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Department of Surgery, University of Florida College of Medicine, Gainesville, FL 32610; and
Canji, Incorporated, San Diego, CA 92121
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1-antitrypsin, adenosine deaminase, and
ornithine transcarbamoylase (1). Gene therapy also is
being applied to acquired diseases with a genetic component, such as
p53 mutations in ovarian and head and neck cancers. In these cases, the
goal of gene therapy is the restoration of normal gene function and
protein expression (2). However, gene therapy has several potential uses beyond the restitution of normal gene function. In 1995, Crystal (3) proposed the use of gene therapy as a novel drug delivery system to express proteins in individual tissues. This approach offers several theoretical and practical advantages over the administration of recombinant proteins. With adenoviral constructs, a single intratracheal or i.v. administration could result in sustained protein expression for periods generally lasting between 7 and 14 days, but occasionally for longer periods (4, 5). In addition, by using tissue-specific promoters or taking advantage of the natural tropism of adenovirus (Ad)3 for bronchial epithelium or for the liver and pancreas, targeted expression in these organs, but not in other tissues, could be achieved. This type of local administration and expression may improve therapeutic efficacies of some proteins, particularly cytokines.
However, clinical development of adenoviral vectors has been limited by
their inflammatory potential and the induction of an acquired immune
response that limits repeat dosing (6). Previous studies
have shown that first-generation adenoviral vectors activate the innate
immune response and stimulate the synthesis of the proinflammatory
cytokine TNF- (4, 5). This increased TNF- production
not only reduces the duration and magnitude of transgene expression
(4, 7), but also is a concern in acute inflammatory
conditions where exaggerated TNF- expression may contribute to organ
injury. There have been only limited studies evaluating the safety and
efficacy of adenoviral constructs in the context of an ongoing
inflammatory process.
In the present report, we have explored the feasibility of adenoviral gene therapy as a drug delivery system for the treatment of two acute inflammatory diseases, necrotizing pancreatitis induced by a choline-deficient, ethionine supplemented (CDE) diet, and zymosan-induced multisystem organ failure (MSOF). We have specifically asked whether adenoviral expression of the anti-inflammatory cytokines human (h) or viral (v) IL-10 could be successfully used in these acute inflammatory processes. We chose these two models because previous studies have shown that administration of IL-10 protein can alter the progression of disease and improve outcome. In particular, Deviere and colleagues (8, 9) have shown that exogenously administered and endogenously produced IL-10 reduces the systemic inflammatory response to experimental pancreatitis. These same investigators have shown that a single administration of IL-10 in patients before endoscopic retrograde cholangiopancreatography reduces the risk of pancreatitis (10). Similarly, Jansen and colleagues (11) have shown that repeated administration of IL-10 also can improve outcome in zymosan-induced MSOF.
The findings presented here suggest that adenoviral transduction can be accomplished during existing inflammatory processes without significant exacerbation of organ injury, and Ad-induced expression of IL-10 is associated with reduced inflammation and organ injury and improved outcome.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A derivative of hAd serotype 5 (12) was used as the source of viral DNA backbone. This modified adenoviral vector backbone contains a deletion of base pairs 355-4021, resulting in a deletion of the E1a, E1b, and protein IX polypeptides. In addition, there is a deletion of base pairs 28,592–30,470 that results in a loss of 1.9 kb of DNA from the E3 region. rAd expressing hIL-10 and vIL-10 cDNA transgenes were constructed by standard homologous recombination methods as described by Graham and Prevec (13). Briefly, hIL-10 and vIL-10 cDNAs containing the full-length translated regions (from pDSRG-IL10 and pcDSR-BCRF1 plasmids, respectively, obtained from K. Moore at DNAX Research Institute, Palo Alto, CA; Ref. 14) were subcloned into the BamHI/XbaI cloning site of the pACN transfer plasmid. The pACN transfer plasmid is based on pBR322 and contains, from 5' to 3', the Ad5 inverted terminal repeat and packaging signal (Ad5 bp 1–358), the hCMV immediate early enhancer/promoter, the Ad2 tripartite leader sequence, a multiple cloning site, and Ad2 bp 4,021–10,457. This plasmid was cotransfected into 293 cells, along with a claI-linearized fragment of the plasmid described above containing the modified hAd5 adenoviral vector backbone (12). Additionally, a rAd containing an empty expression cassette was constructed for use as a control. All of the viral constructs were similar with the exception of the transgene, and the production and purification procedures were identical.
