scholarly journals Viral Insertion Safety in Patients with Glioblastoma Who Received a Novel Lentiviral MGMT-P140K Gene Therapy to Protect Bone Marrow from Chemotherapy: No Dominant Clonal Evolution Observed with Chemo-Selection

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4801-4801
Author(s):  
Hua Fung ◽  
Andrew E Sloan ◽  
Jane Reese ◽  
Basem M. William ◽  
Karen Lingas ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Lentiviral Vector ◽  
Lymphoblastic Leukemia ◽  
Insertion Site ◽  
Retroviral Vectors ◽  
Insertion Mutagenesis ◽  
Drug Selection ◽  
Insertion Sites ◽  
Viral Insertion

Abstract INTRODUCTION: To protect normal bone marrow from chemotherapy in glioblastoma patients, we have developed a novel strategy by introducing a strong DNA repair protein, mutant (P140K) of human methylguanine methyltransferase (MGMT), into patients’ CD34+ hematopoietic progenitors (HPC) by lentiviral gene transfer leading to selective expansion of drug-resistant P140K-MGMT CD34+ cells and their myeloid and immune cell progeny. METHODS: To achieve long-term stable expression of the P140K-MGMT gene, we used a lentiviral vector which integrates into the host genome. However, viral insertion mutagenesis has raised safety concerns; as the previous γ-retroviral vectors were associated with insertion mutations leading to development of acute lymphoblastic leukemia in 20% of treated patients. Nevertheless, new improved lentiviral vector with safe feature of insertion site far away from gene transcription start site has been developed. Here we evaluated the safety of a lentivirus vector under selection pressure of chemotherapy. HYPOTHESIS: Our lentiviral vector is safer than traditional γ-retroviral vectors as evident by lack of early clonal dominance even with a chemo-selection. RESULTS: Three glioblastoma patients were recruited and underwent resection, after which CD34+ HPC were mobilized with filgrastim, isolated by magnetic bead separation (Miltneyi CliniMACS), and transduced ex vivo with a GMP-grade lentiviral P140K-MGMT vector (Lentigen Corp). Subsequently, patients received radiation/temozolomide for 6 weeks and up to five cycles of monthly O6-benzylguanine/temozolomide (BG/TMZ) treatment. As a result, all three patients demonstrated a 5-15 fold selective expansion of P140K-MGMT positive HPC and their progeny granulocyte and mononuclear cells in peripheral blood and a small number of CFUs from bone marrow indicating a drug-selection mechanism. The viral insertion sites in the cells of these three patients were closely monitored in each chemotherapy cycle and the patients were followed for up to 1 year after the last therapy. We mapped a total of 17,308 viral insertion sites (VIS), for patient 1(6,146), patient 2(2,081) and patient 3(9,081) and the unique viral insertion sites (UVIS) was 382, i.e. 135, 76 and 171 for patient 1, patient 2 and patient 3 respectively. Overall, during the drug-treatment period, there were no persistent UVIS. Moreover, at the conclusion of BG/TMZ treatment, the VIS number sharply diminished. CONCLUSION: Gene transfer of LV MGMTP140K vector into hematopoietic progenitor cells did not lead to clonal dominance during or after drug selection. Dose escalation of BG/TMZ will define hematopoietic tolerance and treatment response. Disclosures Embree: Lentigen Technology Inc: Employment. Dropulic:Lentigen Technology Inc: Employment, Patents & Royalties.

