Combinatorial incorporation of enhancer-blocking components of the chicken beta-globin 5'HS4 and human T-cell receptor alpha/delta BEAD-1 insulators in self-inactivating retroviral vectors reduces their genotoxic potential.
Insertional mutagenesis by retroviral vectors has emerged as a serious impediment to the widespread application of hematopoietic stem cell gene transfer for the treatment of hematologic diseases. Here we report the development of a 77-base pair element, FII/BEAD-A (FB), which contains the minimal enhancer-blocking components of the chicken beta-globin 5'HS4 insulator and a homologous region from the human T-cell receptor alpha/delta BEAD-1 insulator. With a new flow cytometry-based assay, we show that the FB element is as effective in enhancer-blocking activity as the prototypical 1.2-kilobase 5'HS4 insulator fragment. When incorporated into the residual U3 region of the 3' long terminal repeat (LTR) of a self-inactivating (SIN) gammaretroviral vector, the FB element was stably transferred to the 5' LTR during reverse transcription, flanking the integrated transgene expression cassette. Notably, using a recently established in vitro insertional mutagenesis assay involving primary murine hematopoietic cells, we found that SIN gammaretroviral vectors, as well as SIN lentiviral vectors, containing the FB element exhibited greatly reduced transforming potential-to background levels under the experimental conditions used-compared with their unshielded counterparts. These results suggest that the FB element-mediated enhancer-blocking modification is a promising approach to dramatically improve the safety of retroviral vectors for therapeutic gene transfer.
Ramezani
Ali
A
Department of Anatomy and Regenerative Biology, The George Washington University Medical Center, Washington, DC 20037, USA.
Hawley
Teresa S
TS
Hawley
Robert G
RG
D000818:Animals; D002645:Chickens; D004742:Enhancer Elements, Genetic; D005347:Fibroblasts / Q000378:metabolism; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics; D051379:Mice; D016296:Mutagenesis; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000235:genetics / Q000378:metabolism*; D012190:Retroviridae / Q000235:genetics; D055544:beta-Globins / Q000378:metabolism*
D016693:Receptors, Antigen, T-Cell, alpha-beta; D055544:beta-Globins
Mol Ther. 2000 Nov;2(5):458-69
11082319
Nat Med. 2006 Apr;12(4):401-9
16582916
Science. 2005 May 20;308(5725):1171-4
15905401
Mol Ther. 2002 May;5(5 Pt 1):589-98
11991750
Gene. 1991 Dec 15;108(2):193-9
1660837
J Thromb Haemost. 2004 Jan;2(1):111-8
14717974
Blood. 2006 Oct 15;108(8):2545-53
16825499
Biotechniques. 2001 May;30(5):1028-34
11355338
Blood. 2005 Dec 1;106(12):3932-9
16109773
Blood. 2005 Oct 1;106(7):2530-3
15933056
Leukemia. 2008 Aug;22(8):1519-28
18496560
Gene Ther. 1994 Mar;1(2):136-8
7584069
PLoS Biol. 2004 Dec;2(12):e423
15550989
Curr Gene Ther. 2001 May;1(1):1-17
12109133
Gene Ther. 2004 Nov;11(21):1568-78
15372067
Cell. 1988 Sep 9;54(6):831-40
2842066
Science. 2003 Oct 17;302(5644):415-9
14564000
Proc Natl Acad Sci U S A. 1997 May 13;94(10):5219-24
9144218
Blood. 2005 Jun 1;105(11):4235-46
15713797
Blood. 2003 Jun 15;101(12):4717-24
12586614
Proc Natl Acad Sci U S A. 1986 May;83(10):3194-8
3458176
Science. 2001 Jan 19;291(5503):447-50
11228144
Mol Ther. 2006 Jun;13(6):1031-49
16624621
J Virol. 1999 Apr;73(4):2886-92
10074136
J Virol. 1982 Feb;41(2):542-56
6281466
Cell. 1993 Aug 13;74(3):505-14
8348617
Mol Ther. 2005 Dec;12(6):1034-42
16226058
Hum Gene Ther. 2005 Apr;16(4):457-72
15871677
Trends Genet. 2001 Sep;17(9):520-7
11525835
Mol Ther. 2008 Mar;16(3):525-33
18195719
J Virol. 2002 Jul;76(14):7209-19
12072520
Mol Ther. 2006 Aug;14(2):245-54
16731046
Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14354-9
10588709
Proc Natl Acad Sci U S A. 1996 Sep 17;93(19):10297-302
8816794
Mol Ther. 2005 Oct;12(4):763-71
16084128
J Clin Invest. 2008 Apr;118(4):1502-10
18357342
Nat Biotechnol. 2006 Jun;24(6):687-96
16732270
Blood. 2004 Jun 1;103(11):4062-9
14976042
Mol Ther. 2007 Apr;15(4):801-9
17299406
Hum Gene Ther. 2006 Mar;17(3):253-63
16544975
Proc Natl Acad Sci U S A. 1997 Jan 21;94(2):575-80
9012826
Proc Natl Acad Sci U S A. 1987 Apr;84(8):2406-10
3031683
Science. 2006 Oct 6;314(5796):126-9
16946036
Proc Natl Acad Sci U S A. 2002 May 14;99(10):6883-8
12011446
Curr Opin Cell Biol. 2005 Jun;17(3):251-6
15901493
Gene Ther. 2006 Apr;13(7):641-5
16355114
Science. 2003 Jun 13;300(5626):1749-51
12805549
Mol Ther. 2008 Apr;16(4):718-25
18334985
Curr Protoc Mol Biol. 2002 Nov;Chapter 16:Unit 16.22
18265303
Blood. 2006 May 15;107(10):3865-7
16439674
Cell. 1999 Aug 6;98(3):387-96
10458613
J Virol. 1987 Mar;61(3):925-7
3806800
Methods Mol Biol. 2008;434:183-203
18470646
Gene Ther. 2006 Nov;13(21):1524-33
16763662