C - Chemistry – Metallurgy – 12 – N
Patent
C - Chemistry, Metallurgy
12
N
C12N 15/18 (2006.01) C07K 14/71 (2006.01) C12N 5/06 (2006.01) C12N 5/08 (2006.01) C12N 15/85 (2006.01) C12N 15/87 (2006.01)
Patent
CA 2241576
The present invention involves 1) efficient non-viral gene transfer into human erythroid progenitors cells and 2) use of a constitutively active human EpoR gene to prolong the lifespan of progenitor cells. CD34+ cells enriched from umbilical cord blood LDMNC are cultured ex vivo at a low density in the absence of supportive stroma for 7 to days in a serum-free defined culture medium with the addition of IL-3, SCF and EPO. At this time, cells expand < 70 fold and hematopoietic progenitors are present at a frequency of 25 BFU-E, 28 CFU-E and 10 CFU-GM per 1 x 10 5 cells as detected by methylcellulose colony forming assay. Approximately 2% are CD34+ while 40% are GlyA+. Hematopoietic cells cultured in this manner are rendered highly susceptible to gene transfer by electroporation and a gene transfer efficiency of >25% is obtained. The lifespan of erythroid progenitors that persist at the point of electroporation can be prolonged through the introduction of a constitutively active form of the human EpoR gene.This invention is useful for (1) genetic modification of hematopoietic stem cells, especially erythroid progenitors and (2) the propagation of erythroid cells in vitro for the production of hemoglobin.
Bell David
Matthews Kathryn
Mueller Susan
Hemosol Inc.
Ridout & Maybee Llp
LandOfFree
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