PMID- 9458243
OWN - NLM
STAT- MEDLINE
DCOM- 19980305
LR  - 20131121
IS  - 1043-0342 (Print)
IS  - 1043-0342 (Linking)
VI  - 9
IP  - 1
DP  - 1998 Jan 1
TI  - Retroviral transduction of human CD34+ umbilical cord blood progenitor cells with 
      a mutated dihydrofolate reductase cDNA.
PG  - 63-71
AB  - Umbilical cord blood cells (UCB) have become a major target population for 
      experimental and clinical studies using transfer of genes involved in inborn 
      enzymatic diseases. Cord blood contains hematopoietic progenitor cells at a high 
      frequency, and expanding these cells ex vivo generates sufficient numbers of 
      hematopoietic precursors for transplantation into adults, e.g., as supportive 
      treatment. As clinical reports about retroviral transduction into UCB cells have 
      not been as encouraging as the first preclinical data, we have established a 
      retroviral transduction system that allows expansion and selection of 
      hematopoietic progenitor cells from UCB. CD34-enriched UCB cells were transduced 
      with a retroviral vector encoding a mutated dihydrofolate reductase cDNA that 
      confers MTX resistance. We observed increased resistance to MTX in transduced 
      granulocyte macrophage-colony forming units (CFU-GM) after co-culture of CD34+ 
      UCB cells with the virus-producing cell line, or after incubation with 
      virus-containing supernatant. The supernatant-based transduction protocol 
      included a prestimulation with recombinant interleukin-1 (rhIL-1), rhkit-ligand, 
      and rhIL-3 to increase the percentage of cells in S phase to greater than 50%. 
      Using this protocol we measured a 72-fold expansion of CFU-GM and a 2.5-fold 
      selective advantage of transduced versus nontransduced progenitor cells after 
      exposure to low-dose methotrexate in liquid culture. Polymerase chain reaction 
      analysis revealed integration of proviral DNA into the majority of transduced 
      colonies before and after ex vivo expansion. The retroviral vector and 
      transduction protocol reported here provides an experimental system for selection 
      and expansion of retrovirally transduced progenitor/stem cells from UCB that may 
      help improve the efficiency of current clinical gene therapy strategies.
FAU - Flasshove, M
AU  - Flasshove M
AD  - James Ewing Laboratory of Developmental Hematopoiesis of the Cell Biology and 
      Genetics Program, Sloan-Kettering Institute for Cancer Research, New York, NY 
      10021, USA.
FAU - Banerjee, D
AU  - Banerjee D
FAU - Leonard, J P
AU  - Leonard JP
FAU - Mineishi, S
AU  - Mineishi S
FAU - Li, M X
AU  - Li MX
FAU - Bertino, J R
AU  - Bertino JR
FAU - Moore, M A
AU  - Moore MA
LA  - eng
GR  - CA 59350/CA/NCI NIH HHS/United States
GR  - NCI-P30-CA08748/CA/NCI NIH HHS/United States
GR  - R0-1 DK-42693/DK/NIDDK NIH HHS/United States
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Hum Gene Ther
JT  - Human gene therapy
JID - 9008950
RN  - 0 (Antigens, CD34)
RN  - 0 (DNA, Complementary)
RN  - 0 (Folic Acid Antagonists)
RN  - EC 1.5.1.3 (Tetrahydrofolate Dehydrogenase)
RN  - YL5FZ2Y5U1 (Methotrexate)
SB  - IM
MH  - Antigens, CD34/*immunology
MH  - DNA, Complementary/*genetics
MH  - Drug Resistance
MH  - Fetal Blood/*cytology
MH  - Folic Acid Antagonists/pharmacology
MH  - *Gene Transfer Techniques
MH  - Genetic Vectors/genetics
MH  - Hematopoietic Stem Cells/*drug effects/physiology
MH  - Humans
MH  - Methotrexate/pharmacology
MH  - Mutation
MH  - Proviruses/genetics
MH  - Retroviridae/genetics
MH  - S Phase/drug effects
MH  - Tetrahydrofolate Dehydrogenase/*genetics
EDAT- 1998/02/11 00:00
MHDA- 1998/02/11 00:01
CRDT- 1998/02/11 00:00
PHST- 1998/02/11 00:00 [pubmed]
PHST- 1998/02/11 00:01 [medline]
PHST- 1998/02/11 00:00 [entrez]
AID - 10.1089/hum.1998.9.1-63 [doi]
PST - ppublish
SO  - Hum Gene Ther. 1998 Jan 1;9(1):63-71. doi: 10.1089/hum.1998.9.1-63.