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Journal of Virology, September 2000, p. 8732-8739, Vol. 74, No. 18
0022-538X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Novel Transcriptional Regulatory Signals in the Adeno-Associated
Virus Terminal Repeat A/D Junction Element
Rebecca P.
Haberman,1,2
Thomas J.
McCown,1,3,4 and
Richard Jude
Samulski1,2,5,*
UNC Gene Therapy
Center,1 Neuroscience
Center,3 Curriculum in
Neurobiology,2 and Departments of
Psychiatry4 and
Pharmacology,5 University of North
Carolina, Chapel Hill, North Carolina 27599
Received 7 March 2000/Accepted 3 June 2000
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ABSTRACT |
Adeno-associated virus (AAV) type 2 vectors transfer stable,
long-term gene expression to diverse cell types in vivo. Many gene
therapy applications require the control of long-term transgene expression, and AAV vectors, similar to other gene transfer systems, are being evaluated for delivery of regulated gene expression cassettes. Previously, we (R. P. Haberman, T. J. McCown, and
R. J. Samulski, Gene Ther. 5:1604-1611, 1998) demonstrated the
use of the tetracycline-responsive system for long-term regulated expression in rat brains. In that study, we also observed residual expression in the "off" state both in vitro and in vivo, suggesting that the human cytomegalovirus (CMV) major immediate-early minimal promoter or other cis-acting elements (AAV terminal repeats
[TR]) were contributing to this activity. In the present study, we
identify that the AAV TR, minus the tetracycline-responsive minimal CMV promoter, will initiate mRNA expression from vector templates. Using
deletion analysis and specific PCR-derived TR reporter gene templates,
we mapped this activity to a 37-nucleotide stretch in the
A/D elements of the TR. Although the mRNA derived from the
TR is generated from a non-TATA box element, the use of mutant templates failed to identify function of canonical initiator sequences as previously described. Finally, we demonstrated the presence of green
fluorescent protein expression both in vitro and in vivo in brain by
using recombinant virus carrying only the TR element. Since the AAV
terminal repeat is a necessary component of all recombinant AAV
vectors, this TR transcriptional activity may interfere with all
regulated expression cassettes and may be a problem in the development
of novel TR split gene vectors currently being considered for genes too
large to be packaged.
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TEXT |
Recombinant adeno-associated virus
(AAV) vectors have been studied in both large- and small-animal models
and demonstrate safe and effective gene transfer (22).
Importantly, AAV vectors transduce tissues for long periods, lasting
over 1.5 years in the muscle and central nervous system of rodents
(27, 37). Such long-term expression suggests the possibility
of permanent gene transfer in human gene therapy. While this is
essential for the correction of genetic diseases, it raises the new
concern of properly regulating the therapeutic gene product.
Appropriate implementation of long-term gene expression requires the
ability to control the expression of virus-delivered transgenes using
either endogenous cell-type-specific promoters or exogenous regulation
systems. One exogenous system, the tetracycline-regulated system, has
been studied extensively in vitro and in vivo and has been shown to
give 100- to 1,000-fold levels of regulation between the off and on
states (11, 15). By incorporating this system in an AAV
vector, we demonstrated control of reporter gene expression in the
brain (13), with a range of regulation of 28-fold in vitro
and 10-fold in vivo. Two other groups have generated tetracycline-regulated AAV vectors that demonstrated control of the
secreted erythropoietin protein after vector injection into rodent
muscle, with similar success (1, 18). While the ability to
obtain regulated gene expression in these studies was significant, the
results were vastly different from those published using transfections in vitro or analysis of tetracycline regulation in transgenic animals
(100- to 1,000-fold). It is clear that many human gene therapy and
experimental situations will require tighter control. Our data suggest
that the decreased regulation is most likely to be due to a high
background level of activity in the "off" state. Since AAV vectors
are derived from minimal cis-acting sequences (145-bp
terminal repeats), only a limited number of components of the vector
could be responsible for this activity. This includes a portion of the
tetracycline-regulated promoter, the cytomegalovirus (CMV) minimal
promoter, and/or the AAV terminal repeats (TR). Background expression
from the minimal promoter can be significant, and new versions of the
tetracycline-regulated system are showing promise in resolving this
concern (10). However, several lines of evidence suggest
that the AAV TR may also be a source of transcriptional elements
(8, 9) that could adversely influence regulation from the
tetracycline-regulated AAV vector.
