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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 820-828
GENE THERAPY
From GenStar Therapeutics Corp, San Diego, CA; and the Gene Therapy
Unit, Baxter Healthcare Corp, Round Lake, IL.
The successful prophylactic treatment of hemophilia A by frequent
infusions of plasma concentrates or recombinant factor VIII (hFVIII)
indicates that gene therapy may be a potential alternative for the
treatment of the disease. For efficient delivery and long-term expression of the hFVIII gene, a novel minimal adenovirus (mini-Ad) vector, MiniAdFVIII, has been developed. The vector is devoid of all
viral genes and carries the full-length hFVIII cDNA under the control
of the human 12.5-kb albumin promoter. The MiniAdFVIII vector was
propagated with the assistance of an ancillary vector in 293 cells and
was purified by CsCl banding. Sustained expression of hFVIII at
physiologic levels (100-800 ng/mL) was achieved in mice after a single
intravenous injection of MiniAdFVIII. The expressed hFVIII had a
structure identical to that of recombinant hFVIII, as determined by
Western blot analysis. The functionality of the protein was confirmed
by the restoration of blood coagulation capacity in MiniAdFVIII-treated
hemophilic mice, as determined by tail clipping observations. Although
antivector or antihuman FVIII antibodies at various levels were
detected, long-term expression of the transgene was observed in the
mice that did not generate antibodies against the transgene product.
The vector DNA persisted in the liver tissues of the mice with
long-term expression. No significant histopathologic findings or
toxicities were observed to be associated with the vector in the
MiniAdFVIII-treated C57BL/6 mice. These results support the further
development of MiniAdFVIII for clinical trials toward the treatment of
hemophilia A.
(Blood. 2000;95:820-828)
Hemophilia A is the most common inherited severe
bleeding disorder. The disease is caused by a deficiency in coagulation
factor VIII (FVIII), affecting approximately 1 in 10 000 males in the population.1 Based on the residual activity of FVIII in
plasma, hemophilia A is categorized as mild, moderate, or severe.
Patients with severe cases have less than 1% of normal plasma FVIII
activity, resulting in frequent spontaneous hemorrhages in muscles and
joints with progressively debilitating effects. Supplements of human FVIII (hFVIII) products such as plasma concentrates or recombinant protein are required to stop episodes of severe bleeding.2 Replacement therapy is effective, especially when it is continued as a
long-term prophylactic treatment. It has been suggested that frequent
dosing of hFVIII protein to maintain low plasma levels of the factor
can convert a severe type to a moderate or mild type with significant
improvement in the prognosis and in joint complications.3
However, the high cost of such a prophylactic practice and the
inconvenience of frequent intravenous (IV) injections call for the
development of a more cost-effective and less traumatic treatment.
For these reasons, gene therapy represents a potential alternative for
treating hemophilia A. Among all ex vivo4-7 and in vivo8-12 studies to date, adenovirus-mediated FVIII gene
delivery has proved to be a promising approach. Intravenous
administration of adenovirus vectors results in the prominent delivery
of the transgene to the liver (Balagué C, unpublished data,
1998).13-15 The liver tropism of the vectors
is particularly useful because liver is the natural site of FVIII
biosynthesis and production.16,17 Early generation
adenoviral (Ad) vectors were used to deliver a B-domain deleted hFVIII
cDNA18 and were shown to mediate hFVIII expression at
therapeutic levels on systemic administration.8-10,12 However, early generations of Ad vector for the treatment of genetic diseases are known to have a limited capacity for carrying heterologous DNA and to induce host immune responses because of the expression of
residual viral genes in the vectors.19,20
To overcome these limitations, new adenoviral vector systems with
complete elimination of viral coding sequences have recently been
developed. These systems have shown greater
application,21-25 and they provide a new tool for in vivo
gene therapy. In this study, we analyzed the clinical potential of a
novel adenovirus vector for delivery of the hFVIII gene. The vector,
called MiniAdFVIII, has minimal viral cis-elements
(< 1 kb), and it carries a 20-kb expression cassette that contains
the full-length hFVIII cDNA coding sequence under the transcriptional
control of the human 12.5-kb albumin promoter. MiniAdFVIII was
characterized in vitro and assessed in vivo for hFVIII expression,
vector DNA status, phenotypic correction, toxicity, and immune
responses in mice. Our results indicated that MiniAdFVIII is able to
generate the sustained expression of hFVIII at human physiological
levels in mice in which anti-hFVIII antibodies did not develop. The
vector-treated hemophilic mice were able to stop bleeding in 30 minutes
after tail clipping, in contrast to no clotting and no survival in
vehicle-treated hemophilic mice. The absence of virus-associated
toxicity at effective doses encouraged the development of this vector
for clinical trials for the treatment of hemophilia A.
