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
Condition: Immunodeficiency
MeSH terms: Immunologic Deficiency Syndromes
Study type: Observational

Summary: 
      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.
    

Detailed description: 
      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. 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. Nucleated blood cells will be collected by apheresis and a
      CD34+-enriched cell population isolated using antibody-based techniques.

      Administration of G-CSF is essential to allow collection of sufficient numbers of primitive
      stem and progenitor cells from normal volunteers. 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. 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. Splenic enlargement has been shown by non-invasive measurements and four cases
      of splenic rupture in normal individuals have been described. A reduction in arterial pO2
      associated with a decrease in the O2 saturation from 98 to 96% has been reported. 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. A fatal crisis occurred in a sibling donor for allogeneic transplantation
      who had hemoglobin SC disease. 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 and foamy virus vector systems with respect to relative
      efficiency of gene transfer into primitive human hematopoietic cells. Various substances
      e.g., Fibroblast Growth Factor -1 and Wnt 3A, 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.
    

Observational model: Case-Only
Time perspective: Prospective
Sampling method: Non-Probability Sample
Study population: 
        Healthy Adult Research Participants
      
Biospecimen description: 
      Blood
    
Keywords: gene transfer