Inclusion of IL-3 during retrovirally-mediated transduction on stromal support does not increase the extent of gene transfer into long-term engrafting human hematopoietic progenitors.
Interleukin 3 (IL-3) supports the survival of multilineage hematopoietic progenitors, and increases the extent of retrovirally mediated gene transfer into colony-forming cells. However, effects from the supraphysiological levels used in ex-vivo expansion and gene-therapy protocols on subsequent differentiation of human progenitors have not been well defined. In the current studies, the extents of retrovirally mediated transduction and lineage development from CD34+ cells cultured ex vivo in the presence or absence of IL-3 were compared. All transductions were performed in the presence of an irradiated stromal support layer, IL-6 and SCF, with and without inclusion of 10 ng/ml IL-3. Following transduction, colony-forming (CFU) assays were done, and the remaining cells were transplanted into cohorts of sibling beige/nude/xid (bnx) mice. Marrow from the mice was harvested 9-10 months post transplantation. The average extent of human CD45+ cell engraftment in the bone marrow and the human hematopoietic lineages recovered from the mice in the +IL-3 and -IL-3 groups did not vary significantly. No deleterious effects on the extent of engraftment, lineage generation, or survival of clonogenic progenitors was observed with inclusion of IL-3 in the transductions performed on stromal support. The percentage of G418-resistant human progenitors recovered from mice was equivalent. The extent of marking by the neo gene in the marrow of the mice was equal in both groups, and inverse PCR revealed that primitive cells transduced in the absence of IL-3 had generated progeny with slightly better clonal diversity than progenitors transduced in the presence of IL-3. These data show that, while transduction of colony-forming progenitors may not always be apparent, primitive human hematopoietic cells can be transduced to significant levels in the absence of IL-3.
Dao
M A
MA
Division of Research Immunology/Bone Marrow Transplantation, Children's Hospital Los Angeles, CA, USA.
Nolta
J A
JA
D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D003114:Colony-Forming Units Assay; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D005822:Genetic Vectors; D006085:Graft Survival; D006410:Hematopoiesis / Q000187:drug effects; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000276:immunology; D006801:Humans; D007377:Interleukin-3 / Q000494:pharmacology*; D051379:Mice; D008819:Mice, Nude; D012190:Retroviridae / Q000235:genetics; D014161:Transduction, Genetic; D014183:Transplantation, Heterologous
D018952:Antigens, CD34; D007377:Interleukin-3