Since the AAV TRs contain all the cis-acting sequences
necessary for replication and packaging of recombinant DNA as well as
for mediating the integration of the viral DNA into the host genome,
they cannot be deleted. These sequences have at least two different
transcriptional activities in tissue culture (8, 9). Flotte
et al. (9) removed the first 83 or 140 nucleotides (nt) of
the AAV TR located adjacent to a TATA box-containing promoter and
reduced expression by about 50% after plasmid transfections into an
epithelial cell line. This suggested that an enhancer activity was
located somewhere in the first 83 nt of the AAV TR. In a second study
(8), they demonstrated that the AAV TR was able to initiate
gene expression in a promoterless construct after plasmid transfection
or after AAV vector infection in vitro. Thus, the TR appears to have
both promoter and enhancer activities in tissue culture. While these
studies identified enhancer and transcriptional activities, further
identification of the critical sequences responsible for these
observations have not been forthcoming, probably due to the difficulty
of characterizing the AAV TR.
The TR contains significant secondary structure (see Fig. 2A), which
may also be responsible for the reported transcriptional activity. Due
to palindromic sequences within the TR, it is the only double-stranded
portion of an otherwise single-stranded AAV genome when packaged
(7). This sequence, which has a G+C content of over 80%,
folds back on itself to form a T-shaped structure composed of two small
palindromes, B and C, contained within a larger
palindrome, A (see Fig. 2A). An additional sequence, the D element, is also part of the TR but is single stranded in
the virion. The D element contains the packaging signal (X. Xiao and R. J. Samulski, unpublished data), and the A
stem contains sequences for an AAV Rep protein binding element (RBE),
while the junction between these two elements defines the terminal
resolution site (trs) required for viral DNA replication
(2, 14, 21, 28). In addition to the general secondary
structure which may attract transcriptional proteins (30,
34) and as initially proposed by Flotte et al. (8), we
identified two sequences in the A (nt 88 to 95) and
D (nt 126 to 133) regions which have significant homology (5 of 7 bases for each) to an initiator sequence (see Fig. 2A). Initiator
sequences are characterized as minimal sequences that are sufficient to
mediate transcript initiation in the absence of a TATA box (25,
26). Flotte et al. (8) suggested that a putative
initiator sequence may generate the promoter activity seen in their
studies with AAV. It is possible that these sequences in the TR are
sufficient to produce transcriptional activation of a downstream gene
in vitro and in vivo. To better characterize these observations, we
initiated studies aimed at defining the role of the TR on the
tetracycline-responsive vectors we used in vivo.
TR-initiated expression.
To better define the reason for
inefficient regulation of our tetracycline-responsive vector, a series
of promoterless plasmids were derived from TR/CMV/GFP by restriction
enzyme digestion (Fig. 1A). TRGFP-p is
identical to TR/CMV/GFP except that it is missing the CMV promoter that
is typically used to express the reporter gene, green fluorescent
protein (GFP). Superfect transfection (1 µg of plasmid to 4 µl of
Superfect reagent [Qiagen]) of TRGFP-p into approximately 2 × 105 HEK 293 cells (data not shown) or 1 × 105 HtTA-1 cells (HeLa cells stably transfected with the
tetracycline transactivator [a gift from H. Bujard]) expressed GFP at
a low level (Fig. 1B). In HtTA-1 cells, TRGFP-p produced detectable expression in 5 to 10% of the cells that showed expression with TR/CMV/GFP and at a lower intensity per cell. To ensure that this activity was not due to other elements (e.g., the simian virus 40 [SV40] intron sequence or plasmid readthrough by the neomycin gene
promoter), TR-pint and TR-pneo were constructed to remove these
sequences, respectively. Transfection of each construct produced gene
expression at a level equal to or slightly greater than that for
TRGFP-p. Thus, the AAV TR in plasmid form (Fig. 2) appears sufficient to produce
low-level transcriptional activity in tissue culture. These
observations corroborated the published results of Flotte et al.