Abstract 257: Bone Marrow Cells Form New Cardiomyocytes in the Infarcted Myocardium

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Fumihiro Sanada ◽  
Junghyung Kim ◽  
Anna Czarna ◽  
Sergio Signore ◽  
Andrea Sorrentino ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Fluorescent Proteins ◽  
Infarcted Myocardium ◽  
Multiple Cell ◽  
Insertion Sites ◽  
Color Combinations ◽  
Viral Insertion ◽  
Marrow Cells

The ability of c-kit-positive bone marrow cells (c-kit-BMCs) to form cardiomyocytes following delivery to the acutely infarcted myocardium is a matter of debate. In an attempt to resolve the controversy, we raised the possibility that c-kit-BMCs represent a functionally heterogeneous pool, containing cells with distinct transdifferentiation potential. To test this hypothesis, c-kit-BMCs were infected with EGFP-lentiviruses and injected in infarcted hearts. At 1 and 2 weeks, the regenerated myocardium was enzymatically digested and EGFP-labeled myocytes, endothelial cells (ECs), fibroblasts, and c-kit-cells were sorted. By employing a PCR-based method of detection of viral integrants, we searched for unique sites of viral insertion in the infected c-kit-BMCs and their progeny. Common insertion sites were found in the DNA of all cell populations, documenting that single c-kit-BMCs transdifferentiated into multiple cell lineages. However, the 31 detected clones shared 9 distinct sites of integration, suggesting that myocytes in different animals were generated by specific subsets of c-kit-BMCs, which retained the ability to transdifferentiate. To strengthen the observation that myocardial regeneration is oligoclonal in nature, freshly isolated FACS-sorted c-kit-BMCs were simultaneously transduced with 3 lentiviruses, each encoding red, green or blue (RGB) fluorescent proteins. Different combinations of inserted vectors resulted in the formation of numerous c-kit-BMC clones of mixed colors. RGB-infected HSCs were injected in infarcted rats; at 4 days, engrafted c-kit-BMCs showed a polyclonal pattern characterized by the presence of cells labeled by most of the color combinations seen in vitro. However, at 2 weeks, only a few of the homogeneously colored colonies seen at 4 days persisted and formed large patches of cells composed of myocytes, ECs, and smooth muscle cells. Our findings document that c-kit-BMCs are functionally heterogeneous and have different ability to survive, engraft, and transdifferentiate in the ischemic heart. At present, clonal c-kit-BMCs are being analyzed by RNA-sequencing for the identification of the unique phenotypical properties of c-kit-BMCs with high transdifferentiation potential.

scholarly journals High-efficiency gene transfer and expression in normal human hematopoietic cells with retrovirus vectors

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 811-814 ◽  
Author(s):  
P Laneuville ◽  
W Chang ◽  
S Kamel-Reid ◽  
AA Fauser ◽  
JE Dick
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Progenitor Cells ◽  
High Efficiency ◽  
Bone Marrow Cells ◽  
Fibroblast Cell ◽  
Retroviral Vectors ◽  
Semisolid Medium ◽  
Bacterial Gene

Abstract Retroviral vectors containing the selectable bacterial gene for G418 resistance (neo) were used to demonstrate gene transfer into primary human bone-marrow progenitor cells. To obtain populations of cells in which a high proportion of cells were expressing the neo gene, several important modifications were made to earlier procedures. Cells from normal donors were infected in vitro, were exposed to high concentrations of G418 for two days in liquid culture to enrich for cells expressing the neo gene, and were plated in semisolid medium. Gene transfer and expression were detected in colonies arising from progenitors of granulocyte-macrophage and erythroid lineages. Survival curves indicated that a high proportion of progenitor cells, approaching 100%, were G418 resistant. Furthermore, addition of growth factors contained in 5637-conditioned medium to the bone marrow improved the recovery of G418-resistant progenitors twofold to threefold. In addition to these biological measurements of gene expression in progenitor cells, significant levels of neo-specific RNA, similar to the levels of RNA expression in the virus-producing fibroblast cell line, were detected in the bone marrow cells after preselection. These results demonstrate that retrovirus vectors can be used successfully to transfer genes at high efficiency into progenitor cells in the human blood-forming system.

scholarly journals Expression of a foreign gene in cats reconstituted with retroviral vector infected autologous bone marrow

