PMID- 23555045
OWN - NLM
STAT- MEDLINE
DCOM- 20130930
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 8
IP  - 3
DP  - 2013
TI  - Knockdown of HPRT for selection of genetically modified human hematopoietic 
      progenitor cells.
PG  - e59594
LID - 10.1371/journal.pone.0059594 [doi]
LID - e59594
AB  - The inability to obtain sufficient numbers of transduced cells remains a 
      limitation in gene therapy. One strategy to address this limitation is in vivo 
      pharmacologic selection of transduced cells. We have previously shown that 
      knockdown of HPRT using lentiviral delivered shRNA facilitates efficient 
      selection of transduced murine hematopoietic progenitor cells (HPC) using 
      6-thioguanine (6TG). Herein, we now extend these studies to human HPC. We tested 
      multiple shRNA constructs in human derived cell lines and identified the optimal 
      shRNA sequence for knockdown of HPRT and 6TG resistance. We then tested this 
      vector in human umbilical cord blood derived HPC in vitro and in NOD/SCID 
      recipients. Knockdown of HPRT effectively provided resistance to 6TG in vitro. 
      6TG treatment of mice resulted in increased percentages of transduced human 
      CD45(+) cells in the peripheral blood and in the spleen in particular, in both 
      myeloid and lymphoid compartments. 6TG treatment of secondary recipients resulted 
      in higher percentages of transduced human cells in the bone marrow, confirming 
      selection from the progeny of long-term repopulating HPCs. However, the extent of 
      selection of cells in the bone marrow at the doses of 6TG tested and the toxicity 
      of higher doses, suggest that this strategy may be limited to selection of more 
      committed progenitor cells. Together, these data suggest that human HPC can be 
      programmed to be resistant to purine analogs, but that HPRT knockdown/6TG-based 
      selection may not be robust enough for in vivo selection.
FAU - Choudhary, Rashmi
AU  - Choudhary R
AD  - Department of Pediatrics, University of Colorado School of Medicine, Aurora, 
      Colorado, United States of America.
FAU - Baturin, Dmitry
AU  - Baturin D
FAU - Fosmire, Susan
AU  - Fosmire S
FAU - Freed, Brian
AU  - Freed B
FAU - Porter, Christopher C
AU  - Porter CC
LA  - eng
GR  - P30 CA046934/CA/NCI NIH HHS/United States
GR  - R21 HL094921/HL/NHLBI NIH HHS/United States
GR  - T32 CA082086/CA/NCI NIH HHS/United States
GR  - HL094921/HL/NHLBI NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20130315
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (Antigens, CD34)
RN  - EC 2.4.2.8 (Hypoxanthine Phosphoribosyltransferase)
RN  - FTK8U1GZNX (Thioguanine)
SB  - IM
MH  - Animals
MH  - Antigens, CD34/metabolism
MH  - Cell Line
MH  - Drug Resistance/drug effects/genetics
MH  - Female
MH  - *Gene Knockdown Techniques
MH  - Hematopoietic Stem Cells/cytology/drug effects/enzymology/*metabolism
MH  - Humans
MH  - Hypoxanthine Phosphoribosyltransferase/*deficiency/*genetics
MH  - Lymphocytes/cytology
MH  - Mice
MH  - Myeloid Cells/cytology
MH  - Thioguanine/pharmacology
MH  - Transduction, Genetic/*methods
MH  - Umbilical Cord/cytology
PMC - PMC3598703
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2013/04/05 06:00
MHDA- 2013/10/01 06:00
PMCR- 2013/03/15
CRDT- 2013/04/05 06:00
PHST- 2012/11/16 00:00 [received]
PHST- 2013/02/15 00:00 [accepted]
PHST- 2013/04/05 06:00 [entrez]
PHST- 2013/04/05 06:00 [pubmed]
PHST- 2013/10/01 06:00 [medline]
PHST- 2013/03/15 00:00 [pmc-release]
AID - PONE-D-12-36288 [pii]
AID - 10.1371/journal.pone.0059594 [doi]
PST - ppublish
SO  - PLoS One. 2013;8(3):e59594. doi: 10.1371/journal.pone.0059594. Epub 2013 Mar 15.