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GENE THERAPY
From the Department of Pediatrics and Genetics, School
of Medicine, Stanford University, CA.
We have developed a new helper-dependent (HD) adenoviral
vector FTC that contains 3 cis-acting
sequences as stuffer DNA: a human fragment of alphoid repeat DNA,
matrix-attachment regions (MARs), and the hepatocyte control
region enhancer. To determine the most robust human coagulation
factor IX (hFIX) expression cassette in an adenovirus, we first tested
different hFIX expression sequences with or without flanking MARs in
first-generation adenoviral vectors. After intravenous infusion of the
vector, serum levels of up to 100 000 ng/mL hFIX (normal level, 5000 ng/mL) were obtained at nontoxic doses. In order to make a direct
comparison, a first-generation and a gene-deleted vector with identical
hFIX expression cassettes were constructed. Both first-generation and
HD adenovirus-treated animals demonstrated a threshold effect in a
dose-response study. With the administration of 2 × 109
transducing particles of either vector, supraphysiological serum levels
of hFIX were obtained, with the highest expression (41 000 ng/mL)
occurring during the first 2 months after injection. The serum factor
IX concentrations, while remaining in the therapeutic range, slowly
declined by 95% over a period of 1 year. At this dose, interleukin-6
and tumor necrosis factor- Adenoviral vectors lacking all viral genes are a
promising tool for safe and efficient gene transfer in vitro and in
vivo.1-3 Adenovirus has several advantages over other
viral-based gene therapy approaches, including the ability to produce
high titers, efficient infection of a broad range of cell types, and
the ability to infect dividing and nondividing cells.4-7
However, further development of adenoviral vectors is necessary to make
them safer and more efficient. First-generation E1-deficient
adenoviruses show toxic effects owing to the production of immunogenic
viral proteins. The Cre-loxP helper-dependent (HD) system was developed to generate recombinant adenoviruses in which all viral coding sequences have been deleted.8,9 The helper virus used in this system provides all adenoviral gene products required for replication and packaging of the vector to be supplied in
trans. The packaging signal of these helper viruses is
flanked by loxP sites to inhibit packaging of the helper-virus genome
in stable Cre recombinase-expressing cells. Recombinant adenovirus
produced by the Cre-loxP HD system allows for transfer of up to 35 kilobases (kb) DNA with long-term expression in
rodents.1,2 Recent studies suggest that the stuffer DNA
used for HD vectors may play a major role in production and persistence
of transgene expression. HD vectors with stuffer DNA of prokaryotic
origin result in transient transgene expression.10
Additionally, it was demonstrated that the nature of the stuffer DNA
cloned into the HD vector affects its replication during production and
the expression levels of the transgene after transduction in
vivo.11
The new HD vector presented herein contains a matrix-attachment
region (MAR) and a centromeric fragment as "stuffer" DNA. MARs are
AT-rich noncoding DNA sequences that often contain topoisomerase II
cleavage sites. MARs are believed to be involved in chromatin loop
formation by anchoring the DNA to the nuclear matrix, a protein network
in the nucleus. Furthermore, MARs may function as insulators for
single-copy expression cassettes12 and are capable of
increasing expression levels from a transgene.13 The
centromeric fragment in our high-capacity vector was a 16.2-kb alphoid
repeat DNA fragment from human chromosome 17, which was shown to
function as an origin of replication in vitro.14,15
First-generation adenoviruses with different human coagulation factor
IX (hFIX) expression cassettes were previously generated, and
expression levels from 1 to 5 µg/mL were detected in
vivo.16-18 The hFIX expression cassettes selected for this
study were based on our recent study using a naked DNA liver delivery
approach,19 which demonstrated that expression cassettes
containing the hFIX minigene, the hepatocyte control region (HCR), and
the apolipoprotein E (ApoE) enhancer as cis-DNA elements
show persistent expression at levels of up to 1 µg/mL hFIX in vivo.