Blood ◽  
1991 ◽  
Vol 78 (1) ◽  
pp. 237-245 ◽  
Author(s):  
CD Jr Lothrop ◽  
ZS al-Lebban ◽  
GP Niemeyer ◽  
JB Jones ◽  
MG Peterson ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Bone Marrow ◽  
Gene Transfer ◽  
Autologous Bone Marrow ◽  
Retroviral Vectors ◽  
Retroviral Vector ◽  
Foreign Gene ◽  
Large Animal ◽  
Low Level ◽  
Autologous Bone

Abstract A Moloney murine leukemia virus based retroviral vector was used to transfer the bacterial neomycin resistance gene (neoR) into feline hematopoietic cells. We reconstituted four cats that had been lethally irradiated with autologous bone marrow that had been infected with the N2 or SAX retroviral vector. Bone marrow cells from all four cats expressed the neoR gene 30 days posttransplant and three of four cats still had the neoR gene and a low level of drug resistant colony- forming unit granulocyte-macrophage after more than 200 days. Two of the four cats unexpectedly developed diabetes mellitus 90 days posttransplantation. The expression of a foreign gene in cats, albeit at a low level, demonstrates that retroviral vectors can be used for gene transfer in noninbred large animal species and may be useful for gene therapy of humans. The development of diabetes mellitus in two of the subjects emphasizes the value of animal models for the study of possible deleterious effects of retroviral vector-mediated gene transfer.

Analysis of Retroviral Vector Insertion Sites after T-Cell Directed Gene Therapy.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 289-289 ◽  
Author(s):  
Akihiro Konno ◽  
G. Jayashree Jagadeesh ◽  
Daniele Moratto ◽  
Marita Bosticardo ◽  
Ingeborg Holt ◽  
...  
Keyword(s):  
T Cell ◽  
Gene Transfer ◽  
Human Genome ◽  
Peripheral Blood ◽  
Retroviral Vectors ◽  
Peripheral Blood Lymphocytes ◽  
Inverse Pcr ◽  
Blood Lymphocytes ◽  
Integration Sites ◽  
Insertion Sites

Abstract Gene transfer into peripheral blood lymphocytes has several potential applications including the correction of genetic diseases and therapeutic approaches for HIV-1 infection and cancer. Integrating gene transfer system based on murine oncoretroviruses are a convenient tool for such strategies. However, the recent occurrence of uncontrolled clonal T cell expansions in two patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has raised the concern of the risk of insertional oncogenesis associated with the clinical use of integrating viral systems. In vitro studies have indicated that murine viral vectors tend to integrate in the vicinity of transcription start regions of the genome, thus providing a possible mechanism for oncogene activation, however, data from clinical gene transfer trials is lacking. We are following patients affected with adenosine deaminase (ADA) deficiency who have received T-lymphocyte-directed, retroviral-mediated gene transfer starting in 1990. The first treated patient received the last infusion of gene-corrected cells 12 years ago, has never shown any sign of lymphoproliferation and still carries ~20% of gene-corrected peripheral blood lymphocytes. We set out to study the integration sites in the cells of this patient with the aim of mapping the regions involved by retroviral integrations, determining their localization with respect to known genes, and assessing whether a preferred pattern could be defined. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified ~860 bona fide insertion sites. Search for homology within the human genome using BLAT returned ~330 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). To evaluate the distribution pattern of these integration sites, 2000 randomly generated data sets of genomic coordinates were assembled and their distribution relative to annotations of the human genome was analyzed. A preliminary comparison of the random distribution to our experimental samples showed that retroviral integrations in cells obtained from the patient were significantly skewed toward regions within 2 kb of genes (p<0.002) and CpG islands (p<0.001). These results suggest that, similar to what observed in murine fibroblast and human cancer cell lines, transcriptionally active regions of the genome may be preferred targets of retroviral vectors in human primary T lymphocytes. At the same time, our observations show that the resulting integration events are compatible with long-term, event-free in vivo survival of gene-modified cells in clinical settings.