Relative expression levels of hIL-10 and vIL-10 have been directly compared after in vitro transduction of cell lines as well as in vivo transduction with the respective adenoviral vectors (15). Transduction of cell lines in vitro with the adenoviral vectors expressing hIL-10 and vIL-10 resulted in similar amounts of protein being secreted into the supernatant. Likewise, i.v. injection of adenoviral vectors expressing hIL-10 and vIL-10 resulted in similar amounts of protein being released into the serum. However, there was a comparatively larger portion of vIL-10 that was retained in the cell lysate portion of in vitro transduced cells and in vivo transduced tissues.
Zymosan A preparation
Zymosan A (lot no. 49H0557), a cell wall component of
Saccharomyces cerevisiae, and endotoxin-free paraffin oil
were obtained from Sigma (St. Louis, MO). Zymosan A was irradiated with
5 kGy 
radiation over 5 h. Endotoxin testing of 1 mg/ml zymosan
A in sterile H2O demonstrated that zymosan A
contained < 5 EU/mg. The irradiated zymosan A was suspended in
sterile paraffin oil at a concentration of 0.6 mg/mouse g body
weight using a sterile technique. The zymosan-paraffin oil solution
then was placed in a Fisher sonicating water bath (50/60 Hz) for 30 min
and a high-frequency sonicator (Fisher Sonic Dismembrator model 300;
Fisher Scientific, Pittsburgh, PA) was used at a setting of 70% for 15
min to further resuspend the zymosan in the paraffin oil. The zymosan
suspension then was placed in a 100°C water bath for 80 min. After
this, the solution was vortexed and drawn into sterile syringes.
Animal preparation
Specific pathogen-free female C57BL/6 mice (20–25 g; The Jackson Laboratory, Bar Harbor, ME) were housed in a Bio-Safety Level 2 barrier facility with unlimited chow and water for the duration of the experiments. All animal studies were approved by the Institutional Animal Care and Use Committee of the University of Florida. The use of adenoviral recombinants in mice was approved by the Institutional Biosafety and the Recombinant DNA Committees of the University of Florida. The laboratory adheres to the "Guiding Principles of Laboratory Animal Care," as promulgated by the American Physiological Society.
Mice were anesthetized with 35 mg/kg body weight of i.p. sodium pentobarbital. A mid-line incision was made in the neck, the trachea was visualized and cannulated with a sterile 30-gauge needle, and 32 µl of buffer (PBS, pH 7.5, containing 3% (w/v) sucrose, 2 mM MgCl2) or buffer containing adenoviral vector (107 particles per animal) was delivered. Mice received an intratracheal instillation of either 107 particles of a recombinant adenoviral construct expressing hIL-10 (Ad/hIL-10), vIL-10 (Ad/vIL-10), or an empty expression cassette (Ad/empty), or buffer alone on day -1 or day 5 in the zymosan model, and on day -1 in the CDE pancreatitis model. In the CDE pancreatitis model, i.v. tail vein injections of 100 µl of buffer or buffer containing 1010 particles of either Ad/hIL-10, Ad/vIL-10, or Ad/empty (n = 15 per group) also were performed on day -1 using a sterile 30-gauge needle.
On day 0, the mice underwent an i.p. injection of 1 ml of the zymosan suspension with a sterile 20-gauge needle or were fed a CDE diet (100 g choline-deficient murine chow supplemented with 0.5 g DL-ethionine). On day 4, the CDE diet was exchanged for normal murine chow. A single i.p. injection of sterile zymosan suspended in paraffin oil leads to the development of a triphasic illness characterized by an initial septic shock-like phase (days 1–3) with an associated mortality of 25–30%, followed by a recovery period (days 4–8), and then ultimately the development of MSOF with an additional 60–70% mortality (days 9–14; Refs. 16 and 17). The CDE diet induces a severe necrotizing pancreatitis in mice as described previously (18, 19).
