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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
By
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.
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ABSTRACT |
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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
THE USE OF retrovirus vectors
for the gene therapy of chain hemoglobinopathies has been limited,
in part, by the restricted size of these vectors1 and the
effect of globin gene and enhancer sequences on vector titer and
stability. In the case of retrovirus vectors for human -globin and
-globin, these problems have been addressed to some degree by
introducing several genetic alterations to the coding sequence and
including enhancer elements from the -globin locus control region
(µLCR)2,3 or the  globin locus enhancer.4
Initial studies with retrovirus vectors for -globin have shown that
the -globin gene introns are required for maximal expression, but
the presence of intron sequences greatly diminished vector
titers.5 Because of the efficient antisickling properties
of fetal hemoglobin (HbF) and the therapeutic impact of even moderate
HbF levels in homozygous thalassemia, we sought to develop an
optimal expression cassette for -globin for inclusion in retrovirus
vectors. Such a cassette should have the following properties: (1) a
combination of promoter and enhancer, which can direct therapeutic
levels of globin gene expression; (2) all unnecessary sequences removed
to provide an acceptable size; and (3) lack of sequences, which
significantly reduce vector titers or contribute to vector instability.
To meet the goals, we studied the sequence requirements and strengths of the A- and -globin promoters, compared activities
of various erythroid-specific enhancer elements, and assessed the
influence of 3' flanking sequences and the internal portion of
the second intron of the A-globin gene. Expression was
measured by plasmid transfection of the murine erythroleukemia cell
line MEL585 and quantitative RNase protection analysis of stable pools.
An optimal expression cassette was also introduced into a murine
leukemia virus (MLV)-based vector, and vector titer, stability, and
expression was assessed.
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MATERIALS AND METHODS |
Plasmid constructs.
All constructs were made in the plasmid vector pBluescript (Stratagene,
La Jolla, CA) using standard molecular cloning procedure.6 The contents of the various constructs are shown in
Table 1 and are as follows: the 2.3-kb
StuI/HindIII fragment (coordinates 39050-41382, GenBank
humhbb): A-globin gene with 382 promoter. The 2.2-kb
ApaI/HindIII fragment (39230-41382): the
A-globin gene with 201 promoter. The 2.1-kb
AvaII/HindIII fragment (39271-41382): the human
A-globin gene with 159 promoter. The 2.1-kb
NcoI/HindIII fragment (39290-41382): the
A-globin gene with 141 promoter. The 753-bp
HindIII fragment (41382-42135): the A-globin
gene 3' enhancer. The 5.0-kb BglII fragment
(60629-65610): the -globin gene with 1560 promoter and
3' enhancer. The 4.2-kb HpaI/BglII fragment
(61372-65610): the -globin gene with 614 promoter and
3' enhancer. The 3.7-kb SnaBI/BglII fragment
(61921-65610): the -globin gene with 267 promoter and
3' enhancer. The 3.6-kb RsaI/BglII fragment
(62060-65610): the -globin gene with 127 promoter and
3' enhancer. The 3.5-kb AvrII/BglII fragment
(62101-65610): the -globin gene with 86 promoter and 3'
enhancer. The 1.5-kb BspHI/BglII fragment (64081-65610)
was deleted from the above -globin gene constructs to remove the
-globin 3' enhancer. The pr.-A-globin hybrid genes were made by
linking either the 127 or 267 -globin promoters
(RsaI 62060 or SnaBI 61921 to NcoI 62238) to
the A-globin gene coding sequence (NcoI to
HindIII, 39483-41382) using the NcoI sites as a
junction point. In the constructs with the internal deletion of
A-globin intron 2, the 714-bp
XhoI(blunt)/HpaI fragment (39960-40674) was removed.
The 2.5-kb µLCR was described previously.7 The -globin
HS-40 core is contained in the 356-bp TaqI/XmnI
fragment (a gift from Douglas R. Higgs, University of
Oxford, Oxford, UK). The HS3 core is contained in a 784-bp PstI
fragment (4348-5132). The 298-bp HS2 core is contained in a
HindIII/AluI (8486-8784) fragment. The enhancers were
oriented in the same direction as transcription, except where noted.
Plasmid transfection.
