I. Introduction
The ability to achieve persistent, regulated, high
levels of transgene expression in vivo
is often necessary for the success of a gene therapy vector. Unfortunately,
with most gene therapy vectors used to date, expression is temporary, often
falling to non-therapeutic or undetectable levels within a few weeks after
treatment. For viral vectors, transience may be due to the immunogenicity of
the vector, resulting in loss of transfected cells with a concurrent reduction
in transgene expression. In the case of non-integrating vectors, viral or
non-viral, transience can result from vector loss as the cells divide.
For both integrating and non-integrating systems,
decreased transgene expression may also be attributable to DNA silencing. For
example, when mouse hepatocytes were transfected in vivo with naked plasmid DNA encoding the AAT cDNA under control
of the cytomegalovirus (CMV) promoter, day 1 expression levels of 500 mg/ml were observed. These levels fell to <10 mg/ml within 3 weeks after transfection (Zhang et al, 2000). Southern analysis of liver DNA showed that plasmid
DNA was maintained extrachromosomally in the liver cells for at least 100 days,
indicating that the decrease in expression was primarily a result of DNA
silencing, rather than vector loss.
We observed similar results (Stoll et
al, 2001) in experiments in which naked plasmid DNA was injected into mouse
hepatocytes via hydrodynamic tail vein injection (Zhang et al, 1999). On the one hand, an extrachromosomal plasmid
carrying the 19 kb genomic SERPINA1
encoding human a1-antitrypsin
(AAT) resulted in expression levels of >300 mg/ml in vivo
that persisted at these high levels for > 9 months. However, similar
constructs carrying the AAT cDNA driven by the RSV promoter gave equivalent day
1 expression levels, but the expression dropped >100-fold within two weeks.
Again, Southern analysis showed that plasmid DNA was maintained
extrachromosomally in these relatively quiescent liver cells. In addition to
the SERPINA1 locus, the successful
genomic AAT vector also possessed sequences from Epstein-Barr virus (EBV) that
can aid in extrachromosomal plasmid maintenance and expression.
Epstein-Barr virus (EBV) is a human herpes virus that
is capable of maintaining its genome extrachromosomally in dividing primate
cells. Maintenance is accomplished by the viral latent origin of replication, oriP, and the EBV nuclear antigen 1, EBNA1, which act together to replicate
the viral genome and retain it in the nucleus (Yates et al, 1984, 1985; Reisman et al, 1985). Plasmids containing EBNA1
and a truncated oriP carrying only
the tandem array of 21 EBNA1 binding
sites (family of repeats) from oriP
for retention, but lacking the oriP dyad
symmetry element for replication, are retained in the nucleus of the cells, but
can replicate efficiently only if the plasmid also contains a functional
mammalian origin of replication, such as the 19 kb SERPINA1 sequence ( Krysan et al, 1989; Heinzel et al, 1991; Stoll et
al, 2001). These same EBV components that provide replication and retention
functions are also associated with transcriptional enhancer and anti-silencing
activity (Reisman and Sugden, 1986; Kaneda et al, 2000). Furthermore, in addition to the replication function
of the genomic SERPINA1 sequence
demonstrated in our previous experiments (Stoll et
al, 2001), the full AAT gene was also able to provide more stable expression in vivo than its equivalent cDNA
sequence, which may be subject to silencing.
Silencing of cDNA vectors may occur because the
transgenes are often driven by viral promoters. It has been observed that many
common viral promoters, such as those from cytomegalovirus (CMV), simian virus
40 (SV40), and Rous sarcoma virus (RSV) often exhibit markedly decreased
activity in mammalian cells in vivo
within a few weeks of transfection, a phenomenon that has been attributed to
inhibition by various cytokines (Paillard, 1997). Gill (2001) recently demonstrated that the use of
the cellular elongation factor 1a (EF1a) and ubiquitin C (UbC)
mammalian promoters gave increased persistence and ~10-fold higher expression
levels of a luciferase reporter gene in lungs, compared to a control construct
that expressed luciferase from the CMV promoter. Quantitative PCR analysis of
plasmid vector in the lung tissue revealed that there were no significant
differences in plasmid copy number in the CMV versus EF1a or UbC promoter vectors.