Therefore, we decided to test these robust expression cassettes in the
context of adenoviral vectors. Importantly, while higher levels of gene
expression have been demonstrated when an HD vector is used in place of
a first-generation vector,1,3 none of these studies
directly compare the same expression cassette in both vector systems.
Construction of plasmid pAdFTC and pAdFTC/hFIX
The plasmid adenoviral vector FTC (pAdFTC) for adenoviral
production is based on the plasmid pDYAL containing a 16.2-kb fragment of alphoid repeat DNA from human chromosome 17.15 The
alphoid repeat DNA was flanked by a 4.2-kb fragment containing the left terminus of adenovirus type 5 and 2 copies of the Ig A shuttle plasmid pBS-P/P, based on pBSK/S (Stratagene) was constructed
with an MCS between a PacI and a PmeI recognition site by changing the KpnI site of pBSK/S to PacI
and the SacI site to PmeI. The SpeI
human FIX minigene fragment from pBSh7hF9mgbpA19 was cloned
into the SpeI site of pBS-P/P. The
PacI/PmeI hFIX fragment was cloned into the
PacI/PmeI site of AdFTC, resulting in
pAdFTC/hFIX.
Production of high-capacity adenoviruses using the HD vector
AdFTC
Titering of HD vectors The number of transducing particles of AdFTC/hFIX in HD vector preparations was determined as followed: Hela cells were infected with different volumes of the HD vector preparation. For comparison, Hela cells were infected at different defined multiplicities of infection (MOIs) with the first-generation adenovirus hFIX (fgAdhFIX) with the same expression cassette to generate a standard curve. Cells were incubated for 3 hours, and the genomic DNA was isolated; this was followed by a Southern blot probed with an hFIX complementary DNA (cDNA) probe (HindIII/EcoRI fragment from pAAVCM2). The intensity of the bands for the HD vector was compared with the standard curve.Generation of first-generation adenoviruses The BamHI site of pHM5 was changed to SpeI, resulting in pHM5SpeI.21 The PstI Ig MAR fragment from pPCRMAR was cloned into pHM5, and the
XbaI chicken lysozyme MAR (ChMAR) fragment from pBS-2x(B-1-X1)13 was ligated into the XbaI site
of pHM5. The SpeI fragment from pBSh7hF9mgbpA was cloned
into the XbaI site of pHM5, and the
I-CeuI/PI-SceI fragment containing the hFIX minigene was
cloned into the I-CeuI/PI-SceI site of pAdHM4.
First-generation adenoviruses were produced and amplified as previously
described.21
Isolation of primary hepatocytes Primary hepatocytes were isolated as described.22 In brief, primary mouse hepatocytes were obtained by collagenase perfusion, and hepatocytes with greater than 90% viability were plated at a density of 6 × 105 on 6-well dishes for the first 6 hours in Dulbecco modified Eagle medium with 10% fetal calf serum and afterward in Williams E medium with 10% fetal calf serum.Blood analysis Mouse serum hFIX antigen levels were determined by enzyme-linked immunosorbent assay (ELISA) assay.23 The normal human serum level was 5000 ng/mL. Serum glutamic-pyruvic transaminase (SGPT) (alanine aminotransferase activity) assays were performed by using a diagnostic kit for colorimetric determination of SGPT (Sigma [St Louis, MO] procedure no. 505-OP). To measure the serum levels of interleukin 6 (IL-6) and tumor necrosis factor-
(TNF-), Pharmingen's BD OptEIA human IL-6 ELISA kit
(catalog no. 555 240) and Pharmingen's BD OptEIA human TNF- ELISA
kit (catalog no. 555 268) were used (San Diego, CA).
Animal studies Mice were injected via tail vein with different amounts of adenoviral vector diluted in phosphate-buffered saline (PBS). To permit comparisons between the present study and those already published, Table 1 converts the number of transducing units per mouse to the total amount of transducing units and viral particles per kilogram body weight.
