scholarly journals Human peripheral blood hematopoietic progenitors are optimal targets of retroviral-mediated gene transfer

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1418-1422 ◽  
Author(s):  
M Bregni ◽  
M Magni ◽  
S Siena ◽  
M Di Nicola ◽  
G Bonadonna ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cancer Patients ◽  
Resistance Gene ◽  
Peripheral Blood ◽  
Large Scale ◽  
Human Peripheral Blood ◽  
High Dose ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem

Abstract Hematopoietic progenitor cells circulate in the peripheral blood (PB) of cancer patients during the recovery phase that follows treatment with high-dose cyclophosphamide followed by hematopoietic growth factor infusion. We report that when PB progenitors were exposed in vitro to filtered supernatant from cell line PA317-N2, producing amphotropic helper-free N2 vector at conventional titers, successful retroviral- mediated transfer of neomycin resistance gene was documented by polymerase chain reaction in 93% of day 14 myelomonocytic colonies. Under the same conditions, gene transfer was achieved in 22% of steady- state bone marrow-derived myelomonocytic colonies. Neo-resistance gene transfer was documented also in a CD34+/cyclophosphamide-resistant precursor to granulocyte-macrophage colonies, an undifferentiated progenitor close to the hematopoietic stem cell. Neither cocultivation with vector-producing cells nor high vector titer were stringent requisites for efficient gene transfer. The large-scale availability of PB hematopoietic progenitors in cancer patients, together with the high gene transfer rate achieved under safe and clinically feasible conditions, support an optimal approach for gene transfer procedures into the human hematopoietic system.

scholarly journals Human peripheral blood hematopoietic progenitors are optimal targets of retroviral-mediated gene transfer

Blood ◽  
1992 ◽  
Vol 80 (6) ◽  
pp. 1418-1422 ◽  
Author(s):  
M Bregni ◽  
M Magni ◽  
S Siena ◽  
M Di Nicola ◽  
G Bonadonna ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Cancer Patients ◽  
Resistance Gene ◽  
Peripheral Blood ◽  
Large Scale ◽  
Human Peripheral Blood ◽  
High Dose ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem

Hematopoietic progenitor cells circulate in the peripheral blood (PB) of cancer patients during the recovery phase that follows treatment with high-dose cyclophosphamide followed by hematopoietic growth factor infusion. We report that when PB progenitors were exposed in vitro to filtered supernatant from cell line PA317-N2, producing amphotropic helper-free N2 vector at conventional titers, successful retroviral- mediated transfer of neomycin resistance gene was documented by polymerase chain reaction in 93% of day 14 myelomonocytic colonies. Under the same conditions, gene transfer was achieved in 22% of steady- state bone marrow-derived myelomonocytic colonies. Neo-resistance gene transfer was documented also in a CD34+/cyclophosphamide-resistant precursor to granulocyte-macrophage colonies, an undifferentiated progenitor close to the hematopoietic stem cell. Neither cocultivation with vector-producing cells nor high vector titer were stringent requisites for efficient gene transfer. The large-scale availability of PB hematopoietic progenitors in cancer patients, together with the high gene transfer rate achieved under safe and clinically feasible conditions, support an optimal approach for gene transfer procedures into the human hematopoietic system.

scholarly journals Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2898-2903 ◽  
Author(s):  
R Henschler ◽  
W Brugger ◽  
T Luft ◽  
T Frey ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Large Scale ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Ex Vivo Expansion ◽  
High Dose ◽  
Peripheral Blood Progenitor Cells ◽  
Limiting Dilution

Abstract CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.