In the zymosan model, Ad/hIL-10-, Ad/vIL-10-, and buffer-treated animals were weighed daily and followed for survival for 18 days (n = 15–24 per group). Additional mice (n = 9 per group) were sacrificed by cervical dislocation 24 h after zymosan administration. Blood was collected via a retroorbital venipuncture with a capillary tube. Liver and lungs were removed and snap-frozen in liquid nitrogen.
In the CDE pancreatitis model, Ad/hIL-10-, Ad/vIL-10-, and buffer-treated animals were followed for survival for 10 days (n = 10). Additional mice (n = 15) were sacrificed by cervical dislocation 66 h after induction of pancreatitis (19). Blood was again collected via retroorbital venipuncture, and liver and lungs were removed en bloc and snap-frozen in liquid nitrogen.
Analytical methods
Murine IL-6, IL-1, hIL-10, and vIL-10 levels in the organ
homogenates and in the serum were measured by sandwich ELISA with
commercially available reagents (mIL-6, hIL-10, and vIL-10 by Endogen,
Woburn, MA, and murine IL-1 and IL-10 by R&D Systems, Minneapolis,
MN). Bioactive TNF- was measured in serum and lung homogenates with
the TNF--sensitive WEHI 164 clone 13 murine fibrosarcoma cell line
(20). A standard curve was generated with hTNF-, and
the sensitivity of the assay was 5–25 pg/ml for serum and 75–375
pg/gram wet weight (gww) for lung.
Pulmonary and hepatic neutrophil sequestration were quantitated in the pancreatitis studies by measuring tissue myeloperoxidase content (5). Pancreatic injury was assessed by measuring serum amylase and lipase with a Vitros system analyzer (Shands Hospital Diagnostic Laboratories, Gainesville, FL).
Real-time quantification of IL-10 mRNA and viral DNA
Total RNA was extracted from 
100 mg of lung tissue with
Tri-Reagent (Molecular Research Center, Cincinnati, OH) per the
manufacturer’s protocol. Total RNA was treated with DNase I
(Boehringer-Mannheim, Indianapolis, IN) to remove residual DNA. The
complete removal of DNA in all RNA samples was confirmed with murine
gapdh PCR. RNA concentrations were determined with OD
260/280 absorbance ratios, and 0.1 µg of total RNA was used for
each RT-PCR. Quantification of mRNA and viral DNA were performed with
real-time quantitative RT-PCR, which uses the 5' nuclease activity of
Taq polymerase (Taqman; PerkinElmer Applied Biosystems,
Norwalk, CT) to detect PCR amplicons. Briefly, in addition to
primers, a target sequence-specific oligonucleotide probe labeled with
a reporter fluorescent dye (FAM (6-carboxy-tetramethyl-rhodamine)) at
the 3' end was added to the PCR. When the probe is intact, the
fluorescence emission of the reporter is quenched because of the
physical proximity of the reporter and quencher fluorescent dyes. The
resulting relative increase in reporter fluorescent dye emission is
monitored in real time during PCR amplification by a sequence detector,
the 7700 sequence detector (PerkinElmer Applied BioSystems). In
addition to Ad/hIL10 and Ad/vIL10, relative quantification of murine
GAPDH RNA and DNA also were performed to ensure the quality of RNA
and DNA.
