroviral supernatant in the presence of interleukin-3, interleukin-6, stem cell factor and autologous stroma over a 72 hour period, 3) G1Gc retroviral supernatant in the presence of interleukin-3, interleukin-6, and stem cell factor over a 72 hour period. These transduced cells will be reinfused into the patient and the patient monitored for toxicities as well as evidence of successful gene transfer and expression. A total of twenty-four patients will be enrolled on the protocol. Patients will be assigned in equal numbers to each of six groups. The two sites participating are the National Institutes of Health and Childrens Hospital of Los Angeles.

Update on the use of nonhuman primate models for preclinical testing of gene therapy approaches targeting hematopoietic cells.

Transfer of genes into hematopoietic stem cells or primary lymphocytes has been a primary focus of the gene therapy field for more than a decade because of the wide variety of congenital and acquired diseases that potentially could be cured by successful gene transfer into these cell populations. However, despite success in murine models and in vitro, progress has been slow, and early clinical trials were disappointing due to inefficient gene transfer into long-term repopulating cells. The unique predictive value of nonhuman primate or other large animal models has become more apparent, and major advances in gene transfer efficiency have been made by utilizing these powerful but expensive and complex systems. This review summarizes more recent findings from nonhuman primate investigations focusing on hematopoietic stem cells or lymphocytes as target populations, and highlights specific preclinical issues, including safety. Results from studies using standard retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors are discussed. Judicious application of these models should continue to be a priority, and advances should now be tested in proof-of-concept clinical trials.