In Vitro Manipulation of Peripheral Blood Progenitor Cell Collections

1998 ◽  
Vol 21 (6_suppl) ◽  
pp. 1-10
Author(s):  
C. Carlo-Stella ◽  
V. Rizzoli
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Peripheral Blood Progenitor Cells ◽  
Stem And Progenitor Cells ◽  
Therapy Strategies ◽  
Mobilized Peripheral Blood

Mobilized peripheral blood progenitor cells (PBPC) are increasingly used to reconstitute hematopoiesis in patients undergoing high-dose chemoradiotherapy. PBPC collections comprise a heterogeneous population containing both committed progenitors and pluripotent stem cells and can be harvested (i) in steady state, (ii) after chemotherapeutic conditioning, (iii) growth factor priming, or (iv) both. The use of PBPC has opened new therapeutic perspectives mainly related to the availability of large amounts of mobilized hematopoietic stem and progenitor cells. Extensive manipulation of the grafts, including the possibility of exploiting these cells as vehicles for gene therapy strategies, are now possible and will be reviewed.

Negative Control Region Is a Critical Element Of Insertional Oncogenesis After Gene Transfer Into Hematopoietic Progenitors With Moloney Murine Leukemia Viruses

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 164-164
Author(s):  
Yasuhiro Ikawa ◽  
Toru Uchiyama ◽  
Guridevi Jayashree Jagadeesh ◽  
Fabio Candotti
Keyword(s):  
Stem Cell ◽  
Gene Transfer ◽  
Viral Vectors ◽  
Negative Control ◽  
Hematopoietic Progenitors ◽  
Hematopoietic Stem ◽  
Cell Immortalization ◽  
The Difference ◽  
Murine Leukemia

Abstract Gene transfer into hematopoietic stem cells has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the successes of clinical trials have been tempered by adverse events in which the use of gamma-retroviral vectors (GV) containing full-length long terminal repeats (LTRs) with strong enhancer activity increased transcription of cancer-related genes, and thereby contributed to development of leukemia. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance. The negative control region (NCR) is a particularly well-conserved sequence among mammalian gamma-retroviruses with demonstrated regulating a transcription activity of GV in hematopoietic cells. This suggests that the NCR might play a crucial role of insertional oncogenesis after gene transfer into hematopoietic progenitors. In a series of safety studies of viral gene transfer constructs, we used an in vitro assay of murine bone marrow (BM) cell immortalization and compared the consequences of hematopoietic stem cell transduction with three different kinds of viral vectors, including Moloney murine leukemia virus- (MMLV), lentivirus- (LV), and foamy virus (FV)-based constructs. To evaluate critical elements for cell immortalization by MMLV vectors, we also tested four different MMLV LTR variants deleted of either 1) most of the two 75-bp repeats associated with the viral enhancer (delE1), 2) all of the two 75-bp repeats and the NCR (delE2), 3) only the NCR (delNCR), or 4) carrying a deleterious mutation of the NCR NFAT motif (ΔNFAT). All vectors carried an internal expression cassette including the eGFP gene under the control of a UCOE (ubiquitously acting chromatin opening element) promoter. In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than six weeks in the presence of interleukin-3 and stem cell factor. Real-time PCR was performed to verify comparable transduction efficiency of bone marrow cells by different vectors. In our analysis of MMLV LTR mutants, full-MMLV and delE1 transduction of 92 and 108 cultures, respectively, resulted in 37 and 37 immortalized lines (40% and 34% immortalization rate, respectively). The difference in immortalization rate between full-MMLV and delE1 was not statistically significant. Transductions using delE2-, delNCR- and ΔNFAT-carrying vectors of 60, 36 and 35 cultures resulted in 10, 3 and 10 immortalized lines (17%, 8.3% and 29% immortalization rate, respectively). The difference between the immortalization caused by delE1 and delE2 vectors was statistically significant (p<0.05). Moreover, the difference between the immortalization caused by full-MMLV and delNCR vectors was statistically significant (p<0.01), while there was no significant difference between the immortalization induced by full-MMLV and ΔNFAT vectors. Transduction of 57 and 34 cultures with LV and FV vectors, respectively, resulted in no immortalized lines. Transductions of 128 cultures with a LV construct carrying the U3 region from the murine stem cell virus LTR as an internal promoter (LV-U3) resulted in 2 immortalized lines which was not statistically different from the results obtained with LV vectors carrying the UCOE internal promoter. These results confirm that GV are prone to causing immortalization of hematopoietic cells and indicate that deletion of the whole viral enhancer sequences may not be adequate to eliminate the insertional oncogenesis risk. Importantly, our data point to the NCR as a crucial element for immortalization and justify additional studies to evaluate its specific role in MMLV-mediated insertional oncogenesis. Finally, our results suggest that vectors based on LV and FV backbones are safer alternatives for clinical gene transfer into hematopoietic stem cells. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Retrovirally marked CD34-enriched peripheral blood and bone marrow cells contribute to long-term engraftment after autologous transplantation