RT-PCR was performed with the Taqman EZ RT-PCR kit (PerkinElmer Applied BioSystems). Reactions were performed in a total volume of 50 µl containing 1.0 µl of total RNA extracted from lungs or cells, 300 mM each of dATP, dGTP, or dCTP, and 600 mM of dUTP, 10 U of recombinant Thermus thermophilus DNA polymerase, 0.5 U of AmpErase uracil N-glycosylase (all from PerkinElmer Applied BioSystems) to eliminate carry-over PCR product contamination, 10 mM of each primer, and 10 mM of probe. The following thermal cycler parameters were optimized for IL10 transgene detection: 2 min at 50°C, 30 min at 60°C, 10 min at 95°C, followed by 40 cycles of 20 s at 95°C and 1 min at 61°C. The primer and probe sequences for RT-PCR and PCR were: Ad/hIL-10 and Ad/vIL-10 forward primer, 5'-AACGGTACTCCGCCACC-3'; Ad/hIL-10 reverse, 5'-CGGCCGCTCGAGTCTAGAC-3'; Ad/vIL-10 reverse, 5'-ATGATGGAGCTCTAGACTCGAGA3'; Ad/hIL-10 and Ad/vIL-10 probe, FAM-TCCGCATCGACCGGATCGGTAMRA; murine GAPDH forward, 5'-GAAGGTGAAGGTCGAGTC-3'; GAPDH reverse, 5'-GAAGATGGTGATGGGATTTC-3'; GAPDH probe, FAM-CAAGCTTCCCGTTCTCAGCC-TAMRA. The PCR thermal profile was 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 60°C for 1 min. Relative quantification of gene expression was performed with a serially diluted RNA isolated from either Ad/vIL-10- or Ad/hIL-10-infected 293 cells. MEQ is a relative quantification unit, using an arbitrarily assigned number. For viral DNA quantification, serially diluted viral DNA was isolated from Ad/hIL-10 or Ad/vIL-10 viruses. All RT-PCR and PCR were done in duplicate and the detection limit of RT-PCR assay is 20 MEQ/mg of tissue and PCR assay is 10 copies/mg of tissue.
Statistical analysis
Data are presented as the mean ± SEM (n = 9–18 animals/group). Student’s t test was used for analyses comparing healthy controls to buffer-treated animals in the zymosan and pancreatitis models. A one-way ANOVA was used to compare animals at the same time point receiving different treatments (buffer, Ad/hIL-10, or Ad/vIL-10), and post hoc comparisons were performed with Dunn’s multiple range test. A one-way ANOVA with repeated measures was used to compare the animals’ weight changes over time in the zymosan model, and pair-wise comparisons were performed by the Fisher least significant differences method. Survival was assessed with Kaplan-Meier analysis. Statistical differences were considered to be significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the initial studies, C57BL/6 mice underwent intratracheal
instillation with either 107 particles of
Ad/hIL-10 or Ad/vIL-10 in 32 µl of buffer or 32 µl of buffer alone
at day -1. On day 0, the animals then were challenged with i.p.
zymosan A in paraffin oil as described. As shown in Fig. 1
A, 30% of the buffer-treated mice died within the first 3
days, and then survival plateaued for an additional 3–5 days.
Thereafter, mortality in the buffer group was progressive with only
25% of the animals surviving 18 days.
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A). To
ensure that the survival benefits that were seen in the Ad/hIL-10 and
Ad/vIL-10 treatment groups were attributable to the expression of IL-10
and not secondary to adenoviral transduction per se, the experiment was
repeated, and buffer-treated mice were compared with mice that received
the intratracheal instillation of an identical adenoviral vector
containing an empty cassette. Mice (n = 18 per group)
were again pretreated with the intratracheal delivery of 32 µl of
buffer containing 107 particles of Ad/empty or
buffer alone, and survival was measured. In the Ad/empty treatment
group, there was an insignificant trend toward decreased survival in
the first phase of the zymosan-induced illness (44% survival by day
4), as compared with mice treated with buffer only (77% survival by
day 4). However, 18-day survival was similar between the mice treated
with the Ad/empty and buffer (22 vs 33%, respectively; Fig. 1B).