(8) using chloramphenicol acetyltransferase reporter gene in
293 cells.
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FIG. 1.
(A) Plasmids used for transfection. All promoterless
plasmids are derived from TR-CMV-GFP by restriction digest deletion of
the regions indicated. TR-CMV-GFP was made from pTRUF 2 (39), a kind gift from N. Muzyczka, by removing the GFP gene
and replacing it with the EGFP gene (Clonetech) using convenient
restriction enzymes. pCMV, cytomegalovirus immediate-early promoter;
int, SV40 intron; pro, TK promoter plus tandem repeats of a
polyomavirus enhancer; neo, neomycin resistance gene. (B)
Photomicrographs of HtTA-1 cells 24 h after transfection of the
indicated plasmid. Exposure times are not equivalent for all
photomicrographs.
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FIG. 2.
(A) Diagram of the TR in genome form as
packaged in the virion (top) and as cloned into a plasmid (bottom). The
TR forms a T-shaped structure containing one continuous strand in the
virion. In the plasmid the TR is double stranded and contains the
A, B, and C regions and their
complements, A', B', and C'. The
location within the terminal repeat, intron, or GFP gene of 5' primers
1 to 7 are also shown. Primer 1 spans  13 to +6 of the GFP gene,
primer 2 spans 3 to +15 of the SV40 intron (sequences 5' of the GFP
gene and the intron are polylinker regions), primer 3 spans nt 139 to
145 of the TR plus 15 nt of the polylinker, primer 4 spans nt 110 to
127 of the TR, primer 5 spans nt 104 to 122, primer 6 spans nt 88 to
105, and primer 7 spans nt 60 to 77. (B) RT-PCR products were amplified
using primer 2 from RNA isolated from transfected 293 cells and
analyzed by agarose gel electrophoresis. Lanes: 1 to 3, TR-pneo (Fig.
1)-transfected cells; 4 to 6, TR/CMV/GFP-transfected cells; 7 to 9, mock-transfected cells that were spiked with TR-pneo DNA at cell
harvest; 1, 4, and 7, untreated mRNA; 2, 5, and 8, RNA samples treated
with S1 nuclease prior to RT-PCR; 3, 6, and 9, RNA samples treated with
NaOH to degrade RNA prior to RT-PCR. The 3' primer paired with primer 2 was located in the GFP gene: GFP-RT2 (nt 145 to 126 of the EGFP gene).
Either GFP-RT2 or GFP-RT1 (nt 19 to 3 of the EGFP gene) was paired with
the 5' primers from panel A, as delineated in Table 1.
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Mapping TR promoter activity.
Localization of the promoter
activity within the TR was investigated initially by primer extension
analysis of the mRNA start site. Our technique produced clear
identification of a CMV promoter start site but inconclusive results
with the TR-generated transcripts, presumably due to low levels of
mRNA. Therefore, reverse transcription-PCR (RT-PCR) analysis of mRNA
from plasmid substrates transfected into 293 cells was initiated to
determine an approximate start site. Products of mRNA amplification
were distinguished from any input DNA amplification by use of an
intron-containing construct. The DNA PCR products also served as an
internal control for the reaction (since they were 100 bp larger than
the spliced mRNA product). A series of primers (Fig. 2) were used to
amplify products of spliced mRNA transcripts and 100-bp-larger products
from unspliced RNA or residual DNA (TR-pneo). Primers located 5' of the
initiation site would produce products only of unspliced length,
indicating amplification of residual DNA and not processed mRNA. mRNA
was extracted using Trizol reagent (Gibco BRL) from 10-cm plates of TR-pneo-transfected 293 cells and Dynal poly-T magnetic beads (as
specified by the manufacturer). The mRNA was then subjected to RT with
poly(T) primer and avian myeloblastosis virus reverse transcriptase for
90 min at 42°C. The RT products were PCR amplified with a series of
5' TR primers and one of two 3' primers located in the GFP gene, under
conditions optimized to detect low transcript numbers (1 U of
Perkin-Elmer Taq polymerase, 0.2 µM each primer, and 0.25 mM each deoxynucleoside triphosphate per 50-µl reaction mixture with
40 cycles of amplification). Gel electrophoresis analysis of the PCR
products revealed both spliced and unspliced products from primers 2 through 6 (Fig. 2B, lanes 1, 4, and 7; Table
1), while mRNA preparations from
mock-transfected cells, spiked with DNA, yielded only unspliced sized
products for these primers. Although the TR has many repetitive
