View Clinical Trial (Medical Research Study)
Peripheral Blood Stem Cells Obtained From Normal Volunteers for Studying Retroviral Vector Mediated Gene Transfer Into Primitive Hematopoietic Cells and Vector Mediated Transgene Expression in Mature Hematopoietic Lineages
| City: |
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Memphis |
| State: |
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Tennessee |
| Zip Code: |
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38105 |
| Conditions: |
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Healthy Volunteers |
| Purpose: |
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These studies are designed to evaluate the relative efficiency of gene transfer into
primitive human hematopoietic cells by comparing lentiviral and foamy virus vectors as
vehicles for transfer and expression of globin genes. Normal volunteers will serve as
research participants. Each will receive a 4 day course of Granulocyte-Colony Stimulating
Factor (G-CSF) after which a peripheral blood apheresis will be performed to recover a
mononuclear cell population enriched in primitive hematopoietic cells. The stem and
progenitor cells will be purified by selection based on expression of the CD34 antigen. The
CD34+ population will be cultured in vitro with various cytokines and transduced with vector
particles. The efficiency of gene transfer will be evaluated in the transduced CD34+
population, in progenitors contained within that population by culture in semisolid media
and in cells capable of establishing human hematopoiesis in immunodeficient mice. The level
of transgene expression will be evaluated in mature hematopoietic lineages that develop in
vitro or in immunodeficient mice.
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| Study Summary: |
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The peripheral blood has been established as a source of hematopoietic stem cells, providing
an alternative source to bone marrow for hematopoietic reconstitution of patients with
oncologic, hematologic, and genetic diseases (1-7). Although apheresis is less efficient
than bone marrow harvesting for collection of large numbers of CD34+ (a marker of early
hematopoietic precursors) cells, apheresis is less traumatic, requires no anesthesia and can
be performed more frequently than bone marrow harvesting. The purpose of this preclinical
research project is to investigate the use of hematopoietic stem cells mobilized into
peripheral blood as targets for retroviral-mediated gene therapy, to compare the efficiency
of transduction of primitive cells with the lentiviral and foamy viral vector systems and to
evaluate the ability to expand those stem cells ex vivo using hematopoietic growth factors
(cytokines). Research participants in this study will be healthy adult volunteers.
The growth factor, Granulocyte-Colony Stimulating Factor (G-CSF), will be given for 4 days
prior to apheresis to mobilize increased numbers of primitive hematopoietic cells from the
bone marrow into the circulation (8,9). Nucleated blood cells will be collected by
apheresis and a CD34+-enriched cell population isolated using antibody-based techniques
(10).
Administration of G-CSF is essential to allow collection of sufficient numbers of primitive
stem and progenitor cells from normal volunteers (4-7, 11). In the dose to be used in this
study, a 15-35 fold increase in the concentration of CD34+ cells occurs in peripheral blood
over the 4 days of G-CSF administration resulting in an apheresis product which contains, on
average, about 1-2 x1010 nucleated cells from which 1-2x108 purified CD34+cells can be
recovered. This number is sufficient to allow an experimental design which includes in
vitro culture, transduction with vector particles and assay of transduced cells in the
immunodeficient mouse model.
The use of G-CSF for mobilization of stem and progenitor cells is a widely used clinical
technique for both autologous and allogeneic transplantation which has proved both safe and
effective (4-7,11). Bone and muscle pain, headache and fatigue are the most common
complications which are typically managed with acetaminophen and rarely require
discontinuation of G-CSF. Transient elevations in alkaline phosphatase and LDH are common.
Follow-up studies have shown prolonged, mild neutropenia, lymphopenia and thrombocytopenia
following G-CSF administration and apheresis (12,13). Splenic enlargement has been shown by
non-invasive measurements and four cases of splenic rupture in normal individuals have been
described (14). A reduction in arterial pO2 associated with a decrease in the O2 saturation
from 98 to 96% has been reported (15). Transient thrombophilia has been inferred based on
changes in blood coagulation parameters.