Evidence for Efficacy and Safety of Lentiviral Mediated Gene Transfer in T Cells and CD34+ Cells from Wiskott-Aldrich Syndrome Patients.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3279-3279
Author(s):  
Samantha Scaramuzza ◽  
Sara Trifari ◽  
Francesco Marangoni ◽  
Silvana Martino ◽  
Ayse Metin ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Gene Transfer ◽  
Lentiviral Vector ◽  
Efficacy And Safety ◽  
Wiskott Aldrich Syndrome ◽  
Cd34 Cells ◽  
Was Gene

Abstract Wiskott-Aldrich Syndrome (WAS) is an X-linked primary immunodeficiency characterized by eczema, recurrent infections, severe hemorrhages and lymphomas. Transplantation of hematopoietic stem cells from HLA-identical sibling donors is a resolutive treatment, but it is available only for a minority of patients. Therapy based on the transplant of genetically correct autologous stem cells could represent a valid alternative approach. We investigated the efficacy and the safety of WAS gene transfer using HIV-based lentiviral vector encoding for WAS cDNA under the control of an autologous promoter (1.6 kb). T cells obtained from WAS patients showed normal level of WAS expression after lentiviral transduction. Transduced T cells showed a correction in TCR-driven proliferation and IL-2 production. Furthermore, a selective growth advantage of transduced T cells was observed in long-term in vitro cultures. Studies in T cell clones generated from transduced WAS CD4+ T cells revealed that 1–2 vector copies were necessary and sufficient to correct T cells function. CD34+ cells, isolated from mobilized peripheral blood and bone marrow of healthy donors, were transduced using WASP or GFP-encoding lentiviral vectors. Cells were cultured in the presence of different cytokines to investigate if WAS gene transfer could have any effect on short and long-term differentiation (CFU-C, LTC-IC and B/NK assays). Transduction resulted in a comparable number of CFU-C and LTC-IC colonies and normal B and NK cells differentiation with respect to untransduced cells. Furthermore, transduction of CD34+ cells isolated from the bone marrow of a WAS patient was performed under optimized culture conditions. Lentiviral gene transfer led to restoration of WASP expression in differentiated cells with copy number ranging from 1 to 5 copies per cell. In conclusion, our data demonstrate that the WAS promoter/cDNA-containing lentiviral vector can efficiently transduce and restore WASP expression in CD34+ cells and T cells from WAS patients. Experiments in the Rag2−/−/γchain- murine model are ongoing to test the efficacy and safety of the WASP transduced CD34+ cells. Together, our studies provide a preclinical basis for the implementation of a gene therapy trial for WAS patients.

scholarly journals Human cord blood cells as targets for gene transfer: potential use in genetic therapies of severe combined immunodeficiency disease.

1993 ◽  
Vol 178 (2) ◽  
pp. 529-536 ◽  
Author(s):  
T Moritz ◽  
D C Keller ◽  
D A Williams
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Severe Combined Immunodeficiency ◽  
Genetic Material ◽  
Retroviral Vectors ◽  
Transduction Efficiency ◽  
Human Cord Blood

Human cord blood (CB) contains large numbers of both committed and primitive hematopoietic progenitor cells and has been shown to have the capacity to reconstitute the lympho-hematopoietic system in transplant protocols. To investigate the potential usefulness of CB stem and progenitor cell populations to deliver new genetic material into the blood and immune systems, we have transduced these cells using retroviral technology and compared the efficiency of gene transfer into CB cells with normal adult human bone marrow cells using a variety of infection protocols. Using two retroviral vectors which differ significantly in both recombinant viral titers and vector design, low density CB or adult bone marrow (ABM) cells were infected, and committed progenitor and more primitive hematopoietic cells were analyzed for gene expression by G418 drug resistance (G418r) of neophosphotransferase and protein analysis for murine adenosine deaminase (mADA). Standard methylcellulose progenitor assays were used to quantitate transduction efficiency of committed progenitor cells, and the long term culture-initiating cell (LTC-IC) assay was used to quantitate transduction efficiency of more primitive cells. Our results indicate that CB cells were more efficiently transduced via retroviral-mediated gene transfer as compared with ABM-derived cells. In addition, stable expression of the introduced gene sequences, including the ADA cDNA, was demonstrated in the progeny of infected LTC-ICs after 5 wk in long-term marrow cultures. Expression of the introduced ADA cDNA was higher than the endogenous human ADA gene in the LTC-IC-derived colonies examined. These studies demonstrate that CB progenitor and stem cells can be efficiently infected using retroviral vectors and suggest that CB cells may provide a suitable target population in gene transfer protocols for some genetic diseases.