sequences, no internal TR amplification (indicated by smaller bands)
was seen with these primers. Primer 7, located in the B/C region of the
terminal repeat, was unable to amplify any products of the spliced or
unspliced size, even with the use of a series of annealing temperatures and a GC-rich sequence optimization reagent (GC melt; Clontech). This
primer is located in a GC-rich region in the middle of the secondary
structure of the terminal repeat (Fig. 2A, virus form). Previous
studies have determined that this secondary structure interferes with
PCR amplification (23, 38) and is probably blocking
amplification from primer 7. Likewise, primers located beyond the
terminal repeat in the plasmid were unable to consistently amplify
products of either spliced or unspliced size, confirming previous
observations (28, 38) and making the additional localization of transcript initiation beyond this secondary structure intractable. To ensure that the spliced products detected with the initial primers
were amplified from RNA, mRNA preparations were treated with 30 U of S1
nuclease or 0.3 M NaOH for 1 h at 37°C in the presence of
glycogen and then ethanol precipitated before being subjected to the RT
step. Since S1 nuclease digestion and NaOH digestion preferentially
degrade single-stranded nucleic acids and RNA, respectively, only
products derived from DNA (unspliced size) were detected after these
treatments (Fig. 2B, lanes 2 and 3; Table 1). This approach confirmed
that mRNA transcripts originated as far back as the 5' end of the
A element (primer 6). However, because of our inability to
amplify any product upstream of primer 6, additional determinations of
any other 5' initiation sequences were not possible using this method.
To further evaluate the TR promoter activity and the minimal sequences
necessary for mRNA expression, PCR amplification was
used to generate
minimal TR/GFP cassettes, which were purified,
transfected into HtTA-1
or 293 cells, and assayed for GFP activity.
A number of the primers
used to map mRNA initiation from plasmid
vector templates, along with
several additional 5' primers paired
with a 3' primer located after the
poly(A) signal of the GFP gene,
were used to generate these
miniexpression cassettes (Fig.
3A).
PCR
products were amplified from plasmid templates (TR-pneo or
TR-pint)
under conditions similar to those for RT product amplification.
These
products were amplified a second time to obtain enough material
for
transfection into duplicate wells of a 12-well plate. After
PCR, the
products were digested with
DpnI, which digests input
plasmid DNA, for 2 h and purified on Sephadex G-25 spin columns
to
remove residual deoxynucleoside triphosphates and primers.
Products were quantified by optical densitometry, and approximately
1.5 pmol of each product was transfected into 293 cells or HtTA-1
cells
using superfect (in quantification experiments, equivalent
amounts [in
picomoles] of each product were used). Two measurements
were used to
determine relative expression: the number of GFP-positive
cells and
relative whole-field fluorescence (Fig.
3A). Templates
containing only
the intron (amplified using primer 2) and no TR
sequences produced a
few visible cells (the number of GFP-positive
cells was 18.9% ± 5.9%
of the number obtained with control primer
5) after transfection (Fig.
3). This expression could be due to
nonspecific or random initiation
within the SV40 intron. However,
the levels significantly increased
(the number of GFP-positive
cells was 91.8% ± 9.1% of the number
with control primer 5) after
transfection of a PCR template that
included sequences of the
TR, specifically the
A/D junction
(primer 4). Since intron-minus
templates generated from primer 4 also
gave positive expression
(Fig.
3B), we concluded that the intron was
not required for this
increase. PCR templates carrying a small portion
of the
D element
(primer 3) produced slightly lower
expression than that obtained
with primer 2 (intron primer). This
observation was very reproducible,
indicating the ability of the intron
sequences to initiate nonspecifically
or the potential interference
from the cellular protein(s) previously
shown to bind the AAV
D element (
17). As an additional control,
PCR
templates generated from a primer containing the GFP start
codon
(primer 1) gave no expression (Fig.