Other rare complications have been reported including the development of inflammatory or
infectious lesions (16). A fatal crisis occurred in a sibling donor for allogeneic
transplantation who had hemoglobin SC disease (17). These rare complications should be put
in the context that G-CSF administration followed by apheresis to recover primitive
hematopoietic cells from normal individuals has been utilized in all major transplant
centers for more than a decade and has generally been safe and without significant
complications. We propose to minimize the risk of serious complications by limiting the age
range of research participants to 18-40 years and excluding those with a history of
inflammatory disease, cardiovascular disease, thrombosis or pulmonary embolization,
inflammatory skin disorders, hematological disease or hemoglobinopathy.
The ability to expand mobilized CD34+ cells in vitro using various cytokine combinations and
culture conditions will be examined using flow cytometry, in vitro colony assays and by
quantitating the concentration of cells that are able to establish hematopoiesis in
immunodeficient mice. Both unexpanded and expanded CD34+ cells will be used in experiments
designed to investigate their suitability as targets for retroviral-mediated gene transfer.
Gene transfer efficiency and functional expression of transferred sequences will be assessed
in the differentiated progeny of CD34+ cells.
We plan to compare the lentiviral (18) and foamy virus (19) vector systems with respect to
relative efficiency of gene transfer into primitive human hematopoietic cells. Various
substances e.g., Fibroblast Growth Factor -1 (20) and Wnt 3A (21), that have been reported
to cause expansion of primitive murine hematopoietic cells will also be evaluated for their
effects of primitive human hematopoietic cells in culture. Another focus is the comparison
of various globin gene vectors with respect to the relative levels of globin gene expression
in differentiating erythroid cells. Finally, the impact of cryopreservation on viability
and engraftment potential of primitive hematopoietic cells will be assessed. This research
proposal will provide preclinical data that will aid in the development of treatment
strategies for genetic diseases, specifically hemoglobin disorders, based on gene transfer
into repopulating stem cells.
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| Criteria: |
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Inclusion Criteria:
- Age greater than or equal to 18 years and less than or equal to 40.
- Meets donation criteria for autologous blood donors per SJCRH Blood Donor Center
standard operating procedure.
- Adequate venous access for apheresis on examination as per the judgment of the Blood
Donor Center nursing staff, physician staff or physician investigators.
Exclusion Criteria:
- Females - lactating.
- Concurrent use of systemic medications that in the judgment of the physician
investigators adversely affect platelet function, such as aspirin or non-steroidal
antiinflammatory agents.
- Any of the following diagnoses (prior or current):
- pulmonary disease.
- inflammatory disorder.
- coronary artery disease.
- stroke (cerebral vascular accident).
- hypertension.
- cardiac arrhythmias.
- venous thrombosis.
- pulmonary embolus
- hematological disease.
- eczema or psoriasis.
Additional eligibility criteria (to be obtained after signing informed consent)
- Body mass index less than 30 kg/m2.
- No hepatomegaly or splenomegaly.
- Hemoglobin greater than or equal to 12.5 g/dL.
- Platelet count greater than or equal to 150,000/mm3.
- Total WBC > 4200/ul and neutrophil count >1800/ul.
- Females - not pregnant (negative serum or urine; to be obtained after signing
informed consent).
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| NCT ID: |
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NCT00758992 |
| Primary Contact: |
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Principal Investigator
Arthur W Nienhuis, MD
St. Jude Children's Research Hospital
Arthur W Nienhuis, MD
Phone: 1-866-278-5833
Email: info@stjude.org
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| Backup Contact: |
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N/A |
| Location Contact: |
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Memphis, Tennessee 38105
United States
Arthur W Nienhuis, MD
Phone: 866-278-5833
Email: info@stjude.org
Site Status: Recruiting |
| Data Source: |
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ClinicalTrials.gov |
| Date Processed: |
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May 24, 2013 |
| Modifications to this listing: |
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