In addition to the reduced transgene silencing
observed when mammalian promoters are used, genomic sequences may provide
additional benefits that lead to increased transgene expression. Studies in transgenic mice have indicated that
introns are essential for stable, high levels of transgene expression. In
comparing transgenic mice generated with cDNA constructs versus full genomic
sequences, the intronless constructs resulted in a lower frequency of
transgenic mice expressing rat growth hormone (rGH), mouse metallothionein I
(mMTI), or human b-globin (hBG) reporter genes, as well as decreased expression levels in
those mice that did have observable expression (Brinster et
al, 1988). Similar results have been observed for AAT and b-lactoglobulin expression constructs in mammary cells
of transgenic mice (Whitelaw et
al, 1991). It is possible that genomic introns contain transcriptional enhancer
sequences that may act on their own to increase transgene expression or may act
in concert with upstream/promoter sequences. These intronic sequences may also
act to help the transgene attain an open chromatin configuration, making it
more accessible to transcription factors. This idea is supported by
observations that deletion of intronic sequences makes transgenes more
susceptible to chromosomal position effects in
vivo than their full genomic counterparts (Webster et al, 1997).
Unfortunately, the large size of most full genes often
precludes their use in vectors. In order to obtain the expression advantages of
intronic sequences, while still minimizing transgene size, heterologous introns
and genomic minigenes have been developed. Palmiter (1991) found that including
only select introns, specifically the first one, in the rGH gene resulted in
transgenic frequencies and expression levels comparable to those achieved when
the full rGH was used. Heterologous introns inserted between promoter and cDNA
gave similar results (Palmiter et
al, 1991). This strategy has been applied to the construction of expression
vectors for therapeutically relevant genes. Miao (2000) constructed a human
factor IX minigene, which included the ApoE hepatic locus control region (HCR),
the hepatocyte-specific AAT promoter, and the human factor IX cDNA, with its
intron A and 3' untranslated region (UTR). This 6.1 kb minigene was reported to
provide therapeutic serum levels of factor IX (0.5 - 2 mg/ml) that were sustained for at least 225 days,
whereas a 2.0 kb AAT promoter-factor IX cDNA construct gave transient
expression that fell to <10 ng/ml within 2 weeks of treatment (Miao et al,
2000, 2001). While this minigene strategy may not be successful for all genes, it
raises the possibility of creating a high-expressing transgene that is capable
of stable expression in vivo, without
the large sizes typical of most mammalian genes.
Another strategy to take advantage of the high
expression capabilities of genomic sequences is to insert a cDNA sequence into
the first exon of a full gene. The theory is that a well-expressed genomic
sequence may provide an open chromatin configuration, transcriptional
enhancers, and/or anti-silencing sequences that may act in cis to provide for stable expression of the heterologous cDNA.
While this approach has been used successfully to achieve expression of
diphtheria toxin (Palmiter et al, 1987) and rat transforming growth factor a (Palmiter et
al, 1991), it has not proven successful for all cDNAs and
genomic sequences tested (Palmiter et al, 1991).
We have previously
demonstrated that the 19 kb full genomic SERPINA1
sequence is capable of giving stable, high levels of expression in vivo when introduced into mouse liver
(Stoll et al,
2001).
Unfortunately, 19 kb is still a rather large sequence. Reduction of the genomic
size without loss of the good expression it provides would be beneficial. To
achieve this result, we minimized the size of the AAT genomic sequence, both
through reduction of flanking sequences and through generation of an AAT
minigene that retains only its first intron and all flanking sequences. We
explored the ability of this genomic sequence to stabilize and boost expression
of a heterologous gene, by inserting a human factor IX minigene (Miao et al, 2000) upstream of the
first exon in SERPINA1. Here we
report the successful reduction in size of the AAT gene, without loss of
expression, as well as the successful stabilization of factor IX expression in vivo when expressed from within the
AAT gene.
II. Materials and methods
A. Vector construction
Plasmids pEF, pEF-AAT,
pEF-cAAT, and pcAAT have been described previously (Stoll et al, 2001). Removal of the EBV components (EBNA1 and family of repeats) from
plasmid pEF-AAT was achieved by digestion with ClaI and vector religation, creating the plasmid pAAT.
Vectors pcAAT2 and pEFcAAT2
are identical to pcAAT and pEF-cAAT, respectively, except that the RSV promoter
has been replaced with the endogenous, hepatocyte-specific AAT promoter. 471 bp
containing the AAT promoter was PCR amplified from vector pF9 (Sclimenti et al, 2003) and cloned into the vector pCR2.1 (Invitrogen, Carlsbad, CA), making
the vector pTA-AATpro. From there it was subcloned as an MluI-HinDIII fragment
into the MluI-HinDIII sites in the plasmid pcAAT, replacing the RSV promoter.