Blood ◽  
1995 ◽  
Vol 85 (11) ◽  
pp. 3048-3057 ◽  
Author(s):  
CE Dunbar ◽  
M Cottler-Fox ◽  
JA O'Shaughnessy ◽  
S Doren ◽  
C Carter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Gene Transfer ◽  
Resistance Gene ◽  
Peripheral Blood ◽  
Marker Gene ◽  
Autologous Transplantation ◽  
High Dose ◽  
Neomycin Resistance Gene ◽  
Neomycin Resistance

We report here on a preliminary human autologous transplantation study of retroviral gene transfer to bone marrow (BM) and peripheral blood (PB)-derived CD34-enriched cells. Eleven patients with multiple myeloma or breast cancer had cyclophosphamide and filgrastim-mobilized PB cells CD34-enriched and transduced with a retroviral marking vector containing the neomycin resistance gene, and CD34-enriched BM cells transduced with a second marking vector also containing a neomycin resistance gene. After high-dose conditioning therapy, both transduced cell populations were reinfused and patients were followed over time for the presence of the marker gene and any adverse effects related to the gene-transfer procedure. All 10 evaluable patients had the marker gene detected at the time of engraftment, and 3 of 9 patients had persistence of the marker gene for greater than 18 months posttransplantation. The marker gene was detected in multiple lineages, including granulocytes, T cells, and B cells. The source of the marking was both the transduced PB graft and the BM graft, with a suggestion of better long-term marking originating from the PB graft. The steady-state levels of marking were low, with only 1:1000 to 1:10,000 cells positive. There was no toxicity noted, and patients did not develop detectable replication-competent helper virus at any time posttransplantation. These results suggest that mobilized PB cells may be preferable to BM for gene therapy applications and that progeny of mobilized peripheral blood cells can contribute long-term to engraftment of multiple lineages.

Ex Vivo Expanded Megakaryocyte Progenitors Generated From Human Mobilized Peripheral Blood CD34+ Stem Cells Can Be Cryopreserved and Transplanted Into NSG Mice to Generate Functional Platelets

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 482-482
Author(s):  
Holger Karsunky ◽  
Robert J. Tressler ◽  
Joy Chananukul
Keyword(s):  
Stem Cells ◽  
Peripheral Blood ◽  
Platelet Transfusion ◽  
Ex Vivo ◽  
High Dose ◽  
Hematopoietic Stem ◽  
Nsg Mice ◽  
Mobilized Peripheral Blood ◽  
Megakaryocyte Progenitors

Abstract Abstract 482 Thrombocytopenia is a common side effect of high-dose chemotherapy that can compromise cancer treatment by requiring treatment delay and/or dose reduction for the patient. Platelet transfusion is typically given to prevent severe hemorrhage. However, several factors including acquisition, banking, and associated risks of bacterial infections and alloimmunization are hampering reliance on platelet transfusion. Growth factors are also used to stimulate proliferation and differentiation of megakaryocytes to increase platelet production, but in severely myelosuppressed patients these have only had modest benefit. The limitations of these two modalities for the treatment of chemotherapy-induced thrombocytopenia indicates that additional treatment approaches are needed. We have developed a novel approach to reconstitute megakaryocytes and platelets in thrombocytopenic patients which is presented here. We have identified a scalable culture system using serum-free medium and a defined cytokine cocktail free of animal products to expand CD34+ hematopoietic stem cells from G-CSF mobilized peripheral blood donors in vitro and direct their development to the megakaryocyte lineage to yield committed human megakaryocyte progenitors (MKPs). These MKPs can be readily cryopreserved while retaining their capacity to generate CFU-MK and platelets in vitro. When infused into NSG mice, ex vivo expanded MKP generate clinically relevant platelet levels of platelets in blood within a few days with sustained platelet levels for several weeks. The platelets generated from MKP in vivo are also functional as assessed by CD62P expression in responses to ADP stimulation in vitro. Our results present a compelling approach for the development of off-the-shelf storable MKPs for the treatment of thrombocytopenia. Disclosures: Karsunky: Cellerant Therapeutics Inc.: Employment, Patents & Royalties. Tressler:Cellerant Therapeutics, Inc.: Employment, Equity Ownership. Chananukul:Cellerant Therapeutics Inc.: Employment, Patents & Royalties.

