title: Development of a modified selective amplifier gene for hematopoietic stem cell gene therapy.
abstract: We have proposed a novel concept, ie selective expansion of transduced cells, to overcome the low efficiency of gene transfer into hematopoietic stem cells. Previously, a fusion gene encoding a chimeric receptor (DeltaGCRER) between the mouse granulocyte colony-stimulating factor receptor (G-CSFR) and the hormone-binding domain of rat estrogen receptor was constructed as a 'selective amplifier gene'. Although the chimeric gene conferred estrogen-inducible proliferation on the transduced Ba/F3 cells, it also mediated differentiation of the retrovirally transduced 32D cells upon estrogen treatment. Since only a growth signal is required for our purpose, we further modified the DeltaGCRER gene to attenuate its differentiation signal. Based on the observation that tyrosine-703 in wild-type G-CSFR plays a pivotal role in transmitting the differentiation signal, phenylalanine was substituted for this residue in DeltaGCRER. When the resultant selective amplifier gene (DeltaY703F-GCRER gene) was expressed in 32D cells, sustained growth was supported by estrogen, while differentiation was suppressed. These cells ceased to grow upon estrogen withdrawal and differentiated with G-CSF treatment. The present findings suggested that DeltaY703F-GCRER may have desirable properties as a selective amplifier for hematopoietic stem cell expansion and gene therapy.
authors.affiliation: Division of Genetic Therapeutics, Center for Molecular Medicine, Saitama, Japan.
authors.forename: K M
authors.initials: KM
authors.lastname: Matsuda
authors.forename: A
authors.initials: A
authors.lastname: Kume
authors.forename: Y
authors.initials: Y
authors.lastname: Ueda
authors.forename: M
authors.initials: M
authors.lastname: Urabe
authors.forename: M
authors.initials: M
authors.lastname: Hasegawa
authors.forename: K
authors.initials: K
authors.lastname: Ozawa
mesh_terms: D000818:Animals; D002450:Cell Communication; D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D048429:Cell Size; D005784:Gene Amplification; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D051379:Mice; D051381:Rats
chemical_list: D016179:Granulocyte Colony-Stimulating Factor