This step created the vector pcAAT2. From that plasmid, the 2.1 kb AATpro-cDNA
fragment was liberated as an XhoI
fragment and cloned into the SalI
site in plasmid pEF, creating plasmid pEF-cAAT2.
Minimization of the AAT
genomic sequence began with removal of some of the flanking sequences. From the
5' upstream sequence, 3.7 kb was removed by digesting pAAT with ClaI and MfeI and religating the vector, creating the vector pAAT(-5'). From
the 3' flanking sequence in pAAT, 970 bp was removed by deleting an AflII-SalI fragment, generating pAAT(-3'). This plasmid was further
reduced by digestion with ClaI and MfeI and religation, generating the
double deletion plasmid pAATD, which is reduced by a total
of 4.7 kb, compared to pAAT.
Cloning of the AAT minigene
vector was performed as follows. The AAT cDNA was removed from pcAAT as a 1.3
kb BamHI-PstI fragment, and cloned into the BamHI-PstI sites of
pBCSK+ (Stratagene, La Jolla, CA), creating the vector pBcAAT. An AflII linker was cloned into its BglI-PstI
sites, generating vector pBcAATii. A 1529 bp fragment of AAT 3' sequence was
PCR amplified and cloned into vector pCR2.1, creating pTA-AAT3', from which the
AAT3' sequence was subcloned as an EcoRI
fragment into the EcoRI sites of
pBcAAT, creating pBcAAT-AAT3'. From this plasmid, a 622 bp AvaI-PstI fragment was
cloned into the AvaI-PstI sites of pBcAATii, generating the
vector pBcAATiii. Next, a 499 bp PstI
fragment from pBcAAT-AAT3' was cloned into pBcAATiii, creating plasmid
pBcAATiv. A 552 bp StuI-AflII fragment from pBcAAT-AAT3' was
cloned into pBcAATiv, generating pBcAATv. Next, a 771 bp BamHI fragment from pAAT was cloned into the BamHI site of pBcAATv, to create the plasmid pBcAATvi. The last
step in creating the minigene was to clone the 2.9 kb minigene from pBcAATvi,
as an AflII-SnaBI fragment, into the AflII-SnaBI sites of vector pAAT. The result
is vector pAATmg, which lacks introns 2-4, resulting in a total reduction of
3.5 kb in gene size.
Vector pAAT-fIXmg was
constructed as follows. A BglII
linker was ligated into the SacI site
of pF9, generating pfIXmgi. Next, a 2.6 kb fragment of the AAT first intron was
PCR amplified, digested with BamHI
and BglII, and cloned into the BglII site of pfIXmgi, creating
pfIXmgii. The AAT promoter was released from pTA-AATpro as a SapI-ClaI
fragment and cloned into the SapI-ClaI sites of pfIXmgii, generating the
vector pfIXmgiii. Finally, the 7.9 kb fIXmg was released from plasmid pfIXmgiii
by digestion with BglII and cloned
into the BglII sites in plasmid pAAT,
resulting in the final vector pAAT-fIXmg.
B. In vivo delivery and analysis.
C57BL/6 mice were injected
over a period of 6 – 9 s with 25 mg of DNA in 1.8 ml
of 0.9% NaCl, by hydrodynamic tail-vein injection (Liu et al, 1999; Zhang et
al, 1999). Serum samples
were periodically obtained by retro-orbital bleed. All animal procedures were
performed under the guidelines set forth by Stanford University and the
National Institutes of Health. The samples were assayed by a polyclonal capture
ELISA assay to measure serum AAT (Song et al, 1998; Yant et
al, 2000) and/or serum fIX (Sclimenti et al, 2003). Some animals were subjected
to partial, surgical hepatectomy (PH) (Chen et al, 2001) and then allowed to recover.