The murine erythroleukemia cell line MEL585 was grown in RPMI-1640
medium (GIBCO-BRL, Grand Island, NY) supplemented with 10%
heat-inactivated defined fetal bovine serum (FBS; Gibco BRL). A total
of 107 cells in log-phase growth were resuspended in 0.5 mL
HEPES-buffered sucrose (272 mmol/L sucrose, 8 mmol/L HEPES, pH 7.4),
chilled in an electroporation cuvette with a 0.4 cm electrode gap,
mixed with DNA, and transfected with a Gene Pulse electroporator
(Bio-Rad, Hercules, CA) at 500 V, 1 µF. Cotransfection was achieved
using 10 µg of linearized experimental plasmid DNA plus 1 µg of
PGK-Neo8 as a selectable marker. Cells were then
transferred to 70-cm2 tissue culture flasks with 20 mL of
growth medium and after 48 hours, G418 was added at a concentration of
0.7 mg/mL active component. G418-resistant pools were used for analysis
after 10 to 14 days. The MEL cell pools contained a large number of
clones so that the variation in expression due to position effects was
minimized.9
Retrovirus vector.
The retrovirus vector construct was generated using the MLV vector
LNSX,1 which expresses Neo using the viral 5' long
terminal repeat promoter. The expression cassette consists of the
356-bp HS-40 enhancer, 127 -globin promoter
(RsaI-NcoI, 62060-62238), and A-globin
gene (NcoI/HindIII, 39483-41382) with the 714-bp
internal deletion of intron 2 described above. This cassette was
inserted in the unique BamHI (blunt) and StuI sites of
LNSX in the opposite orientation with respect to virus transcription.
Retrovirus vector producer lines were generated essentially as
described.1 In short, vector plasmid was used to transfect
the ecotropic packaging line PE5011 by CaPO4
precipitation and after 48 hours, virus supernatant was used to
transduce the amphotropic packaging line PA317.10 After an
additional 24 hours, the transduced PA317 cells were plated at low
dilution with 0.5 mg/mL active G418, and individual drug-resistant
colonies were isolated. Virus titer was assayed by serial dilution and
transfer of G418 resistance to naive NIH3T3 cells as previously
described.11 Cells were maintained at 37°C, 7.5%
CO2 in Dulbecco's Modified Eagle's medium (DMEM;
GIBCO-BRL) supplemented with 10% FBS, 2 mmol/L L-glutamine
(GIBCO-BRL), 1 mmol/L sodium pyruvate (GIBCO-BRL), and 0.1 mmol/L
nonessential amino acids (GIBCO-BRL). Vector-containing supernatant was
collected from semiconfluent plates after a 48-hour culture at
33°C, passed through a 0.44-µm filter, and stored at
70°C. MEL585 cells were transduced by 24-hour culture in
this vector-containing supernatant plus 8 µg/mL polybrene
(hexadimethrine bromide; Sigma Chemical Co, St Louis, MO) at 1 to 2 × 105 cells/mL. The cells were then washed and plated
at limiting dilution in 96-well flat-bottom dishes with 0.6 mg/mL
active G418, and individual clones were expanded after 10 to 14 days
for analysis.
Quantification of globin mRNA.
Transfected or transduced MEL585 cells were induced to differentiate by
culture in 3 mmol/L N,N1-hexamethylene bisacetamide (HMBA;
Aldrich, Milwaukee, WI) and 10 µmol/L hemin (Sigma) starting at 2 to
3 × 105 cells/mL as previously described. Cells were
collected after 3 days, and total RNA was isolated using guanidine
thiocyanate-acid-phenol as described.12 Globin gene
transcripts were quantified by RNase protection as previously
described13 using the following probes: pT7m
linearized with BsaI to give a 206-bp protected fragment within exon 2 of the human -globin gene; pT7A(170)
linearized with BstEII to give a 170-bp protected fragment within exon 2 of the human A-globin gene; and pT7M
(128) linearized with HindIII to give a 128-bp protected
fragment within exon 1 of the murine -globin gene. A total of 500 ng
RNA was hybridized overnight at 48°C with 106 cpm of
each radiolabeled probe. A pilot experiment confirmed that probe was in
excess under these conditions. After digestion with RNase A and T1, the
protected fragments were separated on 6% polyacrylamide-8 mol/L urea
gels, and autoradiography was performed without intensifying screens.
Signal intensities were quantified by Phosphorimager (Molecular
Dynamics, Sunnyvale, CA).
Southern analysis.