3A), supporting the
premise that
transcriptional activation observed in this assay
was due primarily to
upstream sequences in the TR. Since templates
derived from primer 4 included the putative
D element initiator
sequence, two
additional variants of primer 4 carrying severe
mutations of the
canonical initiator sequence as previously described
were tested
(
25,
26). PCR products generated from these mutations
had no
consistent effect on GFP expression, indicating that the
initiator
sequence, as currently identified, is not responsible
for TR transcript
initiation observed in our assays. Likewise,
hybrid templates,
generated using primer 8, which carries the
A element
initiator-like sequence coupled to the insufficient
primer 3 sequence
amplified from an
A element-deleted plasmid
(TR-pneo/mp),
did not increase GFP expression beyond that observed
with primer 3 alone. This suggests that the putative
A element
initiator
is not sufficient to initiate transcription under these
conditions.
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FIG. 3.
(A) 5' PCR primers to the indicated regions were paired
with a GFP poly(A) region primer to amplify products containing
portions of the TR or intron and the entire GFP gene. After
transfection of the PCR products into HtTA-1 cells, relative positive
cell counts and relative field fluorescence were determined from a
random field with a 10× objective (eight fields per well, two wells
per condition). Cell counts were obtained for three independent
experiments and are expressed as a mean percentage of that obtained for
primer 5 ± the standard error. Whole-field fluorescence, averaged
from two of the same experiments, was corrected for cell
autofluorescence by subtracting the background of mock-transfected
cells and are also expressed as a percentage of the intensity obtained
with primer 5. Primer 4 M1 contains nt 110 to 138 of the TR, except
that CCA at nt 128 to 130 was changed to GGG. Primer 4 M2 contains nt
110 to 138 of the TR, except that CT at nt 126 and 127 was changed to
GA. Primer 8 consists of nt 72 to 93 linked to nt 139 to 145 of the TR
plus 15 nt of polylinker (primer 3) and amplified from TR-pneo/mp.
TR-pneo/mp is derived from TR-pneo by digestion with MscI
and PstI, blunting, and then religating the backbone with
the GFP fragment. This plasmid contains only nt 119 to 145 of the TR.
*, two of three experiments with this primer showed no positive cells
(in one experiment, two faint green cells of unknown origin were
detected);  , because whole-field measurements cannot distinguish
between specific GFP fluorescence and cell autofluorescence due to cell
death, these values contain more variability than the cell count
measurement. (B) Photomicrographs of 293 cells 48 h after
transfection of the PCR products illustrated above each photograph.
Exposure times were equivalent. All PCR products were derived using the
same 3' poly(A) signal primer. Plasmid and 5' primers were as follows:
I, TR/CMV/GFP (Fig. 1) with primer 4; II, TR-pneo (Fig. 1) with primer
2; III, TR-pneo with primer 4; IV, TR-pint (Fig. 1) with primer 4.
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We determined that sequences of the TR amplified by primer 4 (nt
109 to 145) are sufficient to express GFP from a linear DNA
template in
a transfection assay. To ensure that these results
were not related to
the total length of the 5' sequences, our
PCR products generated with
primer 8 (Fig.
3A) contained the same
amount of 5' DNA as did templates
generated with primer 4. Even
under these conditions, primer 8-derived
templates did not yield
efficient GFP expression. Although the TR
sequence appeared to
contain a classic initiator sequence, as
determined by sequence
homology of published initiator elements,
results obtained with
PCR templates generated from specific mutant
initiator primers
strongly suggest otherwise. Our study shows that the
AAV-TR element
has RNA initiation capacity, maps to nt 109 to 145, and
does not
use a classic initiator-like element to generate this
activity.
No other obvious sequence motif could be identified as a
candidate
for mRNA initiation; therefore, further experiments are
required
to determine the exact mechanism of mRNA initiation from this
region. While our experiments clearly demonstrate a role for this
sequence in the PCR-based transfection assay, we extended these
observations by testing for GFP expression by generating a
promoter-minus
recombinant AAV and assaying in
vivo.