Propagation and purification of MiniAdFVIII
Analysis of purified MiniAdFVIII
Titering. Viral particles (vp) were determined by absorbance at 260 nm using the conversion factor 1 OD260 = 1012 vp. The number of viral particles was used as a measure of input vector for experimentation. Restriction analysis. Viral DNA from 6 × 1010 vp was extracted and digested with PshAI restriction enzyme, which gives distinct DNA fragments for both vectors. The digested DNA was separated on a 0.7% agarose gel containing EtBr, and the specific bands for MiniAdFVIII (9949 bp) and ancillary (8497 bp) vectors were quantified by densitometry. X-gal staining.
For quantitation of the ancillary vector, which contains the Replication-competent adenovirus assay. A supernatant rescue assay for replication-competent adenovirus (RCA) detection was carried out as previously described.26 FVIII protein and functional assays ELISA. A double-sandwich enzyme-linked immunosorbent assay (ELISA) was designed to quantify the hFVIII protein in cell culture medium or mouse plasma. In the conditions used, the assay was linear up to 100 ng/mL FVIII. The assay was specific for hFVIII and sensitive down to 1.5 ng/mL. Because the mouse samples were diluted 10-fold for testing, the minimum amount of hFVIII detectable in mouse plasma was 15 ng/mL. Briefly, plates were coated with capture antibodies (ESH-5 and ESH-8; 500 ng each per well; American Diagnostica, Greenwich, CT) in carbonate buffer and incubated for 1 hour at 37°C. Plates were washed with 0.05% Tween 20 in PBS and blocked in 50 mmol/L Tris, pH 7.2, 150 mmol/L NaCl, 0.5% gelatin, and 0.05% Tween 20 for 2 hours at 37°C. Mouse plasma samples were assayed in duplicate at 1:10 dilution in blocking buffer. Purified recombinant FVIII (Hyland; Baxter Healthcare, Glendale, CA), prepared in blocking buffer with 1:10 normal mouse plasma (Harlan, Indianapolis, IN), served as the standard. Samples and standards were incubated for 1 hour at 37°C. Detection antibody (sheep antihuman FVIII, horseradish peroxide [HRP]-conjugated; Cedar Lane, Ontario, Canada) was diluted in blocking buffer and incubated for 1 hour at 37°C. After a final washing step, plates were developed with o-phenylenediamine dihydrochloride peroxidase substrate (Sigma, St. Louis, MO) and the optical density at 490 nm was read using an EL 340 Spectrophotometer (Bio-tech Instruments, Winooski, VT). Functional assay. hFVIII activity in cell culture media and mouse plasma samples was detected with a Coatest chromogenic assay (Chromogenix, Molndal, Sweden) modified for microtiter plates. Human plasma (FACT pool reference plasma; George King Biochemicals, Overland Park, KS) was used as a standard, defining 1 U hFVIII as 200 ng/mL and applying the correction factor specified by the manufacturer. Appropriate dilutions of cell culture supernatants and mouse plasma were tested in duplicate, and results were plotted as hFVIII ng/mL. Specific activity analysis. The ratio between the functional units and the quantity of the protein was used to calculate the specific activity as U activity/µg protein. Western blot. For protein analysis of hFVIII from MiniAdFVIII-treated cultured cells, HepG2 cells were infected with 10 000 vp/cell in medium with 10% fetal calf serum, and the supernatant was harvested 3 days after infection. Immunoprecipitation of hFVIII was carried out with the anti-hFVIII:C monoclonal antibody (Hyland; Baxter Healthcare) coupled to agarose beads. The beads were incubated with culture supernatant overnight at 4°C with constant rotation. Pellets were washed 4 times in 50 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 2 mmol/L EDTA, 1% Triton X-100, 0.1% sodium dodecyl sulfate. Proteins were separated by electrophoresis in a 7.5% polyacrylamide gel and transferred to a nitrocellulose membrane. FVIII heavy chain was detected with an HRP-conjugated sheep anti-hFVIII antibody (Cedar Lane). For light chain detection, a biotinylated anti-hFVIII:C antibody (QED Bioscience, San Diego, CA) was used. This was followed by incubation with