Search

Ex-vivo gene therapy has been shown to be an effective method for treating bone defects in preclinical models. As gene therapy is explored as a potential treatment option in humans, an assessment of the safety profile becomes an important next step. The purpose of this study was to evaluate the biodistribution of viral particles at the defect site and various internal organs in a rat femoral defect model after implantation of human ASCs transduced with lentivirus (LV) with two-step transcriptional activation (TSTA) of bone morphogenetic protein 2 (LV-TSTA-BMP-2). Animals were sacrificed at 4-, 14-, 56-, and 84-days post implantation. Treatment groups included 1) standard dose LV-TSTA-BMP-2 2) high dose LV-TSTA-BMP-2, 3) standard dose LV-TSTA-GFP 4) high dose LV-TSTA-GFP and 5) standard dose nontransduced cells. The viral load was assessed at each timepoint in the defect in ten organs and the defect site. Histology of all organs, ipsilateral tibia, and femur were evaluated at each timepoint. There were nearly undetectable levels of LV-TSTA-BMP-2 transduced cells at the defect site at 84 days and no pathologic changes in any organ at all timepoints. Humana ASCs transduced with LV-TSTA may be a safe and effective treatment option when adopted for us in patients.

Paul Volcker was a model public servant, not only his service as Fed Chair, but also in his work on international economic policy at the Treasury, and in his service in many other realms. Three distinguished persons—Sheila Bair, Donald Kohn, and John Taylor—provide their memories of Volcker and reflect upon his legacy.

The extraordinary circumstances of the past few years have led to extraordinary responses by the Federal Reserve and other central banks. These ventures into uncharted waters have heightened political scrutiny to the point of raising concern about future independence. In discussing independence of the Federal Reserve, it is important to separate its regulatory and supervisory functions from its monetary policy function. It is the latter in which the question of independence is most important. History indicates that independent monetary policy has been a powerful deterrent to inflation. This paper outlines the threats to Federal Reserve independence, particularly as it exists from the unconventional policies that it pursued to mitigate the financial crisis. Economists have an important role in making the case that monetary policy remains independent.

Blood and marrow transplantation (BMT) is a standard curative therapy for patients with nonmalignant genetic diseases. Myeloablative conditioning has been associated with significant regimen-related toxicity (RRT), whereas reduced-intensity conditioning regimens have been associated with graft failure. In this prospective pilot trial conducted at 2 centers between 2006 and 2013, we report the outcome of 22 patients with nonmalignant genetic diseases who were conditioned with a novel reduced-toxicity regimen: i.v. busulfan (16 mg/kg), alemtuzumab (52 mg/m2), fludarabine (140 mg/m2), and cyclophosphamide (105 mg/kg). The median age of the study population was 3.5 years (range, 5 months to 26 years). No cases of sinusoidal obstruction syndrome, severe or chronic graft-versus-host disease (GVHD), or primary graft failure were reported. Median time to neutrophil engraftment (>500 cells/μL) and platelet engraftment (>20K cells/μL) were 19 (range, 12 to 50) and 23.5 (range, 14 to 134) days, respectively. The median length of follow-up was 3 years (range, .2 to 6.3). The overall survival rates were 95% at 100 days (95% confidence interval, .72 to .99) and 90% at 6 years (95% confidence interval, .68 to .98). RRT and chronic GVHD are significant barriers to BMT for patients with nonmalignant genetic diseases. This alemtuzumab-based reduced-toxicity regimen appears to be promising with durable engraftment, effective cure of clinical disease, low rates of RRT, and no observed chronic GVHD.

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.