Genomic DNA was isolated by standard methods.6 For copy
number determinations, DNA from transfected cells was digested with
EcoRI, which cuts twice in each plasmid construct. Fluorometry was then used to accurately determine the DNA concentration, and 10 µg of the digested DNA was separated on 1.0% agarose gels and blotted onto nylon filters. The blots were probed with radiolabeled BamHI/EcoRI fragments for exon 2 of either
A-globin or -globin, and a murine -globin probe
from pUCmu (a gift from Margaret H. Baron, Mt Sinai
School of Medicine, New York, NY) as a loading control. Normal human
genomic DNA digested with EcoRI and run in parallel served as a
copy number standard. Signals were quantified on a Phosphorimager. For
the studies with the retrovirus vectors, 10 µg DNA was digested with
KpnI, which cuts once in each of the virus LTRs, separated on
0.8% agarose gels, and blotted onto nylon filters. The blots were
probed with a radiolabeled 923-bp PstI fragment for Neo.
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RESULTS |
Truncations of the A-globin gene promoter.
Our previous studies in transgenic mice carrying µLCRA
gene recombinants showed that truncation of the A-globin
promoter to position 201 relative to the mRNA cap site allows
high-level expression in adult blood (15% to 20% of murine -globin
mRNA per copy), while truncation to 141 (which deletes the CACCC
box at 145 and the GATA-1/Oct-1 motifs around 175) completely abolishes gene expression.14 We have also
shown that the sequence between 382 and 730 of the
A gene promoter contains a position-dependent silencer
of A gene expression.14 These results
suggest that sequences upstream of 382 A-globin
promoter should be omitted from virus vectors to prevent silencing.
To test if the A-globin gene promoter could be further
truncated without impairing the promoter strength,
µLCRA-globin expression plasmids containing the
201 A or the 141 A promoter
truncations, a 382 A promoter truncation, and a
159 A promoter truncation (which leaves the CACCC
box intact, but removes the GATA-1 and Oct-1 motifs) were used
(Fig 1A). These constructs were
cotransfected into MEL585 cells with a neomycin resistance plasmid, and
the indicated numbers of pools were selected under G418. After
induction with HMBA and hemin, total RNA and genomic DNA were isolated,
and the levels of A-globin mRNA and transgene copy
numbers were measured by quantitative RNase protection and Southern
analysis. As summarized to the right of Fig 1A, the construct with the
159 truncation expressed A-globin at an average
35% of endogenous murine -globin on a per copy basis, with a range
of 22.4% to 54.9%. This expression level is similar to that of
constructs containing the 382 or the 201
A-globin promoter truncations (averaging about 29%
each). These results suggest that the CACCC box and more proximal
regulatory elements are required for µLCR-enhanced expression of the
A-globin gene, and that all other distal elements of the
A promoter can be omitted from the constructs of
retrovirus vectors.
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| Fig 1.
Diagrams and expression of constructs containing
the µLCR and A-globin gene with promoter truncations.
(A) Is for constructs containing the normal A-globin
promoter sequence, while (B) is for constructs containing
the indicated G  A point mutation at position 117 of the
A-globin promoter associated with hereditary persistence
of fetal hemoglobin. In the diagrams to the left, the µLCR is
indicated by the thick hatched bar, while the Ag-globin
gene is indicated by the thin filled bar. Exons are indicated by the
filled boxes, the site of transcription initiation (cap site) by the
arrow, while the positions of the individual promoter truncations are
relative to the cap site. To the right of each panel is shown the
number of transfected MEL585 pools included in the analysis, along with
the mean, standard deviation (for data sets containing more than three
pools), and range of expression for each construct. Expression
was determined by quantitative RNase protection and is expressed as a
percentage of the level of endogenous murine -globin mRNA on a per
copy basis.
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Introduction of HPFH 117 point mutation.
Several naturally occurring point mutations in the
A-globin promoter cause hereditary persistence of fetal
hemoglobin, possibly through different mechanisms.15
Previously reported transient and stable transfection assays suggested
that the 117 G A mutation in the
A-globin promoter increases A-globin
expression even in the absence of enhancers.16,17 To
determine whether this point mutation could further elevate A-globin expression in the presence of the µLCR, the
117 G A transistion was introduced into the
µLCRA-globin constructs containing the 201, or
the 382 A-globin promoter truncations described
above, and expression was analyzed in pools of stably transfected
MEL585 cells (Fig 1B).
In constructs carrying the 201 or the 382 truncations,
expression increased about 50% by addition of the 117 G
A mutation (average of 42.3% v 29.9% for the
201 truncation [P = .041] and 49.5%
v 29.4% for the 382 truncation [P = .01]) per
copy of murine -globin indicating that this point mutation only
modestly increases A-globin expression in
µLCR-containing constructs.