Serum samples were obtained periodically by retro-orbital bleed and analyzed by
ELISA. At 51 d post-PH, treated and control animals were sacrificed and the
livers were removed for analysis of plasmid DNA. The liver tissue that was
removed during PH surgery was also subjected to analysis. Liver tissue was
diced and total DNA was prepared using the Blood and Cell Culture DNA Maxi kit
(Qiagen, Valencia, CA). Southern blot analysis was performed as follows. DNA
was digested with ScaI and separated
on a 0.65% agarose gel. The gel was depurinated in 0.25 M HCl, denatured in 0.5
M NaOH, neutralized in 0.5M Tris-HCl (pH 7.0) and transferred to an S&S
Nytran blotting membrane (Schleicher & Shuell, Keene, NH) in 20X SSC
transfer buffer. The membrane was probed with a 484 bp fragment from the
hygromycin resistance gene common to both plasmids, labeled with alkaline phosphatase
enzyme, using the AlkPhos Direct DNA labeling kit (Amersham Pharmacia Biotech).
Hybridization occurred at 55°C for 16 h in hybridization buffer
provided with the kit. Membranes were washed according to protocol and
incubated with CDP-Star Detection Reagent for 4 min. The membrane was then
exposed to Hyperfilm ECL (Amersham Pharmacia Biotech). Southern blot
quantification was performed using Kodak 1D software.
III. Results
A. Role of EBV sequences in extrachromosomal plasmid
expression in vivo
Our previous research showed that the plasmid pEF-AAT,
bearing the 19 kb SERPINA1 genomic
locus encoding human a1-antitrypsin
on a vector with the EBV EBNA1 and
family of repeats retention sequences, was capable of maintaining long-term
high expression levels after transfection into mouse liver (Stoll et
al, 2001). We first wanted to determine what role the EBV components on this
plasmid played in sustaining these high expression levels. EBNA1 and the EBV family of repeats were removed, creating plasmid
pAAT. Naked plasmid DNA of pEF-AAT and pAAT, along with pEF-cAAT and pcAAT
control cDNA plasmids with and without EBV retention sequences (Figure 1A), were injected into the
mouse liver via hydrodynamic tail-vein injection (Liu et al, 1999; Zhang et al, 2000). Serum AAT levels were determined by ELISA. The
results are shown in Figure 1B. Both
genomic vectors were able to provide expression levels ~1000-fold greater than
the cDNA control vectors, which fell to £ 100 ng/ml within 3 weeks post-injection. While the non-EBV plasmid
pAAT was still capable of providing stable, high-levels of expression of AAT
(151 mg/ml at 6 months), the levels provided by pEF-AAT were
~4-fold higher (585 mg/ml at 6 months). This
result indicated that the EBV components provided some expression advantage to
the plasmid pEF-AAT.
The liver is a relatively quiescent tissue, suggesting
that cell division-induced vector loss should not be a significant problem.
This conjecture is supported by observations that plasmid DNA is stably
maintained extrachromosomally in liver cells (Zhang et al, 2000; Stoll et al, 2001). In order to determine if there was indeed a DNA
retention advantage provided by the EBV sequences in pEF-AAT compared to pAAT,
partial hepatectomy (PH) was performed on sample mice injected with each of
these plasmids. Partial surgical hepatectomy involved removal of two-thirds of
the liver, stimulating division and regeneration of the remaining liver tissue.
Under these circumstances, extrachromosomal plasmid DNA is lost from the cells,
unless it contains retention sequences such as those provided by EBNA1 and the EBV family of repeats (Krysan et
al, 1989). Following PH, serum levels of AAT were monitored, and the results are
shown in Figure 2A. Liver genomic
DNA was also analyzed in PH-mice before and after surgery and in control mice (Figure 2B).
The effects of partial hepatectomy on
AAT expression and DNA retention are summarized in Table 1. In mice injected with the EBV vector pEF-AAT, partial
hepatectomy resulted in a 1.7-fold decrease in total plasmid DNA in the liver
and a 4.6-fold decrease in serum AAT levels. Mice injected with the non-EBV
plasmid pAAT showed a 3.5-fold decrease in total liver plasmid DNA and a
7.0-fold decrease in serum AAT levels. These results suggested that the EBV
sequences were playing a role in enhancing the retention and expression of
plasmid DNA in vivo in the presence
of cell division. When in non-dividing tissue, the non-EBV pAAT plasmid
appeared to be capable of maintaining stable, high-levels of expression. For
this reason, and also to eliminate any effects on expression that the EBV
sequences may have, all further experiments were conducted using a non-EBV
extrachromosomal vector.