neutravidin-HRP (Pierce, Rockford, IL). The signal was developed with a chemiluminescence substrate (Enhanced Chemoluminescence; Amersham, Uppsala, Sweden). A Western blot of immunoprecipitated hFVIII was used to analyze plasma samples of MiniAdFVIII-treated mice. Animal studies MiniAdFVIII activity studies. Six- to 8-week-old C57BL/6 and Balb/c mice were purchased from Harlan. Hemophilic mice (exon 16-disrupted FVIII knockout mice) were obtained from the University of Pennsylvania27 and bred in-house. For dose-response studies, groups of 3 mice were injected through tail veins with different doses of MiniAdFVIII. At the designated time points, blood was collected by retro-orbital puncture in tubes containing 0.1 vol of 0.1 mol/L sodium citrate. Cells were removed by centrifugation, and the plasma was tested for hFVIII by ELISA or functional assay. Phenotypic correction studies. Groups of 12 mice were injected on day 1 with 2.5 × 1011 vp of MiniAdFVIII or vehicle (PBS), respectively. On day 3, blood was collected by retro-orbital puncture with a glass capillary tube. Tubes were broken at 1-minute intervals to check for clot formation, and the clotting time was recorded. On day 6, a 2-cm section of the tail was clipped from each mouse to measure the bleeding time (time until bleeding stopped) and the volume of blood shed. Toxicity studies. Groups of 6 C57BL/6 mice were injected through the tail veins with the selected doses of MiniAdFVIII or with vehicle. Three and 14 days after injection, 3 mice from each group were killed. Blood was collected by cardiac puncture, and serum samples were tested for alanine aminotransferase (ALT) levels (Boehringer Mannheim, Indianapolis, IN). Tissues (liver, spleen, kidney, lung, and heart) were fixed in formalin for 24 hours, paraffin embedded, and processed for histopathology. Analysis of vector DNA in vivo. Polymerase chain reaction (PCR) assays with hFVIII-specific primers (Figure 6A) were used for detecting the MiniAdFVIII DNA. Liver tissue (0.25 g per sample) from MiniAdFVIII-treated mice was ground to a powder in a liquid nitrogen-cooled mortar, and DNA was extracted using a Qiagen Tissue Kit (Qiagen, Valencia, CA). Each PCR reaction contained 1 × reaction buffer, 200 µmol/L each deoxynucleotide triphosphate, 2.25 to 4.0 Mg++ (the concentration varied with different primers), 0.4 µmol/L each primer, and 2.5 U Qiagen HotStar Taq polymerase (Qiagen) in a total volume of 50 µL. Amplification was carried out for 1 cycle of 15 minutes at 95°C followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 52°C, and 45 seconds at 72°C, with a final extension of 7 minutes at 72°C. Aliquots of the PCR reactions (15 µL) were analyzed on 1.5% agarose gels. Amplified DNA fragments were semiquantitated by comparing Gelstar-stained band intensities from tissue samples to those for the control plasmid (Gelstar; FMC BioProducts, Rockland, ME). Division of the copies by pg input DNA yielded copies/pg input DNA. These values were normalized to copies/cell equivalent by assuming 6 pg/cell nucleus and adjusting the values accordingly. Analysis of anti-hFVIII and anti-MiniAdFVIII antibodies. ELISA assays were used to determine the antibodies against the hFVIII protein and the MiniAdFVIII vector. The assay plates, coated with either purified hFVIII protein or the ancillary vector in carbonate buffer, were blocked with PBS containing 4% fetal bovine serum for 1 hour at room temperature, washed once, and incubated overnight with serially diluted test plasma samples (100 µL/well). After 5 washes, each well was incubated with 100 µL 1:2000 diluted goat antimouse IgG (H + L) conjugated to HRP (Southern Biotechnology Associates, Birmingham, AL) at 37°C for 1 hour. After 5 washes, 100 µL peroxidase substrate solution (o-phenylenediamine) was added to each well. Color development was monitored for 7 minutes, and the reactions were stopped with the addition of 3 N HCl (50 µL/well). The optical density at 490 nm (OD490) was read using an EL340 spectrophotometer (Bio-Tek Instruments, Winooski, VT).
