STEM CELL TRANSDUCTION OF SLF/FLT-3-LIGAND GENES BY AAV

Project: Research project

Description

Regulation of the earliest stem and progenitor cells is not completely
understood. In this context we propose to test the hypothesis that the
combination of two early acting cytokines: Steel factor (SLF) and the Flt-
3/Flk-2-ligand, which respectively utilize the tyrosine kinase receptors
c-kit and Flt-3-/Flk-2 for their action is important for proliferation and
self-renewal of early stem/progenitor cells. This will be done by using
recombinant(r) adeno-associated virus (AAV) vectors to obtain high
efficiency stable integration of the genes for the soluble and membrane-
bound forms of both ligands into high purified stem/progenitor cells. The
aims are to: 1) construct high titer rAAV vectors containing soluble or
membrane-bound forms of the human (hu) or murine (mu) ligands with
different promoters and selectable markers; 2) evaluate different
phenotypic target cell populations that are highly enriched for stem
and/or progenitor cells (including hu CD34+++ cord blood and marrow cells,
and their subsets distinguished by CD38 and/or HLA-DR antigens and mu Sca-
1+ marrow cells) for their responsiveness to transduction by separate AAV
vectors containing SLF or Flt-3/Flk-2, alone and in combination, and also
to evaluate procedures necessary for optimal transduction of these cells;
and 3) determine the success of the different transduction procedures and
viral vectors on the different phenotypic target cell populations by
utilizing a variety of in vitro and in vivo assays to assess effects on
proliferation, self-renewal and differentiation of the cells. Assays in
vitro include those for high proliferative potential colony forming cells
(HPP-CFC), and immature and mature subsets of multipotential (CFU-GEMM),
erythroid (BFU-E), granulocyte-macrophage (CFU-GM), granulocyte (CFU-G),
and macrophage (CFU-M) progenitors and for long-term culture-initiating
cells (LTC-IC). Assays in vivo will utilize hu-cell inoculated
sublethally irradiated SCID mice and mu-cell inoculated lethally
irradiated syngeneic normal mice. Self-renewal in vitro and in vivo will
respectively be estimated by replating capacity of individual colonies and
transfer of marrow cells from l degree to 2 degrees mouse recipients. It
is anticipated that these studies will help to clarify a role for SLF and
Flt-3/Flk-2- ligand in early hematopoiesis and the use of rAAV vectors for
gene transduction and future gene therapy.
StatusFinished
Effective start/end date8/1/947/31/98

Funding

  • National Institutes of Health
  • National Institutes of Health: $259,133.00
  • National Institutes of Health: $239,583.00
  • National Institutes of Health

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Dependovirus
Stem Cell Factor
Stem Cells
Genes
Ligands
Granulocyte-Macrophage Progenitor Cells
Health Services Needs and Demand
Bone Marrow
Granulocytes
Macrophages
Myeloid Progenitor Cells
Erythroid Precursor Cells
SCID Mice
flt3 ligand protein
Hematopoiesis
HLA-DR Antigens
Fetal Blood
Genetic Therapy
Membranes
Blood Cells

ASJC

  • Medicine(all)