scholarly journals Maintenance of transplantation potential in ex vivo expanded CD34(+)- selected human peripheral blood progenitor cells

Blood ◽  
1994 ◽  
Vol 84 (9) ◽  
pp. 2898-2903 ◽  
Author(s):  
R Henschler ◽  
W Brugger ◽  
T Luft ◽  
T Frey ◽  
R Mertelsmann ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Peripheral Blood ◽  
Large Scale ◽  
Ex Vivo ◽  
Human Peripheral Blood ◽  
Ex Vivo Expansion ◽  
High Dose ◽  
Peripheral Blood Progenitor Cells ◽  
Limiting Dilution

CD34(+)-selected hematopoietic progenitor cells are being increasingly used for autotransplantation, and recent evidence indicates that these cells can be expanded ex vivo. Of 15 patients with solid tumors undergoing a phase I/II clinical trial using CD34(+)-selected peripheral blood progenitor cells (PBPCs) after high-dose chemotherapy, we analyzed the frequency of long-term culture-initiating cells (LTCIC) as a measure of transplantation potential before and after ex vivo expansion of CD34+ cells. PBPCs were mobilized by combination chemotherapy and granulocyte colony-stimulating factor (G-CSF). The original unseparated leukapheresis preparations, the CD34(+)-enriched transplants, as well as nonabsorbed fractions eluting from the CD34 immunoaffinity columns (Ceprate; CellPro, Bothell, WA) were monitored for their capacity to repopulate irradiated allogeneic stroma in human long-term bone marrow cultures. We found preservation of more than three quarters of fully functional LTCIC in the CD34(+)-selected fractions. Quantitation of LTCIC by limiting dilution analysis showed a 53-fold enrichment of LTCIC from 1/9,075 in the unseparated cells to an incidence of 1/169 in the CD34+ fractions. Thus, in a single apheresis, it was possible to harvest a median of 1.65 x 10(4) LTCIC per kg body weight (range, 0.71 to 3.72). In addition, in six patients, large-scale ex vivo expansions were performed using a five-factor cytokine combination consisting of stem cell factor (SCF), interleukin-1 (IL-1), IL-3, IL-6, and erythropoietin (EPO), previously shown to expand committed progenitor cells. LTCIC were preserved, but not expanded during the culture period. Optimization of ex vivo expansion growth factor requirements using limiting dilution assays for LTCIC estimation indicated that the five-factor combination using SCF, IL-1, IL-3, IL-6, and EPO together with autologous plasma was the most reliable combination securing both high progenitor yield and, at the same time, optimal preservation of LTCIC. Our data suggest that ex vivo-expanded CD34+ PBPCs might be able to allow long-term reconstitution of hematopoiesis.

scholarly journals Single Agent Polysaccharopeptide Delays Metastases and Improves Survival in Naturally Occurring Hemangiosarcoma

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Dorothy Cimino Brown ◽  
Jennifer Reetz
Keyword(s):  
Cancer Patients ◽  
Large Scale ◽  
Single Agent ◽  
Treatment Strategies ◽  
World Health ◽  
High Dose ◽  
Survival Times ◽  
Double Blind ◽  
Naturally Occurring