B. Minimization of AAT sequences required for expression in vivo
While
the 19 kb SERPINA1 sequence was not
prohibitively large for use in extrachromosomal gene therapy vectors, it was
still a cumbersome size. It was likely that some of the 8.5 kb of flanking
sequences were not necessary for regulation or enhancement of gene expression.
In order to address this question and possibly minimize the functional size of
this genomic sequence, we began by deleting segments of flanking sequence and
analyzing the in vivo expression of
these deleted vectors. Using convenient restriction enzyme sites, 3.7 kb of 5'
flanking sequence were removed from pAAT to generate pAAT(-5'), 921 bp were
removed from the 3' flanking sequence to create pAAT(-3'), and both deletions
were generated in the same plasmid to produce plasmid pAATD. These plasmids, along with the full gene vector pAAT
and a cDNA vector pcAAT, were injected hydrodynamically into the tail-vein of
mice and serum AAT levels were monitored by ELISA. The results are shown in Figure 3. All genomic vectors were able
to provide high expression levels of AAT, >190 mg/ml at two months post-injection, though vectors
pAAT(-5') and pAATD gave ~2-fold higher
expression levels than pAAT and pAAT(-3').
Control cDNA vectors pcAAT and pEF-cAAT (Figure 1A) were both expressed from the
Rous sarcoma virus (RSV) promoter. It was possible that the AAT genomic
sequence was not necessary at all, and that the high expression was simply the
result of the strength of the AAT promoter. To address this possibility, the
RSV promoter was replaced with the AAT promoter in plasmids pcAAT and pEF-cAAT,
generating the vectors pcAAT2 and pEF-cAAT2, respectively (Figure 4A). Following liver transfection via tail-vein injection, in vivo expression was monitored by
ELISA (Figure 4B). The AAT promoter
was able to provide some transient stability to cDNA expression in vivo. While the RSV-cDNA vectors fell
to nearly undetectable levels within 2 weeks post-injection, the AAT
promoter-cDNA vectors did not fall to these levels until ~5 weeks
post-injection. However, there was no significant difference in expression
levels from the two promoters. Ultimately, expression was lost from all four
cDNA vectors, whereas the genomic sequence in pAAT maintained expression at
>280 mg/ml for the duration of the experiment.
Miao (2000) created a
well-expressed human factor IX minigene that retained only a portion of the
first intron of that gene. We wondered if construction of a similar minigene
for AAT would be equally successful. We created the plasmid pAATmg, which
contained the full 5' and 3' flanking sequences of pAAT, the AAT promoter, and
all exons, but retained only the 6.1 kb first intron (Figure 4A). Serum AAT levels were monitored by ELISA following
tail-vein injection of plasmid DNA into mice, with the results shown in Figure 4C. The 15.5 kb AAT minigene was
not capable of providing stable, high expression levels of AAT in vivo. While day 1 levels of
expression were within 3-fold of each other, expression from pAATmg dropped
~100-fold below that of pAAT within 3 weeks post-injection, and continued to
drop, approaching cDNA expression levels by day 85.
C. Expression of a
heterologous gene from within SERPINA1
Our unsuccessful attempt to
create an AAT minigene indicated that not all genes could be easily minimized
to a size convenient for use in gene therapy vectors, transgenic mice, or
numerous other applications, while still maintaining high expression. Due to
the high expression levels of AAT in vivo
in these and previous experiments (Stoll et al, 2001), we hypothesized that the
full-genomic SERPINA1 might contain
sequences that could enhance expression of a heterologous gene cloned within
it. To demonstrate this idea, a human factor IX minigene (Miao et al, 2000) was cloned into the first
exon of the AAT gene. Miao (2000) reported high levels of expression for this
factor IX minigene that were maintained in
vivo for ≥ 225 days. However, Sclimenti (2003) observed only
transiently high expression levels for this minigene that stabilized at £100
ng/ml after 2-3 months post-injection. We therefore chose this factor IX
minigene (fIXmg), rather than a factor IX cDNA, to clone within SERPINA1 for expression studies,
creating the vector pAAT-fIXmg (Figure
5A). Plasmid DNA was transfected into mouse liver by hydrodynamic tail-vein
injection, and serum fIX levels were monitored by ELISA. In addition to
pAAT-fIXmg, we also tested expression from control plasmids pfIX and pDY-fIX,
which contained the fIXmg on a plasmid with EBV retention sequences (Sclimenti et al, 2003). Results are shown in Figure 5B. As observed by Sclimenti
(2003), the factor IX minigene alone plasmid, pfIX, had high day 1 levels (13 mg/ml)
that fell to <100 ng/ml by 5 months post-injection. In contrast, pDY-fIX
maintained expression ~6-fold higher after 1 year, and pAAT-fIXmg expression of
fIX was even higher at 1.9 mg/ml
at 1 year post-injection. These results indicated that there were sequences
within SERPINA1 that were able to act
in cis to increase expression of
nearby or embedded heterologous genes.
