PMID- 9573006
OWN - NLM
STAT- MEDLINE
DCOM- 19980612
LR  - 20210216
IS  - 0006-4971 (Print)
IS  - 0006-4971 (Linking)
VI  - 91
IP  - 10
DP  - 1998 May 15
TI  - Delayed targeting of cytokine-nonresponsive human bone marrow CD34(+) cells with 
      retrovirus-mediated gene transfer enhances transduction efficiency and long-term 
      expression of transduced genes.
PG  - 3693-701
AB  - Primitive hematopoietic progenitor cells (HPCs) are potential targets for 
      treatment of numerous hematopoietic diseases using retroviral-mediated gene 
      transfer (RMGT). To achieve high efficiency of gene transfer into primitive HPCs, 
      a delicate balance between cellular activation and proliferation and maintenance 
      of hematopoietic potential must be established. We have demonstrated that a 
      subpopulation of human bone marrow (BM) CD34(+) cells, highly enriched for 
      primitive HPCs, persists in culture in a mitotically quiescent state due to their 
      cytokine-nonresponsive (CNR) nature, a characteristic that may prevent efficient 
      RMGT of these cells. To evaluate and possibly circumvent this, we designed a 
      two-step transduction protocol using neoR-containing vectors coupled with flow 
      cytometric cell sorting to isolate and examine transduction efficiency in 
      different fractions of cultured CD34(+) cells. BM CD34(+) cells stained on day 0 
      (d0) with the membrane dye PKH2 were prestimulated for 24 hours with stem cell 
      factor (SCF), interleukin-3 (IL-3), and IL-6, and then transduced on fibronectin 
      with the retroviral vector LNL6 on d1. On d5, half of the cultured cells were 
      transduced with the retroviral vector G1Na and sorted on d6 into 
      cytokine-responsive (d6 CR) cells (detected via their loss of PKH2 fluorescence 
      relative to d0 sample) and d6 CNR cells that had not divided since d0. The other 
      half of the cultured cells were first sorted on d5 into d5 CR and d5 CNR cells 
      and then infected separately with G1Na. Both sets of d5 and d6 CR and CNR cells 
      were cultured in secondary long-term cultures (LTCs) and assayed weekly for 
      transduced progenitor cells. Significantly higher numbers of G418-resistant 
      colonies were produced in cultures initiated with d5 and d6 CNR cells compared 
      with respective CR fractions (P < .05). At week 2, transduction efficiency was 
      comparable between d5 and d6 transduced CR and CNR cells (P > .05). However, at 
      weeks 3 and 4, d5 and d6 CNR fractions generated significantly higher numbers of 
      neoR progenitor cells relative to the respective CR fractions (P < .05), while no 
      difference in transduction efficiency between d5 and d6 CNR cells could be 
      demonstrated. Polymerase chain reaction (PCR) analysis of the origin of 
      transduced neoR gene in clonogenic cells demonstrated that mature progenitors (CR 
      fractions) contained predominantly LNL6 sequences, while more primitive 
      progenitor cells (CNR fractions) were transduced with G1Na. These results 
      demonstrate that prolonged stimulation of primitive HPCs is essential for 
      achieving efficient RMGT into cells capable of sustaining long-term in vitro 
      hematopoiesis. These findings may have significant implications for the 
      development of clinical gene therapy protocols.
FAU - Veena, P
AU  - Veena P
AD  - Division of Hematology/Oncology, Department of Medicine, Indiana University 
      School of Medicine, Indianapolis, IN, USA.
FAU - Traycoff, C M
AU  - Traycoff CM
FAU - Williams, D A
AU  - Williams DA
FAU - McMahel, J
AU  - McMahel J
FAU - Rice, S
AU  - Rice S
FAU - Cornetta, K
AU  - Cornetta K
FAU - Srour, E F
AU  - Srour EF
LA  - eng
GR  - P01 CA59348/CA/NCI NIH HHS/United States
GR  - P50 DK49218/DK/NIDDK NIH HHS/United States
GR  - R01 HL55716/HL/NHLBI NIH HHS/United States
PT  - Comparative Study
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  - Blood
JT  - Blood
JID - 7603509
RN  - 0 (Antigens, CD34)
RN  - 0 (Fibronectins)
RN  - 0 (Interleukin-3)
RN  - 0 (Interleukin-6)
RN  - 0 (Recombinant Fusion Proteins)
RN  - 0 (Stem Cell Factor)
RN  - EC 2.7.1.95 (Kanamycin Kinase)
SB  - IM
MH  - Adult
MH  - Animals
MH  - Antigens, CD34/analysis
MH  - Cell Division
MH  - Cell Separation
MH  - Colony-Forming Units Assay
MH  - Drug Resistance, Microbial
MH  - Fibronectins
MH  - Flow Cytometry
MH  - Gene Expression
MH  - Gene Targeting/*methods
MH  - Genetic Vectors/*genetics
MH  - Hematopoietic Stem Cells/drug effects/metabolism/*virology
MH  - Humans
MH  - Immunomagnetic Separation
MH  - Interleukin-3/pharmacology
MH  - Interleukin-6/pharmacology
MH  - Kanamycin Kinase/*biosynthesis/genetics
MH  - Polymerase Chain Reaction
MH  - Recombinant Fusion Proteins/*biosynthesis/genetics
MH  - Retroviridae/*genetics
MH  - Stem Cell Factor/pharmacology
MH  - *Transfection
EDAT- 1998/06/20 00:00
MHDA- 1998/06/20 00:01
CRDT- 1998/06/20 00:00
PHST- 1998/06/20 00:00 [pubmed]
PHST- 1998/06/20 00:01 [medline]
PHST- 1998/06/20 00:00 [entrez]
AID - S0006-4971(20)54690-9 [pii]
PST - ppublish
SO  - Blood. 1998 May 15;91(10):3693-701.