OBJECTIVE: Inducible and transient expression of transcription factors, growth factors, or mitogenic factors can be used to influence proliferation or differentiation of hematopoietic progenitor/stem cells (HSC). Furthermore, transient expression of proteins with site-specific endonuclease activity, potentially, can support targeted integration of exogenous transgenes into specific sites in the genome, a task that is currently a focus in development of gene therapy vectors. MATERIALS AND METHODS: We constructed a set of helper-dependent adenovirus (Ad) vectors with serotype 35 fiber knob domains (HD-Ad5/35), which can efficiently transduce human CD34(+) cells, particularly subsets with potential stem cell capacity. These vectors contain Tet-inducible expression systems that were shielded by insulators and transcription stop signals to minimize unspecific interference by transcriptional elements present in viral and stuffer DNA. We compared two vectors, containing a fusion between the Krüppel-associated box (KRAB) domain and the tetracycline repressor (HD-Ad5/35.Tet-1) or an autoregulated rtTA (HD-Ad5/35.Tet-2) for regulated transgene expression in Mo7e cells, a model for HSC, and primary human CD34(+) cells. RESULTS: HD-Ad5/35.Tet-1 conferred lower background expression than HD-Ad5/35.Tet-2, although levels of induced gene expression were higher for HD-Ad5/35.Tet-2. In CD34(+) cells, while HD-Ad5/35.Tet-1 allowed for activated gene expression in all transduced cells, induced gene expression from HD-Ad5/35.Tet-2 was restricted to a small subset of CD34(+) cells. Importantly, clonogeneic assays and repopulation studies in nonobese diabetic/severe combined immunodeficient mice showed that both HD-Ad5/35.Tet-1 and -2 vectors mediated induced gene expression in primitive hematopoietic cells. These studies also confirmed that transduction of CD34(+) cells with HD-Ad5/35 vectors is not associated with cytotoxicity, a problem observed with first-generation Ad5/35 vectors. CONCLUSIONS: Both HD-Ad5/35 vector expression systems confer tightly regulated, transient transgene expression in human HSC.
OBJECTIVE: Inducible and transient expression of transcription factors, growth factors, or mitogenic factors can be used to influence proliferation or differentiation of hematopoietic progenitor/stem cells (HSC). Furthermore, transient expression of proteins with site-specific endonuclease activity, potentially, can support targeted integration of exogenous transgenes into specific sites in the genome, a task that is currently a focus in development of gene therapy vectors. MATERIALS AND METHODS: We constructed a set of helper-dependent adenovirus (Ad) vectors with serotype 35 fiber knob domains (HD-Ad5/35), which can efficiently transduce humanCD34(+) cells, particularly subsets with potential stem cell capacity. These vectors contain Tet-inducible expression systems that were shielded by insulators and transcription stop signals to minimize unspecific interference by transcriptional elements present in viral and stuffer DNA. We compared two vectors, containing a fusion between the Krüppel-associated box (KRAB) domain and the tetracycline repressor (HD-Ad5/35.Tet-1) or an autoregulated rtTA (HD-Ad5/35.Tet-2) for regulated transgene expression in Mo7e cells, a model for HSC, and primary humanCD34(+) cells. RESULTS:HD-Ad5/35.Tet-1 conferred lower background expression than HD-Ad5/35.Tet-2, although levels of induced gene expression were higher for HD-Ad5/35.Tet-2. In CD34(+) cells, while HD-Ad5/35.Tet-1 allowed for activated gene expression in all transduced cells, induced gene expression from HD-Ad5/35.Tet-2 was restricted to a small subset of CD34(+) cells. Importantly, clonogeneic assays and repopulation studies in nonobese diabetic/severe combined immunodeficientmice showed that both HD-Ad5/35.Tet-1 and -2 vectors mediated induced gene expression in primitive hematopoietic cells. These studies also confirmed that transduction of CD34(+) cells with HD-Ad5/35 vectors is not associated with cytotoxicity, a problem observed with first-generation Ad5/35 vectors. CONCLUSIONS: Both HD-Ad5/35 vector expression systems confer tightly regulated, transient transgene expression in human HSC.
Authors: V Sandig; R Youil; A J Bett; L L Franlin; M Oshima; D Maione; F Wang; M L Metzker; R Savino; C T Caskey Journal: Proc Natl Acad Sci U S A Date: 2000-02-01 Impact factor: 11.205
Authors: P L Hallenbeck; Y N Chang; C Hay; D Golightly; D Stewart; J Lin; S Phipps; Y L Chiang Journal: Hum Gene Ther Date: 1999-07-01 Impact factor: 5.695
Authors: Lorenz Jager; Martin A Hausl; Christina Rauschhuber; Nicola M Wolf; Mark A Kay; Anja Ehrhardt Journal: Nat Protoc Date: 2009 Impact factor: 13.491
Authors: Maximilian Richter; Daniel Stone; Carol Miao; Olivier Humbert; Hans-Peter Kiem; Thalia Papayannopoulou; André Lieber Journal: Hematol Oncol Clin North Am Date: 2017-10 Impact factor: 3.722
Authors: Kamola Saydaminova; Xun Ye; Hongjie Wang; Maximilian Richter; Martin Ho; HongZhuan Chen; Ning Xu; Jin-Soo Kim; Eirini Papapetrou; Michael C Holmes; Philip D Gregory; Donna Palmer; Philip Ng; Anja Ehrhardt; André Lieber Journal: Mol Ther Methods Clin Dev Date: 2015-01-14 Impact factor: 6.698