title: Comparison of transduction efficiency among various lentiviruses containing GFP reporter in bone marrow hematopoietic stem cell transplantation.
abstract: HIV-derived lentiviral vectors have been used widely to transduce non-dividing cells, such as hematopoietic stem cells (HSCs), in the setting of gene therapy. In this study, we screened lentiviral vectors for their ability to drive expression of the murine MHC class II chaperone, invariant chain (Ii) and a GFP reporter. The vectors included T2A vector with T2A-separated Ii and GFP under the same MSCV promoter, dual-promoter vectors with separate promoters for Ii and GFP (called MSCV or EF1a according to the promoter driving Ii expression), and a vector with EF1a driving a fusion of Ii/GFP (called Fusion vector). T2A and MSCV induced the highest levels of Ii and GFP expression, respectively, after direct transfection of 293T cells. All vectors except the Fusion vector drove expression of functional Ii, based on the enhancement of MHC class II level, which is a known consequence of Ii expression. Comparing the vectors after they were packaged into lentiviruses and used to transduce 293T, we found that MSCV and EF1a vectors mediated higher Ii and GFP expression. In ckit(+) bone marrow (BM) cells, MSCV still induced the highest Ii and GFP expression, whereas EF1a induced only robust Ii expression. Regardless of the vector, both Ii and GFP levels were significantly reduced in BM cells compared to 293T cells. When in vivo expression was assessed in cells derived from MSCV-transduced BM-HSCs, up to 80% of myeloid cells were GFP(+), but no Ii expression was observed. In contrast, transplantation of EF1a-transduced BM-HSCs led to much higher in vivo Ii expression. Thus, among those compared, dual-promoter vector-based lentivirus with the EF1a promoter driving the gene of interest is optimal for murine BM-HSC transduction.
authors: Department of Pediatrics, Program in Immunology, Program in Human Gene Therapy, Stanford University School of Medicine, Stanford, CA, USA.
authors: Nan
authors: N
authors: Wang
authors: Narendiran
authors: N
authors: Rajasekaran
authors: Tieying
authors: T
authors: Hou
authors: Leszek
authors: L
authors: Lisowski
authors: Elizabeth D
authors: ED
authors: Mellins
mesh_terms: D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000235:genetics / Q000378:metabolism; D001853:Bone Marrow / Q000378:metabolism*; D005434:Flow Cytometry; D005786:Gene Expression Regulation; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism; D057809:HEK293 Cells; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II / Q000235:genetics / Q000378:metabolism; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D016688:Mice, Inbred NOD; D020648:Peptide Elongation Factor 1 / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012190:Retroviridae / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014161:Transduction, Genetic
chemical_list: D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D020648:Peptide Elongation Factor 1; C045084:invariant chain; D049452:Green Fluorescent Proteins
references: Mol Ther. 2000 Nov;2(5):458-69
references: 11082319
references: Immunol Rev. 2011 Jan;239(1):45-61
references: 21198664
references: Mol Ther. 2003 Jun;7(6):827-38
references: 12788657
references: Arthritis Rheum. 2013 Mar;65(3):681-92
references: 23233229
references: Science. 2000 Dec 8;290(5498):1876-9
references: 11187040
references: J Stem Cells Regen Med. 2011 Apr 01;7(1):41-53
references: 21743782
references: Blood. 2002 Jan 15;99(2):399-408
references: 11781219
references: J Biol Chem. 2004 Jan 2;279(1):34-41
references: 14559924
references: J Clin Invest. 2003 May;111(9):1357-63
references: 12727927
references: J Clin Invest. 2003 Nov;112(10):1478-80
references: 14617747
references: Rev Med Virol. 2001 Jul-Aug;11(4):205-17
references: 11479927
references: Blood. 2000 Nov 15;96(10):3385-91
references: 11071632
references: Exp Hematol. 2008 Aug;36(8):947-56
references: 18640494
references: J Clin Invest. 2005 Aug;115(8):2268-76
references: 16075062
references: EMBO Rep. 2013 Jan;14(1):73-9
references: 23154467
references: PLoS One. 2012;7(11):e48943
references: 23145033
references: Hum Gene Ther. 2005 Nov;16(11):1241-6
references: 16259557
references: Nat Biotechnol. 2011 Oct 02;29(10):928-33
references: 21964413
references: J Biotechnol. 2011 Dec 10;156(3):218-26
references: 21933686
references: Blood. 2003 Aug 1;102(3):881-6
references: 12676789
references: Gene Ther. 2009 Jan;16(1):111-8
references: 18784749
references: Blood. 2000 Aug 1;96(3):894-901
references: 10910902
references: Blood. 2000 Apr 15;95(8):2499-504
references: 10753827
references: Gene Ther. 2009 Jul;16(7):874-84
references: 19440229
references: Gene Ther. 2000 Sep;7(18):1562-9
references: 11021594
references: Stem Cells. 2001;19(3):236-46
references: 11359949
references: Int Immunol. 2010 Aug;22(8):705-16
references: 20547545
references: Mol Ther. 2009 Aug;17(8):1316-32
references: 19491821
references: J Exp Med. 1999 Jun 7;189(11):1723-34
references: 10359576
references: Curr Gene Ther. 2002 Sep;2(3):355-78
references: 12189721
references: Nat Protoc. 2009;4(4):495-505
references: 19300443
references: Cold Spring Harb Protoc. 2012 Feb 01;2012(2):199-204
references: 22301656
references: Gene Ther. 2008 Mar;15(5):384-90
references: 18283290