Mice receiving the intratracheal administration of Ad/hIL-10 and
Ad/vIL-10 also demonstrated less initial weight loss and more rapid
weight gain, a sensitive clinical marker of murine illness, after
zymosan challenge, as compared with buffer only-treated animals (Fig. 2). Interestingly, the animals treated intratracheally with Ad/hIL-10
appeared to lose benefit in the third phase of illness, as the mice
began to demonstrate weight losses that mirrored the buffer
only-treated mice. In contrast, mice receiving the intratracheal
administration Ad/vIL-10 continued to experience stable weights, or
actually demonstrated weight gain, from days 9 to 18, when MSOF usually
develops in this model (Fig. 2). Both the loss of survival protection
as well as the weight loss seen in the third phase of illness in the
Ad/hIL-10 treatment group occurred around days 9–10.
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Proinflammatory cytokine appearance in the serum, lungs, and liver of the pretreated animals 24 h after zymosan challenge
As shown in Table I, a step-wise reduction in serum IL-1 and IL-6 was seen in mice
treated intratracheally with Ad/hIL-10 and Ad/vIL-10, as compared with
buffer alone, with the greatest reductions seen in the Ad/vIL-10
treatment group (p = 0.05 for IL-6; Table I).
TNF- could not be detected in the serum at any time point in any
animal. Lung IL-6 levels also were significantly reduced in the
Ad/vIL-10 treatment group at 24 h (1.54 ± 0.12 ng/gww in the
Ad/vIL-10 animals vs 6.55 ± 2.97 ng/gww in the buffer only
animals; p < 0.05). Similarly, lung and liver murine
IL-10 levels were significantly reduced in the Ad/hIL-10 and Ad/vIL-10
treatment groups (2.09 ± 0.04 ng/gww for lung Ad/vIL-10 vs
2.72 ± 0.17 ng/gww for lung buffer (p <
0.05) and 10.51 ± 0.37 ng/gww for liver Ad/hIL-10, 10.42 ±
1.3 ng/gww for liver Ad/vIL-10 vs 14.19 ± 0.92 ng/gww for liver
buffer (p < 0.05)).
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, significant neutrophil infiltration (dense, multilobular, nucleated,
cellular infiltrate) of the lung parenchyma was seen in both the buffer
alone- (Fig. 3A) and Ad/hIL-10-treated (Fig. 3B)
mice. However, in stark contrast, the Ad/vIL-10-treated (Fig. 3C) mice had more modest pulmonary neutrophil infiltration
with preservation of more normal lung architecture.
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). In contrast to the
pretreatment studies, mice receiving the intratracheal instillation of
Ad/hIL-10 or Ad/vIL-10 on day 5 after zymosan administration did not
demonstrate any improvement in weight gain (data not shown) or survival
(36% survival (5 of 14) for Ad/hIL-10 and 21% survival (3 of 14) for
Ad/vIL-10) as compared with buffer controls (36% survival (5 of 14);
p > 0.05). Effects of intratracheal and i.v. pretreatment with Ad/IL-10 in the CDE model of pancreatitis
Given the encouraging results seen with an intratracheal pretreatment approach in the zymosan model, similar studies were initially conducted in the CDE pancreatitis model. C57BL/6 mice underwent intratracheal instillation with 107 particles of either Ad/hIL-10, Ad/vIL-10, or buffer alone on day -1, as described. Pancreatitis then was induced and mice were followed for survival, with additional mice sacrificed at 66 h for the measurement of tissue cytokine production and serum amylase and lipase concentrations. Intratracheal pretreatment in this model with hIL-10- or vIL-10-expressing vectors did not lead to an improvement in survival (22% (2 of 9 in the buffer-treated animals), vs 36% (4 of 11 in the Ad/hIL-10-treated animals) and 22% (2 of 9 in the Ad/vIL-10-treated animals); p > 0.05), nor was there any reduction in lung and liver cytokine production or serum amylase and lipase in the Ad/hIL-10 and Ad/vIL-10 treatment groups as compared with buffer controls (data not shown).