IV. Discussion
We previously reported (Stoll et al, 2001) that the plasmid pEF-AAT,
containing the 19 kb SERPINA1 genomic
locus encoding human a1-antitrypsin and the EBNA1 and family of repeat sequences from Epstein-Barr virus, 
Figure 4. SERPINA1 sequence requirements for AAT expression in vivo. (A) Plasmids. pcAAT2 and pEF-cAAT2 contain AAT cDNA driven by the
mammalian hepatocyte-specific AAT promoter, without and with EBV sequences,
respectively. pAATmg contains the AAT minigene, which retains all flanking
sequences, exons, and the first intron of SERPINA1.. (B)
AAT expression levels in mice injected with 25 mg of pAAT (n;
Figure 1B), pEF-cAAT (l; Figure1A), pEF-cAAT2 (¡), pcAAT (s; Figure 1D), or pcAAT2 (ê).
(C) AAT expression levels in mice
injected with 25 mg of pAAT (n), pAATmg (o), or pcAAT2 (ê). (B & C) Blood was
sampled periodically, and serum AAT was determined by ELISA. Error bars
represent the standard error from five mice.
Figure
5.
Effect of genomic sequence on expression of a heterologous fIX minigene. (A) Plasmid maps. (B) Factor IX expression levels in mice. Groups of 5 mice were
injected with 25 mg of pfIX (u), pDY-fIX (n), or pAAT-fIXmg
(s). Blood was sampled periodically, and serum fIX levels
were determined by ELISA. Error bars represent the standard error.
was
able to provide long-term high-level expression of AAT in vivo. The AAT genomic sequence was capable of acting as a
mammalian origin of replication for this extrachromosomal plasmid (Stoll et
al, 2001), and the EBV sequences can act to retain the plasmid in cells.
However, these features were not likely to be relevant in vivo because these experiments were conducted in non-dividing
liver cells.
In order to determine whether the EBV sequences were
providing any advantage in vivo in
expression or retention of the vector, we constructed the genomic/non-EBV
vector pAAT. Vectors with and without EBV sequences both provided stable, high
expression levels in vivo (>150 mg/ml), but the pEF-AAT vector with EBV sequences was
~4-fold higher (Figure 1B).
Following partial hepatectomy in representative mice, we observed an average
3.3-fold decrease in AAT expression in pEF-AAT-injected mice and a 7.0-fold
decrease in serum AAT in pAAT-injected mice (Figure 2A; Table 1).
Southern analysis revealed that partial hepatectomy-induced hepatocyte
replication resulted in loss of plasmid DNA in both the presence and absence of
EBV retention sequences. However, pEF-AAT mice retained twice as much plasmid
DNA as did pAAT mice, which directly correlated with the observed serum AAT
levels in these two different groups of mice. These results indicated that the
EBV sequences were providing increased retention to these extrachromosomal
vectors in dividing tissue in vivo.
The ~4-fold higher expression levels in pEF-AAT mice
vs. pAAT mice pre-PH suggested that the EBV sequences were also providing
expression enhancement activity. Enhancer activity has previously been
attributed to the action of these EBV sequences (Kenney et al, 1998; Langle-Rouault et al, 1998;
Sclimenti et al, 2003). This enhancer function appears to rely on the basal
expression ability of the transgene construct. The EBV sequences provided a
~4-fold boost in expression from the full AAT gene, but were unable to prolong
or increase expression from AAT cDNA constructs. Even when the viral RSV
promoter driving the cDNA was replaced with the human AAT promoter, expression
was only minimally prolonged, with or without EBV sequences (Figure 4B). This result suggests that
either the AAT promoter alone was not able to maintain persistent expression in vivo, or that these cDNA constructs
lacked structural or regulatory sequences that contribute to the persistence of
expression in vivo, as was observed
for the full-genomic construct pAAT.
Using the pAAT vector, we attempted to minimize the
size of the SERPINA1 locus required
for high expression levels in vivo.
