title: Retroviral gene transduction of adult peripheral blood or marrow-derived CD34+ cells for six hours without growth factors or on autologous stroma does not improve marking efficiency assessed in vivo.
abstract: Our previous work in patients undergoing autologous transplant for multiple myeloma (MM) or breast cancer (BC) has shown that retroviral transduction of adult CD34+ cells for 72 hours in the presence of interleukin-3 (IL-3), IL-6, and stem cell factor (SCF) resulted in .01% to 1% long-term marking of peripheral blood and marrow cells (Blood 85:3948, 1995). In this study we compare these previous studies to transduction with no added growth factors, previously shown to result in higher levels of marking in children (Lancet 342:1134, 1993) or transduction in the presence of an autologous stromal layer. Peripheral blood (PB) mononuclear cells were collected via apheresis after high-dose cyclophosphamide and granulocyte colony-stimulating factor. Bone marrow (BM) was also harvested in all patients. One third of both BM and PB collections were enriched for CD34+ cells and transduced with one of two marking vectors containing the neomycin-resistance gene to distinguish cells originating from BM and PB posttransplantation. Cells from 3 MM and 2 BC patients were transduced without growth factors for 6 hours and cells from 2 MM and 2 BC patients were transduced in the presence of autologous marrow stroma. Immediately posttransduction, the percentage of Neo-resistant PB and BM progenitors (colony-forming units) were: 0% to 19% in the 6-hour no growth factor group and 0% to 36% in the autologous stroma group. After conditioning therapy, both transduced and untransduced PB and BM fractions were infused into the patients. Semi-quantitative nested DNA polymerase chain reaction was performed on total, mononuclear, and granulocyte fractions of PB and BM at 1, 3, 6, 9, 12, and 18 months. Poor marking has been observed in both groups, with no consistently positive patients. These results compare unfavorably with our prior experience using growth factors during transduction. Further optimization of transduction conditions and vectors needs to be developed to improve transduction efficiency of adult human repopulating hematopoietic cells.
authors.affiliation: Hematology Branch, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA.
authors.forename: R V
authors.initials: RV
authors.lastname: Emmons
authors.forename: S
authors.initials: S
authors.lastname: Doren
authors.forename: J
authors.initials: J
authors.lastname: Zujewski
authors.forename: M
authors.initials: M
authors.lastname: Cottler-Fox
authors.forename: C S
authors.initials: CS
authors.lastname: Carter
authors.forename: K
authors.initials: K
authors.lastname: Hines
authors.forename: J A
authors.initials: JA
authors.lastname: O'Shaughnessy
authors.forename: S F
authors.initials: SF
authors.lastname: Leitman
authors.forename: J J
authors.initials: JJ
authors.lastname: Greenblatt
authors.forename: K
authors.initials: K
authors.lastname: Cowan
authors.forename: C E
authors.initials: CE
authors.lastname: Dunbar
mesh_terms: D000328:Adult; D001853:Bone Marrow / Q000187:drug effects / Q000473:pathology*; D004352:Drug Resistance, Microbial / Q000235:genetics; D005260:Female; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000187:drug effects / Q000473:pathology*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009355:Neomycin / Q000494:pharmacology; D012190:Retroviridae / Q000235:genetics; D017154:Stromal Cells / Q000187:drug effects / Q000473:pathology / Q000637:transplantation; D014182:Transplantation, Autologous
chemical_list: D009355:Neomycin