Truncations of the -globin promoter.
A similar truncation approach was used to define the minimal promoter
for human -globin gene. As diagrammed in
Fig 2A, the control construct consisted of
the µLCR enhancer linked to a 5-kb BglII fragment containing
a 1560-bp promoter and the 3' enhancer. This construct
contains all of the necessary cis-regulatory elements defined
by transgenic mouse studies.18 Four truncations were tested, including a deletion to 614, a deletion to 267,
which has been used before in retrovirus vectors for
-globin,2,3 a deletion to 127, which includes
both proximal and distal CACCC boxes, and a deletion to 86,
which removes both CACCC boxes.
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| Fig 2.
Diagrams and expression of constructs containing the
µLCR and -globin gene with promoter truncations. (A) Is for
constructs containing the -globin gene extended to include the
3' enhancer sequence, while (B) is for constructs containing the
-globin gene without the 3' enhancer. In the diagrams to the
left, the µLCR is indicated by the thick hatched bar, while the
-globin gene is indicated by the thin open bar. Exons are indicated
by the open boxes, the site of transcription initiation (cap site) by
the arrow, and the positions of the individual promoter truncations are
relative to the cap site. To the right of each panel is indicated
the number of transfected MEL585 pools included in the
analysis along with the mean and range of expression for each
construct.
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As summarized to the right of Fig 2A, the -globin mRNA levels for
the 1560 control averaged 55.4% per copy of murine -globin, with a range of 20.6% to 119.3%. The -globin gene with the
614 and 267 truncations were expressed at similar levels,
averaging 48.7% and 46.0% per copy of murine -globin,
respectively, indicating the absence of either positive or negative
elements within the 267 to 614 region. Expression of the
-globin gene with the 127 truncation was higher, averaging
71.6% (±16.7) per copy of murine -globin. The 86
truncation decreased -globin mRNA expression to 5.0% per copy of
murine -globin gene, suggesting that one or both -globin gene
CACCC boxes participate in the interaction between the -globin
promoter and the µLCR.
Deletion of the -globin 3' enhancer.
All of the -globin gene constructs tested above contain 3'
sequences identified in erythroid cell lines and transgenic mice to
have enhancer activity.19-22 To determine whether the
3' -globin enhancer had any discernible activity in the
presence of the µLCR, this element was deleted in three of the
-globin gene constructs described above (the 1560 control and
the 267 and 127 promoter truncations). As seen in Fig 2B,
the -globin gene having a 127 truncated promoter and deleted
3' enhancer was expressed at 64.4% of murine -globin mRNA per
copy. This level is not statistically different (P = .5) from
the level of the corresponding -globin gene construct with the
3' enhancer (71.6%). Similar results were obtained for the
267 truncation (34.6% v 48.7%) and the 1560 promoter (32.2% v 55.4%). It is noteworthy that the
127 truncated promoter was also better than the longer promoters
in the absence of the -globin 3' enhancer.
Enhancing activity of HS3/HS2 combination.
In the studies described above, the µLCR originally described by
Forrester et al,7 was used as an erythroid-specific
enhancer. This element is an abbreviated version of the natural 22-kb
-globin LCR23 and contains sequences from four of the
erythroid-specific DNase I hypersensitive sites (HS1-4) thought to be
critical for specific enhancer function.23-26 Although the
µLCR retains most of the enhancing activity of the whole
LCR,7 at 2.5 kb, this cassette is too large to include in
conventional retrovirus vectors, which contain a full-length globin
gene and selectable marker. Previous studies of individual HS core
elements suggested that pair-wise combinations may also potentially
provide nearly full LCR activity.18 To test this
possibility, the most evolutionarily conserved sequences from HS2 and
HS3 were combined and tested for enhancer activity as described above
using both A-globin and -globin genes. The 299-bp HS2
fragment contains most of the sequences identified by DNase
footprinting,27 and it is almost half the size of the
732-bp HS2 fragment used in the µLCR. The 784-bp HS3 fragment
contains all of the sequences identified by DNase
footprinting28 and is slightly larger than the 564-bp HS3
fragment used in the µLCR.