AAV-TR vector expression in vivo.
It is clear from the
experiments above that in plasmid or PCR-derived linear DNA form, the
AAV terminal repeat can initiate the transcription of a reporter
gene (the PCR template gives 2.9% of the expression obtained with the
CMV template). However, this expression is only a concern if the TR
promoter activity is also present when delivered in a recombinant
virus. Therefore, virus AAV TR-pneo was generated from TR-pneo by the
University of North Carolina vector core facility by standard methods
(12). AAV TR-pneo was microinjected into two regions of the
brains of rats and analyzed as described by Haberman et al.
(13). GFP expression was seen 2 weeks later in both regions
injected: the hypothalamus and the inferior colliculus (Fig.
4). These results demonstrate that the
AAV TR can generate reporter gene expression from a virus vector
template as well as in plasmid form. More importantly, in vivo
expression indicated that this promoter activity is not limited to
tissue culture cells and may influence gene expression from rAAV
vectors in the intact animal. Based on all our in vivo data established
to date, we typically observed from the TR-only vectors about 2 to 5%
of the level of expression seen with a wild-type CMV vector. This small
amount of activity may arise as a result of AAV DNA integration 3' of
endogenous promoters. However, GFP expression was similar in all cells
in all animals, which would not be expected if the expression was due
to random integration. Supporting this conclusion, AAV has been
demonstrated to last for long periods as episomes in muscle (5,
33). More recently, AAV TR vectors coinfected with enhancer
sequences demonstrated significant expression from episomal templates
in vivo (6, 29).
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FIG. 4.
In vivo expression of TR-driven GFP. Virus made from a
TR-pneo (AAV TR-pneo) was injected into the inferior colliculus and the
hypothalamus of rats; 2 weeks later, the brains were sectioned and
analyzed for GFP fluorescence by fluorescent microscopy. Many cells
from each animal are visible in both regions of the brain.
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Transcriptional activity within terminal repeat sequences of viruses is
not a novel discovery. This activity is essential
for the retrovirus
life cycle (
32) and has been detected in
other DNA viruses
such as adenovirus (
19,
24). Observation
of transcriptional
activity from the AAV TR supports previous
observations seen in rabbit
and monkey lung (
4,
20). The
exact role that this activity
may play in wild-type AAV infection
remains unknown. In addition, it is
not clear if the activity
is restricted to specific cell types or
ubiquitous. We demonstrated,
using plasmid and PCR linear templates,
that the primary sequences
necessary for activity in vitro are
contained in the
D element
and the 3' half of the
A element. However, we did observe transcripts
(only from
RT-PCR) which initiated 5' to the
A/D junction, suggesting
that the GC-rich secondary structure of the terminal repeat may
also
influence gene expression. In addition, neither of the two
putative
consensus initiator sequences, as described in the literature,
appear
to be responsible for the observed activity. Since the
TR contains all
of the essential functions required to generate
AAV vectors (Rep/RBE
binding,
trs resolution, origin of replication,
and
packaging signals), we were not able to test the mini-TR expression
cassettes in the context of a virus. However, our in vivo studies
confirm that TR promoter activity is a very real phenomenon that
can
occur in the intact animal. This observation may partly explain
some of
the published observations for neurotropism of AAV transduction
in the
brain (
3). In addition, a loss of tissue-specific
expression,
when in the context of the AAV vector (N. Muzyczka,
personal communication)
may be related to TR transcriptional activity
and repression.
The implications of TR promoter activity on the use of
AAV vectors
will probably depend on the gene to be expressed, the
promoter
used, and the location of virus transduction in vivo. Our
studies
suggest that the most prominent impact of this activity will be
on the regulation of gene expression. Attempts to reduce expression
completely in transduced cells through the use of regulated
promoters
or cell-type-specific promoters may be influenced by TR
promoter
activity. Current vectors may have to be modified before they
can be used effectively in such
instances.
Although we have not mapped the AAV TR transcriptional activity to the
nucleotide level, this region of the AAV TR has been
extensively
characterized regarding its function in replication
and packaging of
the viral DNA. The
A region contains the AAV
RBE and the
trs, both of which are required for virus replication.