Phenotypic correction studies C57Bl/6 hemophilia B mice were used.24 Three mice per group were injected with 1 × 109, 5 × 108, and 1 × 108 transducing units of AdFTC/hFIX or fgAdhFIX. On day 7, a 0.5-cm section of the tail was clipped from each mouse to measure the bleeding time (time until bleeding stopped).Southern blotting For genomic DNA isolation, the liver was removed and homogenized, and a 100-mg portion was used for DNA extraction as previously described.25,26 Then, 20 µg genomic DNA was digested with HindIII, run on a 0.8% agarose gel, and electrotransferred to a Hybond membrane (Amersham, Buckinghamshire, United Kingdom). The blots were hybridized with an-32]-deoxycytidine triphosphate (dCTP)-labeled cDNA
hFIX probe (HindIII/EcoRI fragment from pAAVCM2),
by means of a random priming kit (Stratagene).
Different hFIX expression cassettes and cis-DNA elements in first-generation adenoviruses In earlier studies, we compared different hFIX expression cassettes by in vivo hydrodynamic delivery of naked plasmid DNAs into mouse liver.27 The 2 most robust expression cassettes contained the ApoE enhancer and HCR, the human alpha antitrypsin promoter (hAAT-p), and the hFIX minigene (which includes a portion of the first hFIX intron and the bovine growth hormone gene polyadenylation signal) with or without the 3' untranslated region (Figure 1A-B) and were subsequently used in the current studies.
To determine if a MAR would affect gene expression, we constructed
different vectors containing the hFIX expression cassette with or
without the 3' untranslated region, and variations of 2 well-characterized MARs: the chicken lysozyme 5'MAR13 and the Ig
Development of an HD vector expressing hFIX We generated a new high-capacity adenoviral vector (AdFTC) that contained 3 cis-acting sequences as stuffer DNA: a human centromeric fragment containing alphoid repeat DNA, IgMAR, and the
HCR for hepatocyte-restricted transgene expression. AdFTC also
contained an MCS that allowed for the production of deleted
adenoviruses with any gene of interest, ranging in size between 4 kb
and 14 kb (to keep the total size of the HD vector between 26 and 36 kb) (Figure 1E). The HD vector, AdFTC, contained the hFIX expression cassette (minigene and 3' untranslated region) that was shown to be the
most robust in the first-generation vector. The resulting HD vector
AdFTC/hFIX (Figure 1F) was produced as described in "Materials and
methods" (the helper-virus contamination after CsCl gradients was
below 0.5%), and the total amount of transducing units was determined.
Comparison of the expression levels of the HD and first-generation adenovirus containing the identical expression cassette for hFIX For all comparisons, the same number of adenoviral transducing particles (determined by Southern blot; see "Materials and methods") were used. The HD and first-generation adenoviruses (fgAdhFIX), which contained identical expression cassettes, were compared in primary hepatocytes in vitro. The data in Figure 3A show the result of transduction experiments performed with AdFTC/FIX at different MOIs while the studies in Figure 3B directly compare AdFTC/hFIX and fgAdhFIX. For example, at an MOI of 100, AdFTC/hFIX-transduced cells had hFIX levels of up to 5400 ng per 6 × 105 cells per day, 5 times more than that obtained with the first-generation vector.
To study adenoviral-mediated expression from these vectors in vivo,
2 × 109 transducing units of AdFTC/hFIX or fgAdhFIX
vector were infused into C57BL/6 mice (Figure
4A). Sustained levels of serum hFIX were
achieved in both groups of animals. Interestingly, serum hFIX was
observed more quickly in animals that received the HD adenovirus. The
highest hFIX expression levels were obtained during the first 2 months
(approximately 41 000 ng/mL for AdFTC/hFIX and approximately 11 000
ng/mL for fgAdhFIX). The expression levels stabilized for the following
4 months at approximately 15 000 ng/mL followed by a slow decline. At
1 year after injection, 5% of the original serum levels of hFIX serum
were detected. No significant increase in liver enzyme (SGPT) levels
were detected in mice treated with AdFTC/hFIX and fgAdhFIX at 1 day or
at later time points after injection (Table
3).