Gene transfer into human hematopoietic stem cells with expression targeted to the maturing myelomonocytic progeny has applications for gene therapy of genetic diseases affecting granulocytes and macrophages. We hypothesized that promoters of myeloid-specific genes that are upregulated with myelomonocytic differentiation would also upregulate expression of an exogenous gene in a retroviral vector. Moloney murine leukemia virus (MoMuLV)-based retroviral vectors using promoters from hematopoietic genes (CD11b, CD18, and CD34) were compared with vectors with viral promoters (MoMuLV long terminal repeat [LTR], cytomegalovirus [CMV], and simian virus 40 [SV40]). Human glucocerebrosidase (GC) cDNA was the reporter gene. HL60 cells were transduced with these vectors and vector-derived GC activity was compared in undifferentiated HL-60 cells and the same cells differentiated into granulocytes using dimethyl sulfoxide or monocyte/macrophages using phorbol myristate acetate. In undifferentiated HL-60 cells, vector-derived GC activity was the highest when it was controlled by the MoMuLV LTR. In HL-60 cells differentiated into granulocytes, vector-derived GC activity transcribed from the CD11b, MoMuLV LTR, and CMV promoters was equivalent to 1.7, 1.5, and 1.5 times the normal endogenous GC activity, respectively, and 0.8, 2.0, and 3.6 times the normal GC activity, respectively, in those differentiated into macrophages. With granulocytic differentiation, the CD11b promoter showed maximal induction in GC activity (8-fold); with macrophage differentiation, the CD11b promoter showed a fourfold induction in GC expression. The CD11b promoter also generated significant levels of GC activity in the myelomonocytic progeny of transduced CD34+ cells. Expression from the CD11b promoter, unlike that from the CMV or the MoMuLV LTR promoters, was relatively myelomonocyte-specific, with minimal expression observed in Jurkat T cells or HeLa carcinoma cells. The induction of expression from the CD11b promoter with differentiation in HL-60 cells correlates with the developmental regulation of the CD11b gene. Retroviral vectors using the CD11b promoter have potential utility for gene therapy of disorders affecting the myelomonocytic lineage.

The cytokine thrombopoietin (Tpo) plays a critical role in hematopoiesis by binding to the extracellular domain and inducing homodimerization of the intracellular signaling domain of its receptor, c-Mpl. Mpl homodimerization can also be accomplished by binding of a synthetic ligand to a constitutively expressed fusion protein F36VMpl consisting of a ligand binding domain (F36V) and the intracellular signaling domain of Mpl. Unexpectedly, in contrast to Tpo stimulation, robust erythropoiesis is induced after dimerization of F36VMpl in human CD34+ progenitor cells. The goal of this study was to define the hematopoietic progenitor stages at which dimerization of intracellular Mpl induces erythropoiesis and the downstream molecular events that mediate this unanticipated effect. Dimerization (in the absence of erythropoietin and other cytokines) in human common myeloid progenitors and megakaryocytic erythroid progenitors caused a significant increase in CD34+ cells (p < .01) and induced all stages of erythropoiesis including production of enucleated red blood cells. In contrast, erythropoiesis was not seen with Tpo stimulation. CD34+ cell expansion was the result of increased cell cycling and survival (p < .05). Microarray profiling of CD34+ cells demonstrated that a unique transcriptional pattern is activated in progenitors by F36VMpl dimerization. Ligand-inducible dimerization of intracellular Mpl in human myeloerythroid progenitors induces progenitor expansion and erythropoiesis through molecular mechanisms that are not shared by Tpo stimulation of endogenous Mpl. Disclosure of potential conflicts of interest is found at the end of this article.

NON-TECHNICAL ABSTRACT The possibility of transferring a “sensitivity” gene into a growing brain tumor has been investigated. The purpose is to make the tumor sensitive to a type of chemotherapy th...

The role of the homeobox gene HOXA5 in normal human hematopoiesis was studied by constitutively expressing the HOXA5 cDNA in CD34(+) and CD34(+)CD38(-) cells from bone marrow and cord blood. By using retroviral vectors that contained both HOXA5 and a cell surface marker gene, pure populations of progenitors that expressed the transgene were obtained for analysis of differentiation patterns. Based on both immunophenotypic and morphological analysis of cultures from transduced CD34(+) cells, HOXA5 expression caused a significant shift toward myeloid differentiation and away from erythroid differentiation in comparison to CD34(+) cells transduced with Control vectors (P =.001, n = 15 for immunophenotypic analysis; and P.0001, n = 19 for morphological analysis). Transduction of more primitive progenitors (CD34(+)CD38(-) cells) resulted in a significantly greater effect on differentiation than did transduction of the largely committed CD34(+) population (P =.006 for difference between HOXA5 effect on CD34(+) v CD34(+)CD38(-) cells). Erythroid progenitors (burst-forming unit-erythroid [BFU-E]) were significantly decreased in frequency among progenitors transduced with the HOXA5 vector (P =.016, n = 7), with no reduction in total CFU numbers. Clonal analysis of single cells transduced with HOXA5 or control vectors (cultured in erythroid culture conditions) showed that HOXA5 expression prevented erythroid differentiation and produced clones with a preponderance of undifferentiated blasts. These studies show that constitutive expression of HOXA5 inhibits human erythropoiesis and promotes myelopoiesis. The reciprocal inhibition of erythropoiesis and promotion of myelopoiesis in the absence of any demonstrable effect on proliferation suggests that HOXA5 diverts differentiation at a mulitpotent progenitor stage away from the erythroid toward the myeloid pathway.