Unlike the zymosan model, which has a well-described associated
hemorrhagic lung injury (16, 17), the pathology seen in
the CDE model of pancreatitis is primarily restricted to the pancreas
and liver (18, 19). With lung myeloperoxidase levels as an
indicator of neutrophil infiltration, there was not an associated lung
infiltration in mice with CDE-induced pancreatitis (0.41 ± 0.07
U/gww in healthy animals vs 0.41 ± 0.04 U/gww in buffer-treated
mice with pancreatitis). Therefore, a systemic gene therapy approach
then was used in this model taking advantage of the natural tropism of
the Ad for hepatocytes and pancreatic epithelial cells. On day -1,
C57BL/6 mice underwent i.v. delivery of 1010
particles of Ad/hIL-10, Ad/vIL-10, or buffer alone, followed by
induction of CDE pancreatitis on day 0. Mice were sacrificed
(n = 15) 66 h after induction of CDE pancreatitis.
Although a survival benefit was not observed with an i.v. approach
(44%; 7 of 16 in buffer-, Ad/hIL-10-, and Ad/vIL-10-treated animals),
significant reductions in the liver and pancreatic injury and liver
inflammation were seen in the Ad/hIL-10 and Ad/vIL-10 treatment groups
as compared with buffer alone. As shown in Fig. 4, a significant reduction in serum amylase also was seen in the
Ad/hIL-10 treatment group (7,302 ± 500 U/L serum for the
Ad/hIL-10 treatment group vs 10, 674 ± 857 U/L serum for the
buffer only group; p = 0.03; Fig. 4A). Serum
lipase also was reduced in both the Ad/hIL-10- and Ad/vIL-10-treated
animals as compared with buffer (7, 389 ± 1,058 U/L serum and 7,
137 ± 1,363 U/L serum vs 10, 414 ± 1,315 U/L serum,
respectively; Fig. 4B). Similarly, as shown in Table II, liver inflammation was markedly reduced. Liver IL-1 levels were
reduced significantly in both groups of animals pretreated i.v. with
Ad/hIL-10 and Ad/vIL-10. Additionally, liver IL-6 levels and
myeloperoxidase activity were significantly reduced in the Ad/vIL-10
treatment group (Table II). The only cytokine that was increased in the
lung after CDE pancreatitis was IL-1 (1.36 ± 0.82 ng/gww in
buffer-treated mice with pancreatitis vs <0.50 in healthy murine
lungs). Ad/vIL-10-treated mice with pancreatitis demonstrated baseline
levels of lung IL-1 (<0.50 ng/gww) as compared with buffer-treated
animals with pancreatitis; p < 0.05).
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. As seen in our previous studies (5), vIL-10
concentrations were markedly higher in the tissues as compared with
hIL-10 levels, whereas hIL-10 concentrations were more readily detected
in the systemic circulation, despite transduction with otherwise
identical viral vectors.
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, liver IL-1 and IL-6 levels
and myeloperoxidase activity were similar or greater than the levels
seen in the buffer-treated animals with pancreatitis (Table II). Serum
amylase and lipase levels also were not significantly different between
the two treatment groups (data not shown). This suggests the beneficial
effects seen in the mice treated with Ad/hIL-10 and Ad/vIL-10 were
attributable to the expression of IL-10, and not to the transduction
with the Ad recombinant. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Nevertheless, the use of Ad-based gene therapy offers several significant advantages over the administration of recombinant proteins. Most apparent is the observation that a single instillation of Ad results in the sustained expression of the transgene for extended periods of time. Expression with adenoviral vectors is rapid, with protein appearance usually occurring within hours. Peak expression occurs within 1–3 days after adenoviral instillation (5). For recombinant proteins like IL-10, which have a relatively short half-life that necessitates repeated or continuous administrations, adenoviral delivery offers a significant theoretical and practical advantage. We have previously shown that a single intratracheal injection of Ad/hIL-10 or Ad/vIL-10 results in expression exceeding 10 and 42 days, respectively (15).
In addition, adenoviral gene therapy offers the opportunity to target specific tissues for local expression. Because Ad has tropism for pulmonary epithelial cells (21) as well as for hepatocytes (22) and pancreatic epithelial cells (23), high local concentrations can be achieved in the absence of high systemic concentrations. In healthy mice, we observed that after intratracheal administration of Ad/hIL-10 or Ad/vIL-10, tissue levels of protein were often 100-1000 times higher than plasma levels (5, 15). vIL-10 appears to have a strong propensity for remaining in the tissues where it is expressed.