One potential explanation for the 95% decline in hFIX expression
levels was the development of antibodies against the transgene hFIX.
However, we performed a reverse ELISA27 and found that no
antibodies against hFIX were present (data not shown). To further investigate the source of the 95% decline in hFIX expression levels over the course of 1 year, we analyzed the vector DNA from genomic liver DNA of recipients. Three C57Bl/6 mice per group were injected with 2 × 109 transducing particles of either AdFTC/hFIX
or fgAdhFIX, and at both 5 days and 1 year after injection,
liver genomic DNA was isolated and a Southern blot was performed. We
found that the copy number of the transduced adenoviral vector DNA per
cell significantly dropped from about 4.5 copies per cell at 5 days
after injection to fewer than 0.51 copies per cell at 1 year after
injection (Figure 5).
Cytokine responses To compare the early innate immune responses, we measured serum concentrations of IL-6 and TNF- at 6, 18, 36, and 96 hours after
adenoviral delivery. Mice were injected with 2 × 109
transducing units of the first-generation vector fgAdhFIX or the HD
adenoviral vector AdFTC/hFIX. As a negative control, PBS was injected.
As a positive control for a robust IL-6 response, we injected
1 × 1010 transducing units of the first-generation vector Ad.hAAT, which expresses alpha 1-antitrypsin
cDNA under the transcriptional control of the Rous sarcoma virus
promoter.26 At a dose of 2 × 109
transducing units, serum IL-6 concentrations were higher for fgAdhFIX
compared with the HD vector AdFTC/hFIX (Figure
6A). The first-generation vector had a
peak IL-6 level at 18 hours after injection (up to 2200 pg/mL IL-6).
AdFTC/hFIX injection resulted in a slight increase in IL-6 levels
compared with the vehicle control. In mice that received
1 × 1010 transducing units of Ad.hAAT, serum IL-6 levels
reached 7500 pg/mL (Figure 6A). After 96 hours, IL-6 serum
concentrations returned to normal levels in all groups.
The peak serum TNF- Dose-response curve for AdFTC/hFIX and fgAdhFIX To determine the lowest amount of the high-capacity AdFTC/hFIX and first-generation fgAdhFIX vectors required to generate therapeutic serum levels of hFIX (approximately 50 ng/mL), a dose-response analysis was performed. Mice were injected with AdFTC/hFIX or fgAdhFIX at 5-fold dose increments ranging from an MOI of 10 (1 × 109 transducing particles) to 0.01 (1 × 106 transducing particles).Although for both vectors there was a threshold dose response,
gene expression became detectable at a lower dose in the HD adenovirus-treated animals (Figure 7).
On day 7 after injection, up to 29 000 ng/mL serum hFIX was measured
in animals receiving an MOI of 5 and 10 or 5 × 108 and
1 × 109 transducing particles of AdFTC/hFIX. At the
lower MOIs (MOI 1 and 0.5), therapeutic levels with serum hFIX
concentrations of up to 1200 ng/mL were still detected. In contrast,
the first-generation adenoviral vector resulted in serum hFIX
concentrations of 500 ng/mL and 150 ng/mL hFIX at MOIs of 10 and 5, respectively, which is about 58 times less than that obtained with the
HD vector at the same MOIs. At the lower MOIs (MOI 1 and 0.5),
no therapeutic serum levels of hFIX (less than 50 ng/mL) were detected
with the use of the first-generation fgAdhFIX vector at 7 days after
injection.