Autologous leukemia cells engineered to express immune-stimulating molecules may be used to elicit antileukemia immune responses. Gene delivery to human B-precursor acute lymphoblastic leukemia (ALL) cells was investigated using the enhanced green fluorescent protein (EGFP) as a reporter gene, measured by flow cytometry. Transfection of the Nalm-6 and Reh B-precursor ALL leukemia cell lines with an expression plasmid was investigated using lipofection, electroporation, and a polycationic compound. Only the liposomal compound Cellfectin showed significant gene transfer (3.9% to 12% for Nalm-6 cells and 3.1% to 5% for Reh cells). Transduction with gibbon-ape leukemia virus pseudotyped Moloney murine leukemia virus (MoMuLV)-based retrovirus vectors was investigated in various settings. Cocultivation of ALL cell lines with packaging cell lines showed the highest transduction efficiency for retroviral gene transfer (40.1% to 87.5% for Nalm-6 cells and 0.3% to 9% for Reh cells), followed by transduction with viral supernatant on the recombinant fibronectin fragment CH-296 (13% to 35.5% for Nalm-6 cells and 0.4% to 6% Reh cells), transduction on human bone marrow stroma monolayers (3.2% to 13.3% for Nalm-6 cells and 0% to 0.2% Reh cells), and in suspension with protamine sulfate (0.7% to 3.1% for Nalm-6 cells and 0% for Reh cells). Transduction of both Nalm-6 and Reh cells with human immunodeficiency virus-type 1 (HIV-1)-based lentiviral vectors pseudotyped with the vesicular stomatitis virus-G envelope produced the best gene transfer efficiency, transducing greater than 90% of both cell lines. Gene delivery into primary human B-precursor ALL cells from patients was then investigated using MoMuLV-based retrovirus vectors and HIV-1-based lentivirus vectors. Both vectors transduced the primary B-precursor ALL cells with high efficiencies. These studies may be applied for investigating gene delivery into primary human B-precursor ALL cells to be used for immunotherapy.

We sought to define optimal conditions for retroviral-mediated transduction of long-lived human hematopoietic progenitors from bone marrow and peripheral blood. CD34+ cells were transduced by the LN and G2 retroviral vectors in the presence or absence of stromal support and with or without cytokine addition. After transduction, a portion of the cells was plated in methylcellulose colony-forming assay, with or without G418, to assess the extent of gene transfer into committed progenitors. The remaining cells from each experiment were transplanted into immunodeficient mice to allow analysis of transduction of long-lived progenitors. Human colony-forming cells contained within the murine bone marrow were analyzed after engraftment periods of 2 to 11 months. Cells were plated in a human-specific colony-forming assay with and without G418 to assess the extent of transduction of primitive progenitors. Individual human colonies were also analyzed by polymerase chain reaction for the presence of provirus. Bone marrow progenitors were efficiently transduced only when stroma was present, whereas mobilized peripheral blood progenitors were effectively transduced in the presence of either stroma or cytokines. Inclusion of the cytokines interleukin-3, interleukin-6, and stem cell factor did not further augment the extent of gene transfer in the presence of a stromal support layer. Additionally, human CD34+ progenitors from bone marrow or mobilized peripheral blood that had been transduced for 3 days in the absence of stroma failed to produce sustained, long-term engraftment of bnx mice. Mice transplanted with the same pools of human progenitors that had been transduced in the presence of stroma for 3 days had significant levels of human cell engraftment at the same timepoints, 7 to 11 months after transplantation. Our data show loss of long-lived human progenitors during 3-day in vitro transduction periods in the absence of stromal support. Therefore, the presence of bone marrow stroma has dual benefits in that it increases gene transfer efficiency and is essential for survival of long-lived human hematopoietic progenitors.