However, widespread use of Ad gene therapy has been limited by its
inherent proinflammatory properties. Both intratracheal and i.v.
administration of Ad induces a proinflammatory response in the lung and
liver, characterized by increased TNF- expression (4, 7). Indeed, we have shown that clearance of adenoviral vectors
is a direct function of the magnitude of the innate immune response and
is dependent on TNF- expression (5). Mice lacking TNF
signaling pathways often have extended adenoviral expression (4, 7).
In clinical trials with adenoviral vectors, dose-limiting inflammation has been a significant concern. The use of adenoviral vectors for cystic fibrosis has been hampered by mucosal inflammation, radiographic evidence of airway infiltration, and flu-like symptoms (24, 25). Similarly, i.v. administration of high-dose Ad to a patient with ornithine transcarbamoylase deficiency resulted in death due to fulminant hepatic and MSOF (26, 27).
However, the amount of Ad required to express a secreted protein like IL-10 in pharmacologic quantities in the target tissue is usually quite small. Theoretically, only a limited number of cells need to be transduced and secreting the protein to achieve a tissue-wide, bystander response. In fact, these preliminary studies suggest that doses as low as 107 particles in the lung produce a significant therapeutic response, and doses up to three logs higher in the lungs of healthy mice have not produced any histological evidence of inflammation despite achieving nanogram per gww concentrations of the transgene product (5).
Not surprisingly, there were significant differences in the response to
adenoviral gene therapy that appeared to be dependent on the
experimental injury, the timing and mode of delivery, and whether
hIL-10 or vIL-10 were expressed. It is important to clarify that the
use of Ad gene therapy, per se, will not obviate the difficulties
associated with treating the infected or inflamed patient, which
includes the need for pretreatment. Both models were chosen because of
past successes with recombinant protein (8, 9, 10, 11, 28). We
were initially surprised that intratracheal instillation of Ad/IL-10
would produce such significant improvements in outcome with the zymosan
model, but not with acute pancreatitis. However, it became obvious that
a significant component of the mortality secondary to zymosan
administration was a hemorrhagic pneumonia (Fig. 3), whereas we saw no
evidence of lung inflammation in mice after CDE-induced pancreatitis,
as determined histologically or by lung myeloperoxidase content. In
previous studies with the CDE model, we observed increased expression
of the proinflammatory cytokines TNF- and IL-1 not only in the lung,
but also in the liver and pancreas (29). Because Ad has
such a propensity for the liver, and to a lesser extent, the pancreas,
we repeated the studies with the i.v. administration of the adenoviral
vectors to target those organisms more specifically. In this case, we
saw significant reductions in the magnitude of the inflammatory
response in the liver, and the degree of pancreatic injury, although
survival was not significantly improved.
Timing also appeared to be critical. Although pretreatment of mice with
the intratracheal instillation of Ad produced significant improvements
in outcome in the zymosan model, delaying treatment for five days until
the second phase of the disease did not improve survival. These
findings are consistent with the observations of Jansen et al.
(11), who also found that although systemic pretreatment
with rIL-10 was effective in improving outcome, posttreatment was not.
However, other investigators have demonstrated a survival benefit when
rIL-10 therapy was delivered to the mice when they developed symptoms
of MSOF in the third phase of the zymosan-induced illness
(30). The lack of an improvement in outcome with a
posttreatment approach in the present studies as compared with the
studies of Ferrer and his colleagues (30) is likely
secondary to differing routes of administration, intratracheal vs
systemic. The histology in Fig. 3 would strongly suggest that lung
inflammation is a major component of the early response, whereas
systemic inflammation not amenable to targeted lung expression may
contribute significantly to the later mortality.