We observed an ~2-fold increase in expression when 3.7 kb of 5' upstream
sequence was deleted from the vector, either alone or in combination with 921
bp deleted from the 3' end (Figure 3).
It is possible that the 5' flanking sequence may contain a repressor sequence
that results in enhanced expression when it is removed from the SERPINA1 genomic sequence. However,
expression levels were still remarkably high and stable for all four of these
genomic vectors, indicating that a reduced genomic sequence of 14.4 kb was
sufficient for attaining good expression of AAT in vivo.
Further minimization of the genomic sequence, through
creation of an AAT minigene deleted of all but the first intron, resulted in an
~100-fold decrease in expression in vivo
(Figure 4C). However, an alternative
AAT minigene, retaining all introns except the first one and flanked by b-lactoglobulin sequences, has been shown to express
AAT at high levels in mice transgenic for the construct (Whitelaw et al, 1991; Clark et al, 1993). Considering these two different findings together
suggests that there are sequences within the last three exons that are
necessary for efficient AAT expression in
vivo, though the nature of these expression modulation sequences is
unclear. It is possible that one or more of the deleted introns contain
transcriptional enhancers. Alternatively, the first intron may contain
transcriptional repressor sequences, which are naturally modulated by sequences
in the downstream exons, removal of which in our minigene resulted in
suppression of expression. Also, it may be that the additional exons are
required to allow stabilization and/or proper processing of the mRNA. While
several labs have observed high expression levels from minigenes containing
only a first intron (Palmiter et al, 1991; Miao et al, 2000), our results suggest that not all genes are amenable
to such simple minimization. Creation of genomic minigenes may require analysis
of multiple intron combinations to identify a minigene that provides a suitable
expression profile in vivo.
While SERPINA1
is 19 kb long, thus prompting our attempts to minimize the amount of sequence
sufficient for high expression levels in
vivo, it is still a relatively small gene. Some therapeutically relevant
genes are much larger, such as factor VIII (~185 kb), cystic fibrosis transmembrane
regulator (~230 kb), and dystrophin (~2.4 Mb). Unfortunately, cDNA constructs
often provide only transient expression in
vivo. It has been observed that a cDNA sequence cloned within another
full-length gene can benefit from the genomic sequence in cis, allowing for stable expression of the cDNA (Palmiter et
al, 1987; Palmiter et al, 1991). To test this idea for the AAT gene, we cloned the
human factor IX minigene (Miao et al, 2000) into SERPINA1
between the promoter and the initiation codon. We observed an ~50-fold increase
in fIX expression when the fIX minigene was placed within the AAT gene,
compared to a vector containing only the minigene (Figure 5B). The AAT gene enhanced expression of the fIX minigene to
levels ~9-fold higher than EBV sequences enhanced fIX expression (pDY-fIX; Figure 5B). It therefore appears that
the AAT genomic sequence is capable of increasing and maintaining expression of
a heterologous minigene. It seems likely that these AAT sequences will be
capable of exerting a similarly beneficial effect on other cDNA or minigene
constructs. Since AAT is expressed only in the liver, it is possible that this
effect will be limited to expression from that tissue. However, if the benefit
is the result of a tissue-independent enhancer activity or a structurally open
chromatin configuration of the genomic sequence, it may be extendable to other
tissues, provided that a suitable promoter drives the heterologous cDNA.
While we have demonstrated that some minimization of SERPINA1 genomic size did not affect its
in vivo expression, there was a limit
to the amount and choice of sequences that could be removed without affecting
expression. For example, the last three introns of SERPINA1 appeared to be much more important than the first intron
alone, as evidenced by the difference in in
vivo expression from these two different minigene constructs (Figure 4C) (Whitelaw et al, 1991), though our pAATmg construct actually retained 1.8 kb
more genomic sequence. A factor IX minigene containing only the first intron
has been shown to provide adequate expression levels in vivo (Miao et al,
2000). It is therefore important to note that the sequences required for
efficient expression in vivo are
likely to differ from gene to gene. These results demonstrate the value of
utilizing genomic sequences for in vivo
expression of a transgene in a gene therapy system.
Acknowledgments
This work was supported
by NIH grants HL69737 to MPC and HL64274 to MAK.
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), pEF-cAAT (l), or pcAAT (s). Blood was sampled periodically,
and serum AAT levels were determined by ELISA. Error bars represent the
standard error from five mice.