As diagrammed in Fig 3, the
A-globin gene used in these studies contained the
382 promoter and 117 G A mutation, while the
-globin gene contained the 267 promoter and no 3'
enhancer. The A-globin cassette linked to the HS2/HS3
enhancer was expressed at 36.9% ± 8.5% per copy of murine
-globin, compared with an average 49.5% for the same cassette
linked to the µLCR (Fig 3A). In the case of the -globin gene,
linkage to the HS2/HS3 enhancer led to an average expression of 57.3% ± 19.5% per copy of murine -globin, compared with an average
34.6% for the same cassette linked to the µLCR (Fig 3B). In both
cases, the differences were not statistically significant (P = .07 for A-globin and P = .13 for -globin),
indicating that the 1.0-kb HS2/HS3 composite retains most of the
enhancing activity of the larger 2.5-kb µLCR, regardless of whether
it is linked to genes for A- or -globin.
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| Fig 3.
Diagrams and expression of constructs designed to compare
enhancing activity of the µLCR and HS3/HS2. (A) Is for constructs
containing the Ag-globin gene (thin filled bars) with the
382 promoter and G A transition at position 117, while
(B) is for constructs containing the -globin gene with the 267
promoter and no 3' enhancer (thin open bars). Exons are indicated
by the boxes, and the sites of transcription initiation (cap sites) are
shown by the arrows. The µLCR is indicated by the thick hatched bar,
while the HS3/HS2 enhancer core fragments are indicated by heavy and
light stippling, respectively. To the right of each panel, the
number of transfected MEL585 pools included in the analysis is shown
along with the mean, standard deviation, and range of expression for
each construct as described in the legend to Fig 1.
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Enhancing activity of -globin HS-40.
The HS-40 element from the -globin locus functions as a classical
enhancer, but it is unable to confer copy number-dependent expression
to a linked gene in a stable transfection assay and in transgenic
mice.29-32 The 356-bp HS-40 fragment used here contains the
core DNase I hypersensitive site and multiple binding motifs for
erythroid and ubiquitous trans-acting factors.30,32
To further define the specific function of this enhancer fragment, the
series of plasmid constructs diagrammed in
Fig 4 were generated and tested for
expression in stably transfected MEL585 pools. Placing the HS-40
fragment in the same orientation with an A-globin gene
containing a 382 promoter (Fig 4A) enhanced expression to 16.5% ± 6.2% per copy of murine -globin, compared with 19.5% ± 5.9% for the same construct containing the HS-40 fragment in the
opposite orientation. Similar experiments were performed with the HS-40
fragment linked to the -globin gene cassette with the 267
promoter and no 3' enhancer (Fig 4B); when positioned in the same
orientation as transcription, the HS-40 fragment enhanced expression to
43.0% ± 17.0% per copy of murine -globin, compared with 45.6% ± 19.9% for the same construct containing the HS-40 fragment in
the opposite orientation. These results suggest that the HS-40 enhancer
is orientation independent. Comparison of the findings of Figs 4A and B
with Fig 1A and B also show that the enhancing activity of HS-40 is
statistically indistinguishable from that of the µLCR. When two
copies of the HS-40 fragment were linked to the 382
A-globin gene, expression increased to 27.6% ± 4.9% per copy of murine -globin. Although higher than the
equivalent vector with one copy of the HS-40 fragment (19.5% ± 5.9%; P = .038), it is not clear whether the enhancing
activity is additive.
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| Fig 4.
Diagram and expression of constructs designed to test the
enhancing activity of HS-40 and novel cassettes for
A-globin. (A) Is for constructs containing the
A-globin gene (thin filled bar) with the 382 promoter
and various arrangements of the HS-40 enhancer, (B) is for constructs
containing the -globin gene with the 267 promoter (thin open bar)
and various arrangements of the HS-40 enhancer, and (C) is for
constructs containing hybrid expression cassettes with the -globin
promoter, various versions of the A-globin gene, and a
single copy of the HS-40 enhancer in the same orientation. Exons are
indicated by the boxes, the sites of transcription initiation (cap
sites) by the arrows, and the segments of the -globin promoter and
A-globin intron 2, which were deleted, are indicated by
a thin line. The HS-40 enhancer is indicated by the thick bars with
graded fill, and the orientation is shown underneath by an arrow. To
the right of each panel is indicated the number of transfected MEL585
pools included in the analysis, along with the mean, standard
deviation, and range of expression for each construct as described in
the legend to Fig 1.
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Further modifications of the A-globin
gene.
As described above, the strength of the A-globin gene
promoter is about half of that from the -globin gene, regardless of the enhancer used. To increase A-globin expression, we
generated the series of hybrid genes diagrammed in Fig 4C in which the
promoter for -globin was linked to 3' transcription cassette
for A-globin. This was done using a naturally occurring
NcoI restriction site present at the translational start codon
of both the A- and -globin genes.