Due to a heightened awareness of the potential for adverse events during human gene therapy trials, it is important to document the safety of the vectors and to examine the potential for malignancy arising from insertional mutagenesis from integrating vectors, using detailed in vivo analyses. In the current studies, we assessed the potential for generation of replication-competent retrovirus (RCR) and insertional mutagenesis causing adverse events, in human cells transplanted into immune deficient mice. Both retroviral and lentiviral vectors were assessed, in a total of 630 recipient mice. Human hematopoietic and mesenchymal stem cells carrying two different Moloney-based vectors were co-transplanted into beige/nude/XID and NOD/SCID mice that are unable to reject cells that become transformed. A total of 481 mice transplanted with human cells transduced by Moloney-based vectors, and an additional 149 mice that had received human cells transduced by HIV-based lentiviral vectors were monitored daily for adverse events for 2–18 months post-transplantation. An “adverse event” was logged in the experiment, the mouse was immediately sacrificed, and an autopsy was performed if ANY signs of ill health were observed, including any of the following: weight loss, hunching, lethargy, rapid breathing, skin discoloration or irregularities, bloating, hemi-paresis, enlarged lymph nodes or visible solid tumors under the skin. The presence of tumors and organ or lymph node abnormalities, or progression to monoclonal expansion in hematopoietic cells were specifically sought. The organs, marrow, blood, and serum were banked. Tissue sections were prepared from all organs (and visible tumor, if present). DNA was isolated from marrow, organs, blood, and any tumors to determine whether vector elements were present. Of the 630 recipient mice, 117 had some evidence of adverse effects ranging from development of leukemia to scarred skin or discolored organs detected upon autopsy. Human leukemic cells were found infiltrating the murine liver, spleen, blood and lymph nodes in 4 of these 117 mice. No vector DNA was present. 37 of the mice had developed solid tumors, determined to be of murine origin with no vector DNA present or RCR detected in their serum or tumor tissue. Despite the injection of 459 million transduced cells throughout the experiments, the engineered human MSC never gave rise to solid tumors. None of the serum samples or affected tissues from these mice had vector integrants or produced RCR in the marker rescue assay. In summary, 4 of the 117 total adverse events were due to the spontaneous development of human leukemia and the remainder of the events were due to development of cancerous states in the murine tissues, with no evidence of retroviral vector involvement. There was no evidence of insertional mutagenesis causing human leukemia or solid tumors in any mouse. No RCR were detected in 117 serum samples analyzed by vector rescue assay. No adverse events were caused by the vectors and no mouse tested had HIV p24 capsid protein in their serum. The current studies provide an in vivo system to assess potential risk from RCR and from insertional mutagenesis when retroviral or lentiviral vectors are considered as candidates for human gene therapy.

There are multiple applications for expansion of Hematopoietic Stem Cells (HSC) and progenitors in transplantation. Gene therapy for most hematopoietic diseases requires selective expansion of genetically corrected HSC to achieve therapeutic effects. Obtaining sufficient number of HSC (CD34+) is a limitation for use of cord blood (CB) in transplantation. Delayed immune recovery following HSC transplantation is associated with increased morbidity and mortality. Murine lymphoid recovery can be hastened by co-transplantation of Common Lymphoid Progenitors (CLP) with HSC, however, the rarity of human CLP in harvested products preclude their clinical use. We hypothesized that human HSC and CLP can be selectively and reversibly expanded by expression of a fusion protein comprised of the intracellular signaling domain of Thrombopoietin receptor (mpl), linked to a specific binding domain (F36V) for the chemical inducer of dimerization AP20187 (CID) (ARIAD Pharmaceuticals). Upon binding of CID to F36V, mpl signaling occurs. A lentiviral vector expressing the fusion protein (F36V-mpl) and a marker gene (GFP) was constructed and efficiently transduced and expressed in human CB HSC (CD34+CD38-CD7-) and CLP (CD34+CD38-CD7+). CID-induced mpl signaling in transduced human CLP maintained robust generation of total, B and NK cells for > 60 days, in cytokine free lymphoid cultures (N=4). Under these conditions transduced HSC cultures (N=4) maintained robust generation of total, B, NK cells for >120 day. Transduced HSC continued to generate clonogenic myelo-erythroid progenitors for > 120 days in ELTC-IC assay (N=4). These cytokine free in vitro assays indicate that CID-induced mpl signaling in human HSC and CLP induced prolonged survival and proliferation of transduced progenitors. Most of the cells generated in the presence of CID expressed GFP (mean 86% GFP+), indicating selective proliferation of transduced cells. Rapid decline in the number of cells expressing GFP was noticed upon withdrawal of CID, indicating reversible activation of mpl signaling in the transduced cells. In contrast, transduced human HSC and CLP cultured without CID, proliferated poorly and differentiated rapidly; viable cells were lost by day 22 of culture. To study whether cells stimulated by CID remained immunophenotypically and functionally primitive after dividing, cell divisions were tracked by labeling with the membrane dye PKH26. Proliferation index of transduced HSC was consistently higher in the presence of CID than in its absence. Human CD34+ cells that had undergone 3 divisions in the presence of CID and in absence of any cytokines, maintained a primitive CD34+ immunophenotype in vitro. In contrast, cells that divided in the absence of CID lost CD34 expression. Cells cultured ± CID were isolated after 3 divisions (based on PKH26 staining) and transplanted in equal numbers (40,000 cell/mouse) into sublethally ablated NOD/SCIDb2m −/− mice to assess function. Only CID-expanded cells were able to engraft, producing B lymphoid and myeloid progeny. Bone marrow harvested from the engrafted animals (N=3) contained clonogenic human myelo-erythroid progenitors (confirmed by Alu PCR of human specific CFU); cells cultured without CID failed to engraft. These studies show that CID-induced mpl signaling expands functionally primitive multipotent, engrafting human progenitors. This is a potential approach to selectively and reversibly expand transduced primitive human progenitors for use in cell therapy.

