Inflammatory bowel disease (IBD) is characterized by altered immunoregulation
and augmented intestinal synthesis of nitric oxide. The purpose of this
study was to determine the effects of exogenous IL-4, introduced by a recombinant
human type 5 adenovirus (Ad5) vector, on the tissue injury associated with
an experimental model of colonic immune activation and inflammation. Colitis
was induced in rats by the intrarectal administration of trinitrobenzene
sulfonic acid (TNB) dissolved in 50% ethanol, and control rats received
saline via the same route. 1 h later, all rats were randomized into two
groups. The first group was injected intraperitoneally (ip) with 3.0 x
10(6) plaque forming units (PFUs) of Ad5 transfected with murine interleukin-4
(Ad5IL-4) and the second group was injected ip with the same amount of
Ad5 expressing the Escherichia coli Lac Z gene (Ad5LacZ). One-half of the
colitic and control rats were injected again with 3.0 x 10(6) PFUs of Ad5IL-4
or Ad5LacZ on day 3 of the 6-d study. When introduced once or twice via
the peritoneal route into control rats, Ad5LacZ was localized to the serosal
lining of the peritoneal cavity, the diaphragm and the liver on day 6.
One or two injections of Ad5IL-4 into rats also produced measurable levels
of circulating IL-4. TNB-colitis in both Ad5LacZ-treated groups was associated
with pronounced elevations in serum IFN-gamma, and mucosal ulceration of
the distal colon. Myeloperoxidase and inducible nitric oxide synthase II
(NOS II) synthetic activity were also increased by 30- and fivefold, respectively,
above control levels in the distal colon. However, two injections of Ad5IL-4
into colitic rats caused the overexpression of IL-4, and significantly
inhibited tissue damage, serum and colon IFN-gamma levels and myeloperoxidase
activity in the distal colon. In addition, NOS II gene expression and NOS
II nitric oxide synthesis was significantly inhibited. No therapeutic effect
was observed in rats injected once with Ad5IL-4. Thus, IL-4, introduced
by Ad5, is therapeutic during acute inflammation in the rat colon. The
therapeutic effect of IL-4 was associated with an inhibition of inducible
nitric oxide expression and a reduction in nitric oxide synthesis.
Gene Ther 1997 Nov;4(11):1181-1188
[Medline record in process]
The most limiting factor regarding the use of TNF alpha in tumour therapy is systemic toxicity. The expression of membrane-bound (nonsecreted) TNF alpha within a tumour may serve to reduce systemic toxicity while retaining anti-tumour activity. Two adenovirus (Ad) vectors were constructed: (1) Ad-mTNF-wt expressing wild-type murine TNF alpha; and (2) Ad-mTNF-MEM expressing a mutant non-secreted (membrane-bound) form. Only the Ad-mTNF-wt vector induced high levels of TNF alpha secretion in transduced cells (approximately 400 ng/10(6) cells), however, both vectors induced efficient cell surface expression as detected by FACS. These vectors were used in tumour immunotherapy trials in a murine transgenic breast cancer model. High serum concentrations of mTNF alpha (approximately 1 ng/ml) were detected only in Ad-mTNF-wt-treated mice, while both vectors induced substantial disruption of tumour pathology. The wt TNF vector was highly toxic, killing 12 of 16 mice at a dose of 5 x 10(8) p.f.u., whereas the Ad-mTNF-MEM vector showed low toxicity killing three of 27 at the same dose. Both vectors induced partial, and in some cases, permanent tumour regressions, with cured mice displaying protective immunity and specific CTL activity against the tumour. These results indicate that the use of a nonsecreted form of TNF alpha can result in a relatively large reduction in systemic toxicity with little or no reduction in antitumour activity.