Using the -globin HS-40 enhancer, 267 -globin promoter,
and conventional A-globin cassette, expression levels
were 39.1% ± 14.8% per copy of murine -globin. This expression
level was increased to 73.9% ± 20.9% per copy of murine
-globin when the -globin promoter was truncated to 127.
This is statistically higher than the activity of a recombinant
containing the HS-40 and a 267 -globin promoter (P = .008). These data indicate that the HS-40 can efficiently enhance
-globin gene expression through the -globin promoter, and that
truncation of the -globin promoter to 127 can increase expression of a linked -globin gene in a fashion similar to that observed with the intact -globin genes (Fig 2A).
We also used this system to investigate the enhancing ability of a
3' A-globin regulatory element, which was
initially identified through a transient transfection
assay.33 Although this element was unable to enhance
A-globin gene expression in transgenic mice, it did
confer copy number-dependent expression, suggesting that it may help
stabilize the interaction between LCR and A gene
promoter.34 When the 3' A-globin
regulatory element was added to the recombinant containing the HS-40
enhancer, 127 -globin promoter, and A-globin
transcription cassette, expression averaged 45.3% ± 16.4% per
copy of murine -globin, significantly less (P = .007) than the 73.9% ± 20.9% observed for the same vector without the
3' A-globin regulatory element.
HS-40/pr.-A
construct with intron deletion.
Because of the size limits inherent in conventional retrovirus vectors,
we also used the HS-40/pr.-A construct to
test the effect of a large internal deletion in intron 2 of the
A-globin gene (Fig 4C). This deletion removes 714 bp
from the center of the intron, but leaves intact splice donor and
acceptor sites. When this deletion was introduced into a hybrid
construct containing the HS-40 enhancer, 127 -globin
promoter, and the A-globin gene, expression levels
averaged 166% ± 81.5% per copy of murine -globin. This was
statistically greater (P = .02) than the 73.9% ± 20.9% observed for the same vector with an intact second intron.
To confirm this result, an additional nine MEL585 clones transfected
with this construct were isolated. Expression of
A-globin mRNA in these clones averaged 104.5% per copy
of murine -globin, with a range of 43.0% to 233.1% and a standard
deviation of ± 53.4%. Previous studies9 have shown
that such large variations in expression are characteristic of MEL cell clones.
Testing the
HS-40/pr.-A( 2)
expression cassette in a retrovirus vector.
To determine whether similar expression levels could be achieved via
retrovirus-vector mediated transduction, the cassette described above
was incorporated into the MLV vector LNSX. As seen in
Fig 5A, this cassette was inserted in the
opposite orientation with respect to transcription to prevent splicing
of the genomic transcript and consisted of a single copy of the HS-40
enhancer, the -globin gene promoter truncated to position 127,
and the genomic elements of the A-globin gene starting
with the first exon and containing the large internal deletion of
intron 2. Producer lines were generated using the amphotropic packaging
line PA317 and screened for functional titer by serial dilution of
virus supernatant and transfer of G418 resistance to NIH3T3 cells. The
best of 12 clones screened gave a titer of 3 × 106
colony-forming units (CFU) per mL, which is essentially equivalent to
the of 5 × 106 CFU/mL achieved with the parental
vector in parallel. These data indicate that the various elements of
the hybrid expression cassette do not adversely effect virus titers, a
major prerequisite for the generation of therapeutic vectors.
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| Fig 5.