The 2008 World Health Organization World Cancer Report describes global cancer incidence soaring with many patients living in countries that lack resources for cancer control. Alternative treatment strategies that can reduce the global disease burden at manageable costs must be developed. Polysaccharopeptide (PSP) is the bioactive agent from the mushroomCoriolus versicolor. Studies indicate PSP has in vitro antitumor activities and inhibits the growth of induced tumors in animal models. Clear evidence of clinically relevant benefits of PSP in cancer patients, however, is lacking. The investment of resources required to complete large-scale, randomized controlled trials of PSP in cancer patients is more easily justified if antitumor and survival benefits are documented in a complex animal model of a naturally occurring cancer that parallels human disease. Because of its high metastatic rate and vascular origin, canine hemangiosarcoma is used for investigations in antimetastatic and antiangiogenic therapies. In this double-blind randomized multidose pilot study, high-dose PSP significantly delayed the progression of metastases and afforded the longest survival times reported in canine hemangiosarcoma. These data suggest that, for those cancer patients for whom advanced treatments are not accessible, PSP as a single agent might offer significant improvements in morbidity and mortality.

Immunoablative Therapy with Autologous Stem Cell Transplantation in the Treatment of Poor Risk Multiple Sclerosis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5519-5519
Author(s):  
Tomas Kozak ◽  
Eva Havrdova ◽  
Jiri Pitha ◽  
Karolina Mayerova ◽  
Gregora Evzen ◽  
...  
Keyword(s):  
Multiple Sclerosis ◽  
Stem Cell ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Peripheral Blood ◽  
Repeated Measure ◽  
High Dose ◽  
Hematopoietic Stem

Abstract The immunoablative therapy with hematopoietic stem cell transplantation is tested in patients with intractable form of autoimmune diseases including multiple sclerosis (MS). Thirty one patients with secondary rapidly progressive MS were included in the phase I/II clinical trial involving the high dose chemotherapy with autologous peripheral blood progenitor cell (PBPC) support. Twenty seven patients underwent high dose conditioning BEAM. T cell depletion in vitro was performed in 18 grafts. Nine patients with not purged graft received in vivo ATG 4mg/kg i.v. D+1, D+2 after transplantation. Median follow-up is 48 months (24–72). Median EDSS (Expanded Disability Status Scale) of grafted patients at the time of inclusion was 6.5 (5.0–7.5), median EDSS of grafted patients at the last follow up was 7,0 (5.5–10.0). Two patients out of 27 (7 %) remain improved significantly (by ≥ 1.0 point on EDSS), 3 patients (11 %) are improved not significantly (by 0.5 point). Five patients (19 %) are stable in their EDSS. Seven patients (26 %) gained their disability significantly (by ≥ 1.0 point on EDSS) despite the treatment, one of them died 31 months after the transplantation from disease progression (EDSS 10.0). Ten patients (37 %) worsened not significantly (by 0.5 point) on their EDSS. Patients who clearly stabilized their disability or remain improved represent 37 %. The increase of EDSS at the 48 months (m) of the follow up is significatnt (Wicoxon’s, repeated measure ANOVA, t-test), however, is not at 36, 60 and 72 m respectively. The development of disability between the group that was grafted with in vitro purged graft and the group with ATG i.v. was not significant (Wilcoxon’s, Mann-Whitney). 20 patients stabilized their MRI finding, in 2 patients decreased number and size of lesions, 5 patients worsened their MRI. No mortality has been observed in this cohort. However, the toxicity of the transplantation differed in each individual; two serious events involving respiratory tract have been observed. Changes in lymphocyte subsets in peripheral blood were followed before and after the treatment. The percentage and absolute count of CD4+ cells and CD4+CD45 as well as IRI were significantly decreased 12 months after the transplantation Conclusion: Almost 40 % of patients with otherwise intractable rapidly progressive MS remained stable or improved in their disability with median follow up 48 months after immunoablative therapy. The value of this type of therapy should be investigated in the randomized trial. The study was sponsored by Grant IGA No NF/6560-3 from Ministry of Health of the Czech Republic.

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