The
Rep protein binds to the terminal repeat binding element and
nicks the
DNA downstream at the
trs during replication of the
genome
(
28). The nucleotide requirements for terminal resolution
have recently been mapped to a region that spans the
A/D
junction
(
2). One could try to mutate the TR in an effort to
abolish
the promoter activity; in fact, single-nucleotide mutations can
be made without eliminating nicking of the correct site, but small
decreases in nicking efficiency could have drastic effects on
the
ability to produce high-titer recombinant virus. In addition,
spacing
mutations have been generated, indicating that the correct
distance
from the RBE to the
trs is crucial for AAV replication
(
28,
36). Results obtained with deletion mutations through
the
D sequence stress the importance of this region in
packaging
(
35; Xiao and Samulski, unpublished).
Given these data, it is
unlikely that elimination of the TR promoter
activity by traditional
mutagenesis analysis will yield viable AAV
vectors. Such mutations
will probably impair viral replication and/or
packaging. Since
the sequences at the
A/D junction are
highly conserved between
AAV serotypes 1 to 4 and 6, with lower
conservation for type 5
(
2), TR transcriptional activity may
exist for all serotypes.
Therefore, an underlying role for this
activity may be essential
for the virus life cycle (e.g., initiating
wild-type virus replication
or promoting templates suitable for
integration). Regardless of
the function in wild-type virus, this is
clearly an uncontrolled
activity that is part of the current AAV
vectors.
It is of interest that the many strategies aimed at resolving the small
packaging constraint of AAV have not considered the
impact of TR
promoter activity. Numerous studies are now suggesting
the use of AAV
"split vectors" to overcome the size limitation
(
6,
16,
29). The typical approach with these systems depends
on one
vector carrying the 5' half of a specific gene flanked
by the promoter
and splice donor and another vector carrying the
remaining 3' portion
of the gene flanked by the splice acceptor
and a poly(A) signal.
Expression of a functional gene product
occurs only after recombination
or reannealing through a common
TR sequence from the appropriate two
split gene vectors. While
this and other similar strategies effectively
double the packaging
size of AAV, our TR data would indicate that such
approaches may
also increase the risk of expressing truncated gene
products from
TR transcriptional elements. This could lead to
expression of
aberrant proteins that may have transdominant negative
activity,
elicit an immune response, or interfere with pathways for
normal
secretion of the therapeutic protein (e.g., factor IX). Since
the current AAV vectors are noted for safe, long-term expression
with a
lack of immune response, these new vectors may have to
incorporate
other components to eliminate the risk of unwanted
expression from the
TR.
Although the elimination of AAV TR promoter activity by mutation is not
likely to yield viable virus, the use of insulator
sequences adjacent
to the TRs may provide an alternative. Insulator
sequences that block
the transcriptional activation of downstream
promoters by enhancers
have been tested in other viral vectors
(
31). Although
adding insulator sequences to an already space-constrained
vector may
seem counterproductive, the significant benefit of
greater control of
gene expression will be necessary in some circumstances.
Small
insulator sequences (approximately 200 bp) have been identified
that
may resolve the size constraint in rAAV vectors (
31).
Alternatively,
insertion of a poly(A) signal between the TR and the
cassette
of interest may blunt the unwanted expression via the TR. It
appears
that a number of approaches can be tested to eliminate residual
transcriptional activity from the TR, and these new TR expression
minus
vectors should have a significant impact on the ability
to utilize
exogenous regulation systems and cell-type-specific
promoters within an
AAV vector
context.
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ACKNOWLEDGMENTS |
This work was supported by The Epilepsy Foundation (fellowship to
R.P.H.), NINDS grant NS35633 to T.J.M., and NIH grant DK51880 to R.J.S.
We thank the UNC vector core facility for production of recombinant AAV
vectors. In addition, we recognize Barrie Carter's contribution to the
discovery of promoter-like activity of the AAV TRs.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: CB 7352, 7119 Thurston Bowles, UNC Gene Therapy Center, University of North Carolina, Chapel Hill, NC 27599. Phone: (919) 962-3285. Fax: (919) 966-0907. E-mail: rjs{at}med.unc.edu.
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