Phenotypic correction in hemophilic mice after injection of AdFTC/hFIX and fgAdhFIX To test for phenotypic correction of the bleeding diathesis, C57Bl/6 hemophilia-B mice were injected with 1 × 108, 5 × 108, or 1 × 109 transducing particles of AdFTC/hFIX, or 1 × 109 transducing particles of fgAdhFIX. At 3 days after injection of the HD adenovirus, hFIX serum concentrations ranged from 34 000 ng/mL at the highest dose to 3300 ng/mL at the lowest dose (Table 4). On day 6 after injection, a tail-clipping assay was performed. Bleeding times were substantially reduced from no clotting (longer than 30 minutes) in vehicle-treated mice to 3.1 ± 0.5 minutes for AdFTC/hFIX-treated mice, values similar to those found in normal animals. Therefore, these results demonstrate that low doses of HD adenovirus AdFTC/hFIX were sufficient to correct the bleeding diathesis in hemophilic mice. Human factor IX expression levels in C57Bl/6 hemophilic mice (demonstrated in Figure 4B) showed an expression profile similar to that of normal C57Bl/6 mice (Figure 4A). As observed in normal C57Bl/mice (Figure 7B), there was a dose-threshold effect for the first-generation adenoviral vector, but the threshold level was lower than in normal C57Bl/6 mice (5 × 108 transducing units).
For gene expression studies in vivo, remarkably similar levels of transgene expression were found in the expression cassette that encodes hFIX with the use of both a first-generation and a new HD vector deleted for all viral coding sequences. This was in contrast to an HD adenoviral vector that contained the complete human alpha 1-antitrypsin gene and a first-generation vector that contained the corresponding cDNA, where large differences in the absolute amount and persistence of gene expression were observed.1 In that case, the same expression cassette was not used, and the differences in the promoter and gene/cDNA sequences probably played a major role in the difference in expression patterns. The hFIX expression cassette used in this study resulted in supraphysiological hFIX expression levels of up to 100 000 ng/mL at a low MOI from either a first-generation or a gutless adenovirus; this level was about 10 times higher than reported in other studies using adenovirus as a vehicle for hemophilia-B gene therapy.16-18 The slow decline in gene expression over a period of 8 months is not unlike that observed in nonhuman primates infused with an HD vector that contained the human alpha 1-antitrypsin gene.30 The Ig In this study, we demonstrate an in vivo dose-threshold effect for an HD adenoviral vector. More importantly, the dose threshold for the HD adenovirus was approximately 5 × 108 transducing particles or 4 times lower than a first-generation adenovirus (Figure 7). Bristol et al31 recently published an in vivo dose-threshold effect for a third-generation adenovirus (E1, E2A, and E3 deleted). The threshold level of about 1 × 1010 to 2 × 1010 particles equals our observed threshold level for the first-generation adenovirus of about 2 × 1010 particles. Using a low titer of adenovirus for gene therapy is important to decrease the potential toxicity from helper virus contaminants and/or vector capsids. Most of the current HD adenoviral vectors contain 0.1% to 0.5% contaminating adenoviral helper-virus particles, which might contribute to cell-mediated immune response and/or cytotoxicity. One potential explanation for differences in the threshold level for the first-generation and the gutless adenoviral vector is related to the de novo expression of adenoviral genes from a first-generation adenoviral vector.32-35 Lozier et al29 showed that in rhesus macaques, liver toxicity from a first-generation adenovirus expressing hFIX is dose dependent. Because of the low dose required for efficacy, our new high-capacity adenovirus with an optimized expression cassette holds great promise for gene therapy, since it should significantly reduce toxicity from helper-virus genomes and possible toxicity from capsids. The threshold effect is probably due at least in part to uptake and
degradation of viral particles by nonparenchymal Kupffer cells in the
liver26,36-38 that then directly and indirectly result in