Preparation of purified MiniAdFVIII The MiniAdFVIII genome contains a 20-kb hFVIII expression cassette consisting of a 12.5-kb human albumin promoter,28 an SV40 intron, a 7.2-kb full-length hFVIII cDNA, and an SV40 polyadenylation signal (Figure 1A). In addition, a 6.9-kb human albumin genomic fragment was included to increase the viral genome to an optimal packageable size.21,29 The only Ad sequences remaining in the vector are the inverted terminal repeats, the packaging signal, and a 400-bp noncoding fragment of E4. MiniAdFVIII was generated by co-transfecting 293 cells with the plasmid form of MiniAdFVIII and the ancillary virus DNA, as previously described.21 Viruses were co-propagated in 293 cells. MiniAdFVIII was purified and characterized as described in "Methods." A total viral DNA digestion from a typical purified MiniAdFVIII preparation is shown in Figure 1B. Comparison of the actual digestion pattern with the expected patterns for MiniAdFVIII and ancillary vector provides information on the structural integrity and purity of MiniAdFVIII at the DNA level. In Figure 1B, the absence of the expected band from the ancillary vector indicated the ancillary vector is present at levels below the detection limit for this assay. Because the ancillary vector carries a -gal reporter, a more
sensitive X-gal staining assay was applied to analyze the contamination
level of the ancillary vector in the MiniAdFVIII preparation. In a
typical lot of purified MiniAdFVIII, the X-gal positively stained cells
were observed at a frequency of 1 of 10 000 to 100 000 negatively
stained cells when a multiplicity of infection of 10 vp/cell was used
(data not shown). Taken together, our data indicate that the purified MiniAdFVIII preparation contains less than 0.1% of the contaminating ancillary vector. The supernatant rescue assay was also applied to
determine the level of RCA contamination in preparations of MiniAdFVIII. No RCA was detectable in 109 vp of
MiniAdFVIII.
Characterization of hFVIII produced in vitro To assess the MiniAdFVIII activity in vitro, 293 cells were infected with the vector at different vp/cell ratios, and the supernatant of the treated cells was analyzed using a Coatest (Chromogenix) chromogenic assay (Figure 2A). The hFVIII activity was detected in the supernatant of the MiniAdFVIII-infected cells in a dose-dependent fashion, indicating that the vector was able to transduce hFVIII and that the hFVIII protein encoded by MiniAdFVIII was biologically active. To analyze further the structure of the hFVIII protein produced by MiniAdFVIII, HepG2 cells were infected with the vector, and the conditioned medium was used for immunoblot analysis (Figure 2B). Detection with a specific antibody to the heavy chain revealed several protein species ranging in size from 100 kd to 200 kd. A unique band of 80 kd was detected with specific antibodies to the light chain. The pattern obtained from infected cells was comparable to that of recombinant FVIII (Hyland-Immuno; Baxter Healthcare). The slight difference in the intensity of the bands of heavy chain (Figure 2B; HC) may reflect a difference in posttranslational processing in different cell lines because the recombinant FVIII was produced from Chinese hamster ovary cells. Taken together, the results demonstrate that hFVIII produced in vitro by the MiniAdFVIII-infected cells is biologically active and has the expected protein structure.