scholarly journals Analysis of optimal conditions for retroviral-mediated transduction of primitive human hematopoietic cells

Blood ◽  
1995 ◽  
Vol 86 (1) ◽  
pp. 101-110 ◽  
Author(s):  
JA Nolta ◽  
EM Smogorzewska ◽  
DB Kohn
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Peripheral Blood ◽  
Retroviral Vectors ◽  
Optimal Conditions ◽  
Hematopoietic Progenitors ◽  
Immunodeficient Mice ◽  
Stromal Support ◽  
Colony Forming Assay ◽  
Mobilized Peripheral Blood

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.

Leukemogenic Cascade Induced by a Constitutively Active Neurotrophin Receptor, ΔTrkA.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1442-1442
Author(s):  
Zhixiong Li ◽  
Johann Meyer ◽  
Mathias Rhein ◽  
Olga Kustikova ◽  
Cornelia Rudolph ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinases ◽  
Bone Marrow Cells ◽  
Lymphoblastic Leukemia ◽  
Insertion Site ◽  
Leukemic Cells ◽  
Neurotrophin Receptor ◽  
Target Cells ◽  
Receptor Signaling ◽  
Lymphoid Leukemia

Abstract Various neurotrophins (NT) are expressed in the hematopoietic microenvironment to deliver their signals through TRK receptor tyrosine kinases. Although previous reports suggested a transforming potential of activated TRK signaling in hematopoiesis, the target cells and underlying mechanisms are largely unknown. In this study, we investigated in vivo leukemogenesis of ΔTrkA, a mutant of TRKA isolated from a patient with acute myeloid leukemia (AML). Retroviral expression of ΔTrkA in myeloid 32D cells induced AML at ~4 weeks after transplantation into syngeneic C3H/Hej mice (n=11). C57BL/6J mice (n=15) transplanted with ΔTrkA-transduced primary bone marrow cells developed myeloid and/or lymphoid leukemia with infiltration in multiple organs including bone marrow, spleen, liver, lung, and central nervous system. While activation of a tyrosine kinase generally is not sufficient to cause AML, surprisingly, 7 out of 15 C57BL/6J mice developed a polyclonal AML with a latency of <12 days. This strongly suggests that ΔTrkA is sufficient to block differentiation and promote proliferation of short-term repopulating myeloid progenitor cells. This simultaneous interference with two complementary leukemogenic pathways has not been reported for other oncogenic tyrosine kinases. However, as half of the mice survived this disorder, ΔTrkA does not seem to confer self-renewal properties to transformed blasts. Supporting this hypothesis, transplantation into lethally irradiated secondary recipients did not re-induce polyclonal AML, but rather gave rise to clonal acute lymphoblastic leukemia (ALL) after a latency of >78 days. This suggests that ΔTrkA can also transform long-term repopulating cells with a lymphoid potential. Retroviral insertion site analyses and spectral karyotyping revealed that induction of ALL by ΔTrkA required additional genetic lesions: Leukemic cells showed retroviral insertions in proto-oncogenes Bcl11a and Bcl11b, among others. Characterization of signal transduction demonstrated that PI3K and mTOR-raptor were crucial components of the transforming pathway induced by ΔTrkA. Phospholipase D was an important contributing factor, whereas STAT and MAP kinase pathways were not involved. In summary, our findings reveal potent transforming properties of altered NT receptor signaling in leukemia induction, which are in many respects distinct from other oncogenic tyrosine kinases. We would suggest to further evaluate the role of NT receptor signaling in leukemia pathogenesis, prognosis and treatment.

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