Diagram and analysis of retrovirus vector. (A) The
retrovirus vector HS40-6 was generated using the MLV-based vector
LNSX1 indicated at the top, which expresses Neo from the
promoter in the 5' LTR. The optimal expression cassette was
inserted in the opposite orientation with respect to viral
transcription. This cassette consists of a single copy of the HS-40
enhancer (graded fill), the -globin gene promoter (open thin bar)
truncated to position 127, and the genomic elements of the
A-globin gene (closed thin bar) starting with the first
exon (filled boxes) and containing the large internal deletion ( ) of
intron 2. Heavy arrows, sites of transcription initiation; sd/sa,
vector splice donor/acceptor sites;  , packaging signal; pA,
polyadenylation sites; K, KpnI restriction sites used for
Southern analysis. (B) DNA was prepared from clones of
vector-transduced MEL585 cells and analyzed for intact provirus by
digestion with KpnI (which cuts once in each LTR) and probing
for Neo. Controls include DNA from untransduced MEL585 cells (U) and
the producer clone (P) used to generate virus supernatant. The expected
position of intact provirus is indicated to the left of the panel with
an arrow. The limited signal for clone no. 9 was due to a loading
error. (C) RNase protection analysis for A-globin
expression in the 12 MEL585 clones transduced with the retrovirus
vector. The positions of the protected fragments for
A-globin (170 bp, exon 2) and murine -globin (128 bp,
exon 1) are indicated to the left of the panel. Two novel protected
fragments in samples no. 2, 7, and 10 are indicated by asterisks. (D)
The protected fragments in (C) were quantified by Phosphorimager, and
expression of the transduced A-globin cassette is
reported as a percentage per copy of endogenous murine -globin. For
clones no. 2, 7, and 10, the contribution from the secondary bands are
indicated by the hatched portion of the bar. Clones no. 6 and 8 are
marked with () to indicate they contain deleted provirus.
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Supernatant from the best producer clone was used to transduce MEL585
cells, and individual clones were isolated by limiting dilution and
selection with G418. These clones were then analyzed for intact
provirus by Southern blotting and for expression of A-globin by RNase protection. As seen in Fig 5B, 10 of
12 clones contained fully intact provirus. In clone no. 6, there was
some intact provirus, but most of the provirus appeared to contain an
internal deletion estimated to be about 0.5 kb. In the case of clone
no. 8, most of the provirus appeared to have a deletion of about the
same size as in clone no. 6, as well as a faint band about 1 kb larger
than the intact vector.
After induction, RNase protection analysis was performed on 12 MEL585
clones (Fig 5C). Excluding the clones with obvious provirus deletions,
A-globin mRNA expression averaged 62.7% ± 31.7% per copy of murine -globin, with a range of 24.2% to
114.7%. Two extra bands were observed in the RNase protection analysis
of three clones (nos. 2, 7, and 10) indicating abnormal splicing. The
average A-globin mRNA expression in the remaining seven
clones was 74.8% ± 30.1%. This level of expression is
statistically indistinguishable from the 104.5% ± 53.4% observed
for the plasmid construct containing the same cassette in the set of
nine transfected MEL585 clones (P = .20), suggesting
that expression of this cassette is not affected by elements from the
retrovirus vector. High level expression of A-globin
protein was confirmed in the virus vector-transduced MEL585 clones
described in Fig 5 by immunofluorescent staining and flow cytometry
(data not shown). These data show the ability of this vector to
generate high functional titers and express A-globin at
high levels in the erythroleukemia cell line MEL585.
| |
DISCUSSION |
Effective gene therapy of -chain hemoglobinopathies will require
vectors, which are capable of expressing the transferred globin gene at
near physiologic levels at the appropriate stage of erythroid
differentiation. Initial studies with retrovirus vectors for human
-globin showed that an extended -globin promoter and coding
sequences alone were not sufficient to achieve high levels of
expression.5 Although the promoters from the - and -globin genes have been intensively studied for many years, their relative strengths in isolation remain unclear, due in part to differences in experimental systems and construct components. We made a
series of truncations in both the A- and -globin
promoters and coupled them to the same µLCR enhancer. The natural
globin coding sequences, rather then heterologous reporter genes, were
used to assure the presence of any critical cis-regulatory
elements within the transcribed regions, and to allow for the direct
assessment of authentic globin gene transcripts. Maximal expression was
achieved in this system even after deleting several upstream motifs,
including the binding sites for GATA-1 and Oct-1 present around
position 175 of the A-globin promoter. The high
level of expression from the truncated promoters may be due, in part,
to the relative proximity of the µLCR enhancer in these constructs.
In the case of the plasmid constructs used here, the µLCR is already
located directly adjacent to the promoters. This possibility is
supported by studies in mice, where expression of an
A-globin transgene linked to LCR elements in a plasmid
was found to persist into adulthood,13 compared with the
normal developmental silencing observed when the
A-globin gene and LCR were separated on cosmids or yeast
artificial chromosomes.35,36
Globin gene expression in vivo is absolutely dependent on
locus-specific enhancers such as the -globin LCR or the -globin HS-40 element. The -globin LCR consists of five DNase I
hypersensitive sites. Three of these sites, HS2, HS3, and HS4 have
shown enhancing activity, while the enhancing activity of HS1 has only
been observed in combination with other HS elements.37 The
core elements of these sites have been mapped to about 200 to 300 bp
each using a combination of DNase footprinting, evolutionary
conservation, and functional assays (reviewed in Stamatoyannopoulos and
Nienhuis38). The core sequence of the -globin HS-40
enhancer has likewise been mapped to about 300 bp.30 These
cores share several common features, including binding sites for
erythroid-specific transcription factors such as GATA-1 and NF-E2, as
well as for ubiquitous factors, such as the GT motifs. This similarity
is mirrored in the relative enhancing ability of these elements. As
shown in the studies reported here, there is no significant difference
between the enhancing activity of the µLCR, the combination of HS2
and HS3, or the HS-40 core when MEL cells are used as target cells.