cytokine responses. We found higher serum levels of IL-6 and TNF- Antibodies directed against the transgene product can cause a slow decline in transgene expression levels as shown for factors VIII and IX.2,27,42-44 However, we eliminated anti-hFIX antibodies as the explanation for the 95% decline in hFIX expression levels. Because the vector DNA copy number dropped significantly over the course of 1 year with each vector (Figure 5), we speculate that the natural turnover of hepatocytes and/or development of a cell-specific immune response directed against viral antigens contained within the vector particle are most likely responsible. A cell-mediated immune response was previously demonstrated in another HD adenoviral vector.2 Presumably, the viral antigens derived from the particles are processed by antigen-presenting cells prior to their recognition by the antigen-dependent immune cells. We demonstrated long-term expression of the transgene hFIX from an HD and first-generation adenovirus in C57Bl/6 mice. The hFIX transgene in the recombinant adenoviral vectors was driven by the liver-specific hAAT promoter and liver-specific enhancers. Pastore et al45 showed that the tissue specificity of a promoter can influence long-term expression of the transgene. For example, a humoral immune response directed against the transgene product occurred when a ubiquitous but not a liver-specific promoter was used in a first-generation vector. Additionally, it was found that variations in recombinant adenoviral genome persistence and the length of transgene expression from mouse hepatocytes are dependent on the mouse strain.46,47 Hepatocyte-restricted gene expression might explain the observed high and long-term hFIX expression levels not only from a high-capacity vector but also from a first-generation adenoviral vector. Recent studies suggest that the stuffer DNA in the HD vector can have
an effect on transgene expression.10,11 Our HD vector AdFTC/hFIX contains a 16.2-kb human centromeric fragment, an Ig
We would like to thank Leonard Meuse and Hui Xu for technical assistance. We gratefully thank Jeffrey Chamberlain (University of Washington, Seattle) for providing C7-Cre cells and the helper virus for adenoviral production. We further thank Michele Calos (Stanford University, Stanford) for providing the subclone pDYAL with the alphoid repeat DNA fragment.
Submitted August 2, 2001; accepted December 31, 2001.
Supported by National Institutes of Health grant R01 DK49022. A.E. is a recipient of a postdoctoral fellowship by the Deutscher Akademischer Austauschdienst in cooperation with the Dr Mildred Scheel Cancer Foundation and is a current recipient of the Judith Graham Pool Fellowship of the National Hemophilia Foundation.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mark A. Kay, Department of Pediatrics and Genetics, School of Medicine, Stanford University, Stanford, CA 94305; e-mail: markay{at}stanford.edu.
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An adenoviral vector deleted for all viral coding sequences results in enhanced safety and extended expression of a leptin transgene.
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1998;95:7866-7871 4. Wickham TJ, Roelvink PW, Brough DE, Kovesdi I. Adenovirus targeted to heparan-containing receptors increases its gene delivery efficiency to multiple cell types. Nat Biotechnol. 1996;14:1570-1573[CrossRef][Medline] [Order article via Infotrieve]. 5. Kozarsky KF, Wilson JM. Gene therapy: adenovirus vectors. Curr Opin Genet Dev. 1993;3:499-503[CrossRef][Medline] [Order article via Infotrieve]. 6. Benihoud K, Yeh P, Perricaudet M. Adenovirus vectors for gene delivery. Curr Opin Biotechnol. 1999;10:440-447[CrossRef][Medline] [Order article via Infotrieve]. 7. Stratford-Perricaudet LD, Levrero M, Chasse JF, Perricaudet M, Briand P. Evaluation of the transfer and expression in mice of an enzyme-encoding gene using a human adenovirus vector. Hum Gene Ther. 1990;1:241-256[Medline] [Order article via Infotrieve]. 8. Parks RJ, Graham FL. A helper-dependent system for adenovirus vector production helps define a lower limit for efficient DNA packaging. J Virol. 1997;71:3293-3298[Abstract].
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Top
Abstract
Introduction
). For the
first-generation adenoviruses shown in Figure 
and 
), Figure
and 
), and Figure 
and 
), an MOI of 2 (
= 5 × 108 transducing particles;