Retroviral vectors based on the Moloney murine leukemia virus (MoMuLV) are currently the most commonly used vehicles for stable gene transfer into mammalian hematopoietic cells. But, even with reasonable transduction efficiency, expression only occurs in a low percentage of transduced cells and decreases to undetectable levels over time. We have previously reported the modified MND LTR (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted) to show increased expression frequency and decreased methylation in transduced murine embryonic stem cells and hematopoietic stem cells. We have now compared expression of the enhanced green fluorescent protein (eGFP) from a vector using the MoMuLV LTR (LeGFPSN) with that from the modified vector (MNDeGFPSN) in mature hematopoietic and lymphoid cells in the mouse bone marrow transplant (BMT) model. In primary BMT recipients, we observed a higher frequency of expression from the MND LTR (20% to 80%) in hematopoietic cells of all lineages in spleen, bone marrow, thymus, and blood compared with expression from the MoMuLV LTR (5% to 10%). Expression from the MND LTR reached 88% in thymic T lymphocytes and 54% in splenic B lymphocytes for up to 8 months after BMT. The mean fluorescence intensity of the individual cells, indicating the amount of protein synthesized, was 6- to 10-fold higher in cells expressing MNDeGFPSN compared with cells expressing LeGFPSN. Transduction efficiencies determined by DNA polymerase chain reaction of vector copy number were comparable for the 2 vectors. Therefore, the MND vector offers an improved vehicle for reliable gene expression in hematopoietic cells.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disease characterized by immunodeficiency and severe thrombocytopenia in affected males, but no demonstrable clinical abnormalities in carrier females. Through analysis of the methylation patterns of X-linked genes that display restriction fragment length polymorphisms (RFLPs), we studied the pattern of X-chromosome inactivation in various cell populations from female relatives of patients with WAS. The peripheral blood T cells, granulocytes, and B cells of eight obligate WAS carriers were found to display specific patterns of X-chromosome inactivation clearly different from these of normal controls. Thus, carriers of WAS could be accurately identified using this analysis.

Two human melanoma cell lines were transduced with the human interleukin (IL)-7 and IL-2 genes using retroviral-mediated gene transfer. Stable, high-level cytokine expression was achieved. The in vitro growth of transduced tumors was unaltered. Neither of the IL-2- transduced melanoma cell lines grew in athymic mice, whereas one IL-7- transduced melanoma line showed retarded in vivo growth. This is consistent with animal studies suggesting a predominantly T-cell response to IL-7-transduced tumors and a more nonspecific response to IL-2-transduced tumors. Both IL-7- and IL-2-transduced melanoma cell lines could induce cytotoxic lymphocytes in mixed lymphocyte-tumor cultures. The expression of putative melanoma antigens (MAGE)-1 and MAGE-3 was unaltered by cytokine transduction. In one cell line, IL-7 transduction resulted in a marked inhibition of the immunosuppressive peptide transforming growth factor (TGF)beta 1. The results allow a comparison of immunobiologic properties of IL-7- and IL-2-transduced human melanoma cell lines in consideration of their use in genetically engineered tumor vaccines. IL-7 transduction results in stable cytokine expression and phenotypic alterations that appear to be favorable for enhanced immunogenicity and it deserves clinical testing.