Studies of MiniAdFVIII activity in mice To assess the gene transduction efficiency of MiniAdFVIII and the subsequent hFVIII production in vivo, a dose-response study of the vector was performed in C57BL/6 mice. Groups of 3 mice were injected through the tail veins with different doses of MiniAdFVIII. At various time points, plasma was collected and assayed for hFVIII expression by ELISA (Figure 3A). Doses of 4 × 1011 and 2 × 1011 vp MiniAdFVIII-mediated hFVIII expression in mice at levels above the human physiologic levels (more than 200 ng/ml). Accordingly, 6 × 1010 vp produced relatively lower levels of hFVIII (approximately 100 ng/mL), and at 2 × 1010 vp, hFVIII levels were below the assay detection limit. The expression of hFVIII in the mice persisted for 308 days (last time point tested), though a gradual decline in hFVIII expression was observed after postinfection day 160.
Assessment of MiniAdFVIII immunogenicity in mice To understand the immunogenicity of MiniAdFVIII and the nature of the variations observed in the duration of hFVIII expression in the MiniAdFVIII-treated mice, anti-hFVIII as well as anti-Ad antibodies were analyzed by ELISA. Those mice with the shortest duration of hFVIII expression had humoral immune responses to the transgene product. In all cases, the rise in antibody titer inversely correlated with the level and duration of hFVIII expression (Figure 4A). Administration of MiniAdFVIII induced a stronger and faster humoral immune response in Balb/c mice. In these mice the titers of the anti-hFVIII antibodies rose sharply within 1 week after injection of the vector (data not shown).
Characterization of hFVIII produced in vivo The hFVIII protein secreted into the plasma of injected mice was analyzed by immunoprecipitation and Western blotting (Figure 5) using antibodies specific to hFVIII. As in the in vitro assay, both heavy and light chains were detected with molecular masses of 200 kd and 80 kd, respectively, indicating correct posttranscriptional processing of the human protein in the murine host.
Study of MiniAdFVIII efficacy in hemophilic mice
Evaluation of MiniAdFVIII safety in mice
Biodistribution and status of MiniAdFVIII DNA in vivo
This article describes the successful in vivo gene transfer of a
cDNA that encodes a full-length hFVIII polypeptide using a minimal Ad
vector. The vector, MiniAdFVIII, is completely devoid of adenoviral
coding sequences and carries a 20-kb expression cassette consisting of
the full-length hFVIII coding sequence flanked by a human albumin
promoter and genomic sequences. MiniAdFVIII is co-propagated with a
complementing ancillary vector in an AdE1-expressing cell
line.21 The presence of a partially deleted packaging
signal in the ancillary vector allows the preferential packaging of
MiniAdFVIII, which in turn results in high titers of the vector.
We thank the CRTS department at Baxter Healthcare Corporation (Round
Lake, IL) for excellent technical assistance and expertise in the
evaluation of the histopathology data, with special thanks to Ray Ortiz
(CRTS) for his diligence in tissue processing and immunostaining. We
also thank the Animal Care staff for help with animal injections and
blood collections, Patrice Tremble for valuable advice, and Dr William
Raschke for critical reading of the manuscript.
Submitted December 22, 1998; accepted September 15, 1999.
Reprints: Wei-Wei Zhang, GenStar Therapeutics Corporation,
10835 Altman Row, Suite 150, San Diego, CA 92121; e-mail: wzhang{at}genstartherapeutics.com.
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.
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Top
Abstract
Introduction
) allows the MiniAdFVIII
vector to replicate and package efficiently in cells coinfected with
the ancillary vector and expressing adenovirus E1 early proteins.
(arrow) Direction of transcription. The ancillary vector is an
E1-substituted adenovirus with a partially deleted packaging signal.
(B) Detection of ancillary vector in MiniAdFVIII preparations. Total
viral DNA from 6 × 10
