|
|
 Previous Article | Table of Contents | Next Article 
Blood, Vol. 93 No. 7 (April 1), 1999:
pp. 2208-2216
Development of Viral Vectors for Gene Therapy of  -Chain
Hemoglobinopathies: Optimization of a  -Globin Gene Expression
Cassette
Qiliang Li,
David W. Emery,
Magali Fernandez,
Hemei Han, and
George Stamatoyannopoulos
From the Department of Medicine, the Division of Medical Genetics,
University of Washington, Seattle.
Progress toward gene therapy of  -chain hemoglobinopathies has
been limited in part by poor expression of globin genes in virus
vectors. To derive an optimal expression cassette, we systematically analyzed the sequence requirements and relative strengths of the A - and -globin promoters, the activities of various
erythroid-specific enhancers, and the importance of flanking and
intronic sequences. Expression was analyzed by RNase protection after
stable plasmid transfection of the murine erythroleukemia cell line,
MEL585. Promoter truncation studies showed that the
A-globin promoter could be deleted to  159
without affecting expression, while deleting the -globin promoter to
127 actually increased expression compared with longer fragments.
Expression from the optimal -globin gene promoter was consistently
higher than that from the optimal A-globin promoter,
regardless of the enhancer used. Enhancers tested included a 2.5-kb
composite of the -globin locus control region (termed a µLCR), a
combination of the HS2 and HS3 core elements of the LCR, and the HS-40
core element of the  -globin locus. All three enhancers increased
expression from the -globin gene to roughly the same extent, while
the HS-40 element was notably less effective with the
A-globin gene. However, the HS-40 element was able to
efficiently enhance expression of a A-globin gene linked
to the -globin promoter. Inclusion of extended 3' sequences
from either the -globin or the A-globin genes had no
significant effect on expression. A 714-bp internal deletion of
A-globin intron 2 unexpectedly increased expression more
than twofold. With the combination of a 127 -globin promoter, an A-globin gene with the internal deletion of intron 2, and a single copy of the HS-40 enhancer, -globin expression averaged
166% of murine -globin mRNA per copy in six pools and 105% in nine clones. When placed in a retrovirus vector, this cassette was also
expressed at high levels in MEL585 cells (averaging 75% of murine
-globin mRNA per copy) without reducing virus titers. However,
recombined provirus or aberrant splicing was observed in 5 of 12 clones, indicating a significant degree of genetic instability. Taken
together, these data demonstrate the development of an optimal
expression cassette for -globin capable of efficient expression in a
retrovirus vector and form the basis for further refinement of vectors
containing this cassette.
 CiteULike  Connotea  Del.icio.us  Digg  Reddit  Technorati What's this?
This article has been cited by other articles:
|
|
|
|
 
Q. Li, D. W. Emery, H. Han, J. Sun, M. Yu, and G. Stamatoyannopoulos
Differences of globin transgene expression in stably transfected cell lines and transgenic mice
Blood,
April 15, 2005;
105(8):
3346 - 3352.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Suwanmanee, H. Sierakowska, G. Lacerra, S. Svasti, S. Kirby, C. E. Walsh, S. Fucharoen, and R. Kole
Restoration of Human beta -Globin Gene Expression in Murine and Human IVS2-654 Thalassemic Erythroid Cells by Free Uptake of Antisense Oligonucleotides
Mol. Pharmacol.,
September 1, 2002;
62(3):
545 - 553.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. W. Emery, E. Yannaki, J. Tubb, T. Nishino, Q. Li, and G. Stamatoyannopoulos
Development of virus vectors for gene therapy of beta chain hemoglobinopathies: flanking with a chromatin insulator reduces gamma -globin gene silencing in vivo
Blood,
August 28, 2002;
100(6):
2012 - 2019.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. E. SABATINO, N. E. SEIDEL, A. P. CLINE, S. M. ANDERSON, P. G. GALLAGHER, and D. M. BODINE
Development of a Stable Retrovirus Vector Capable of Long-Term Expression of {gamma}-Globin mRNA in Mouse Erythrocytes
Ann. N.Y. Acad. Sci.,
June 1, 2001;
938(1):
246 - 261.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. M. Kowolik, J. Hu, and J.-K. Yee
Locus Control Region of the Human CD2 Gene in a Lentivirus Vector Confers Position-Independent Transgene Expression
J. Virol.,
May 15, 2001;
75(10):
4641 - 4648.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Y. Hanazono, K. Terao, and K. Ozawa
Gene Transfer into Nonhuman Primate Hematopoietic Stem Cells: Implications for Gene Therapy
Stem Cells,
January 1, 2001;
19(1):
12 - 23.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
D. E. Sabatino, N. E. Seidel, G. J. Aviles-Mendoza, A. P. Cline, S. M. Anderson, P. G. Gallagher, and D. M. Bodine
Long-term expression of gamma -globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma -globin gene fused to the ankyrin-1 promoter
PNAS,
November 2, 2000;
(2000)
230453097.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
G. Lacerra, H. Sierakowska, C. Carestia, S. Fucharoen, J. Summerton, D. Weller, and R. Kole
Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients
PNAS,
August 15, 2000;
97(17):
9591 - 9596.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. E. Rubin, P. Pasceri, X. Wu, P. Leboulch, and J. Ellis
Locus control region activity by 5'HS3 requires a functional interaction with beta -globin gene regulatory elements: expression of novel beta /gamma -globin hybrid transgenes
Blood,
May 15, 2000;
95(10):
3242 - 3249.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. E. Sabatino, N. E. Seidel, G. J. Aviles-Mendoza, A. P. Cline, S. M. Anderson, P. G. Gallagher, and D. M. Bodine
Long-term expression of gamma -globin mRNA in mouse erythrocytes from retrovirus vectors containing the human gamma -globin gene fused to the ankyrin-1 promoter
PNAS,
November 21, 2000;
97(24):
13294 - 13299.
[Abstract]
[Full Text]
[PDF]
|
|
|
| |