Finally, there were clear quantitative differences in the response to
hIL-10 and vIL-10. The biological activity of vIL-10 is thought to
represent a subset of activities associated with cellular IL-10. vIL-10
possesses profound anti-inflammatory and immunosuppressive
activities, similar to those possessed by hIL-10. However, although
hIL-10 is known to inhibit IFN- production, MHC class II expression,
T cell proliferation, and B cell IgE production (13, 31),
hIL-10 also has immunostimulatory properties that vIL-10 lacks. hIL-10
can act as a stimulatory factor for immature and mature thymocytes,
mast cells, and B cells (32, 33), whereas vIL-10 lacks
these properties (13, 34). These differing properties of
hIL-10 and vIL-10 are thought to be attributable to a single amino acid
substitution in vIL-10 (35). These contrasting immune
properties may account for the differing biological responses that are
seen in the Ad/hIL-10 and Ad/vIL-10 treatment groups in the zymosan and
pancreatitis models.
A more simple explanation may rest in the differing pharmacokinetics
and pharmacodynamics between Ad/hIL-10 and Ad/vIL-10. In a recent
report, we observed that intratracheal instillation of Ad/vIL-10
resulted in peak lung concentrations that were 10–100 times greater
than peak hIL-10 levels seen with Ad/hIL-10 transductions
(15). vIL-10 appeared to be sequestered more effectively
in tissues than hIL-10, whereas hIL-10 appeared more readily in the
systemic circulation. In addition, the duration of IL-10 expression was
markedly longer in the mice receiving intratracheal instillations of
Ad/vIL-10 than in those receiving Ad/hIL-10. hIL-10 expression lasted
only 10–14 days, whereas vIL-10 expression lasted in excess of 42 days
in healthy animals (15). This latter phenomenon may
explain the failure of Ad/hIL-10 to produce sustained survival through
the third phase of the zymosan-induced illness (MSOF). Zymosan mice
pretreated with Ad/hIL-10 started to lose weight and die 9 to 11 days
after transduction, whereas mice pretreated with Ad/vIL-10 sustained
their body weight and maintained survival. The increased lung vIL-10
concentrations that are typically seen after intratracheal delivery of
Ad/vIL-10 as compared with Ad/hIL-10 may also explain the improvement
in lung histology seen in Fig. 3.
In conclusion, the results of the present studies demonstrate that adenoviral gene therapy expressing hIL-10 and vIL-10 can be safely administered during acute inflammatory events and can improve outcome and reduce inflammation. Gene therapy offers a novel approach for the delivery of protein-based therapies in acute inflammation and can effectively target individual tissues. As gene therapy becomes a more acceptable mode of treatment, it is anticipated that adenoviral-based therapies will become available as a drug delivery system. Coupled with gene therapy, cytokine-modulating therapies like IL-10 represent an attractive therapeutic approach for the treatment of the acutely inflamed patient.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Lyle L. Moldawer, Department of Surgery, Room 6116, Shands Hospital, University of Florida College of Medicine, P. O. Box 100286, Gainesville, FL 32610. E-mail address: moldawer{at}surgery.ufl.edu
3 Abbreviations used in this paper: Ad, adenovirus; CDE, choline deficient, ethionine supplemented; h, human; v, viral; MSOF, multisystem organ failure; gww, gram wet weight.
Received for publication February 15, 2001. Accepted for publication May 8, 2001.
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plays a central role in immune-mediated clearance of adenoviral vectors. Proc. Natl. Acad. Sci. USA 94:9814. receptor signaling and IL-10 gene therapy regulate the innate and humoral immune responses to recombinant adenovirus in the lung. J. Immunol. 164:443. decreases inflammation and prolongs adenovirus gene expression in lung and liver. Hum. Gene Ther. 9:1875.[Medline]
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; 47%; #, p <
0.05) and Ad/vIL-10 (
; 73%; *, p < 0.001),
as compared with mice treated with buffer alone (•; 25%).
B, Early mortality (days 1–4) was modestly increased in
the mice pretreated with the Ad/empty, as compared with buffer alone
(p > 0.05 by Fisher’s exact test). However,
18-day survival was not significantly different in the animals
pretreated with Ad/empty (
; 22%) as compared with mice pretreated
with buffer alone (•; 33%; p = 0.3).