However, studies in transgenic animals have shown important differences
between the functions of the -globin LCR and the -globin HS-40.
The LCR is capable of opening and maintaining the chromosome structure, as evidenced by the copy number-dependent expression of genes linked to
this element.23 The HS-40, on the other hand, is unable to
confer copy number-dependent expression to a linked gene.31 Moreover, the expression levels of genes linked to the HS-40 in transgenic mice decrease with age,39 implying that HS-40
element cannot resist heterochromatin spreading. It is critical to
determine whether globin gene expression cassettes incorporating the
HS-40 element are capable of efficient, long-term expression in vivo.
An unexpected finding in this study was the effect of internal
sequences from A-globin intron 2 on gene expression.
Aside from its role in splicing, no other enhancing or suppressing
activity has previously been ascribed to sequences from this intron.
Initial studies with reporter genes in the absence of the LCR suggested
that an enhancing activity may be present in the second intron of the
-globin gene.20,21 However, such activity was not
detected in subsequent studies where the LCR was included in the
constructs.18 In retrovirus vectors for -globin, a full
deletion of intron abolished expression even in the presence of LCR
sequences,40,41 while expression could be restored by only
deleting an internal portion of this intron.2 In the case
of retrovirus vectors for -globin, a full deletion of intron 2 (needed to achieve high titers) resulted in a twofold decrease in
expression.5 The 714-bp internal deletion of
A-globin intron 2 reported here resulted in 2.3-fold
higher expression compared with the same construct with a full-length
intron 2. Whether the increased expression is due to increased
transcript stability, increased rate of transcription, or a facilitated
interaction between the enhancer and the promoter remains to be determined.
We have previously found a very large variation in globin gene
expression among MEL cell clones tranfected with plasmids9 or YACs containing the -globin locus,42 and we have
concluded that this line cannot be used for studying the function of
the LCR or the sequences, which protect the globin genes from the effects of the position of integration. In contrast to single clones,
pools composed of more than 50 clones are useful for expression studies
because the variation between individual clones is normalized when a
large number of clones are contained in a pool.9 Ideally, studies of globin gene expression cassettes should be performed using
primary cells in transgenic mice, but this approach is impractical when
a large number of constructs have to be functionally characterized. As
shown here, when MEL cell pools containing a large number of clones are
used, meaningful data can be obtained.
We felt it was important to determine whether retrovirus vector
sequences have any intrinsic properties, which may function to suppress
expression of the globin gene in the HS-40/pr-A(
intron 2) cassette. We found no statistically significant difference in
expression between cells transfected with the plasmid construct and
cells transduced with a retrovirus vector containing the
HS-40/pr-A( intron 2) cassette. This observation
suggests that the retrovirus vector sequences need not necessarily
impair globin gene expression, and that the HS-40 enhancer and 127
-globin promoter may function independently of the promoters and
enhancers of the virus LTR.
| |
ACKNOWLEDGMENT |
We thank Betty Mastropaolo, XiaoChun Wang, Mike Mikiska, and Mike
Knibbe for technical support. We would also like to thank Kenneth R. Peterson for providing the 117 G A
A-globin promoter, A. Dusty Miller for providing the
retrovirus vector LNSX and the packaging cell lines PA317 and PE501,
Douglas R. Higgs for providing the -globin HS-40 enhancer fragment,
and Margaret H. Baron for providing the mouse -globin plasmid.
| |
FOOTNOTES |
Submitted August 19, 1998; accepted November 11, 1998.
Supported by Grant No. HL 53750 from the National Institutes of Health,
Bethesda, MD.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to George Stamatoyannopoulos, MD,
Professor of Medicine, Head, Division of Medical Genetics,
University of Washington, Box 357720, Seattle, WA 98195; e-mail:
gstam{at}u.washington.edu.
| |
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ABSTRACT
INTRODUCTION