PMID: 9425441, UI: 98086578
Gene Ther 1997 Oct;4(10):1069-1076
Adenovirus (Ad) vectors are being intensively studied as vehicles for cancer gene therapy. We have been exploring the benefits of direct intratumoral injection of Ads expressing cytokines for immunotherapy. Our previous work demonstrated that therapy using a vector expressing interleukin-12 (AdmIL-12.1) produced regressions in approximately 80% of treated tumors supporting further preclinical investigations. Recent reports have shown that immunity to Ad can be a major limiting factor in Ad-mediated gene transfer. As most animal studies with Ad vectors have involved nonimmune hosts, it remains difficult to predict how effective these treatments will be in humans, where the majority of individuals have had previous exposure to Ad. To address this question, we compared the effectiveness of the AdmIL-12.1 cancer therapy in naive and Ad-immune mice. We found that both groups responded equally well to treatment and that the response to AdmIL-12.1 in both groups resulted in the generation of CTL reactive against tumor antigen indicating that antitumor immunity was achieved. Peak transgene expression in the tumor was only reduced by 2.4 fold in Ad-immune animals compared with nonimmune mice. It was also observed that in naive animals, the virus disseminated from the site of the tumor following injection and by 72 h substantial transgene expression was detected in peripheral organs, most notably the liver. Transgene expression in the liver of Ad-immune animals was reduced by greater than 1000-fold relative to that in naive mice. These results strongly support the clinical utility of Ad-based cancer gene therapy and suggest that Ad immunity may be advantageous in that it is not a complete block to gene transfer in the tumor and it greatly reduces virus dissemination.
Stimulation of antitumor immune mechanisms is the primary goal of cancer
immunotherapy, and accumulating evidence suggests that effective alteration
of the host-tumor relationship involves immunomodulating cytokines and
also the presence of costimulatory molecules. To examine the antitumor
effect of direct in vivo gene transfer of murine interleukin 12 (IL-12)
and B7-1 into tumors, we developed an adenovirus (Ad) vector, AdIL12-B7-1,
that encodes the two IL-12 subunits in early region 1 (E1) and the B7-1
gene in E3 under control of the murine cytomegalovirus promoter. This vector
expressed high levels of IL-12 and B7-1 in infected murine and human cell
lines and in primary murine tumor cells. In mice bearing tumors derived
from a transgenic mouse mammary adenocarcinoma, a single intratumoral injection
with a low dose (2.5 x 10(7) pfu/mouse) of AdIL12-B7-1 mediated complete
regression in 70% of treated animals. By contrast, administration of a
similar dose of recombinant virus encoding IL-12 or B7-1 alone resulted
in only a delay in tumor growth. Interestingly, coinjection of two different
viruses expressing either IL-12 or B7-1 induced complete tumor regression
in only 30% of animals treated at this dose. Significantly, cured animals
remained tumor free after rechallenge with fresh tumor cells, suggesting
that protective immunity had been induced by treatment with AdIL12-B7-1.
These results support the use of Ad vectors as a highly efficient delivery
system for synergistically acting molecules and show that the combination
of IL-12 and B7-1 within a single Ad vector might be a promising approach
for in vivo cancer therapy.
J Clin Invest 1997 Aug 15;100(4):768-776
Transforming growth factor (TGF)-beta1 has been implicated in the pathogenesis of fibrosis based upon its matrix-inducing effects on stromal cells in vitro, and studies demonstrating increased expression of total TGF-beta1 in fibrotic tissues from a variety of organs. The precise role in vivo of this cytokine in both its latent and active forms, however, remains unclear. Using replication-deficient adenovirus vectors to transfer the cDNA of porcine TGF-beta1 to rat lung, we have been able to study the effect of TGF-beta1 protein in the respiratory tract directly. We have demonstrated that transient overexpression of active, but not latent, TGF-beta1 resulted in prolonged and severe interstitial and pleural fibrosis characterized by extensive deposition of the extracellular matrix (ECM) proteins collagen, fibronectin, and elastin, and by emergence of cells with the myofibroblast phenotype. These results illustrate the role of TGF-beta1 and the importance of its activation in the pulmonary fibrotic process, and suggest that targeting active TGF-beta1 and steps involved in TGF-beta1 activation are likely to be valuable antifibrogenic therapeutic strategies. This new and versatile model of pulmonary fibrosis can be used to study such therapies.
Mol Med 1997 Aug;3(8):519-529
BACKGROUND: The pleiotropic cytokine interleukin-6 mediates its multiple
effects at the cell level through a multimeric receptor consisting of a
binding protein (gp80) and a signal transducer (gp130). A soluble form
of gp80 (sIL-6R or gp55) is found released from the surface of cells and
appears to possess interleukin-6 (IL-6) agonist activity. Increases in
circulating levels of sIL-6R have been reported in different pathological
conditions but the precise role of this protein in vivo remains unknown.
MATERIALS AND METHODS: The cDNA encoding the extracellular domain of the
rat IL-6R (sIL-6R) with an appropriate leader sequence has been cloned
into the E1 region of an adenovirus vector under the control of the hCMV
promoter (Ad5.sIL-6R). RESULTS: Infection of different human or rodent
cell lines with Ad5.sIL-6R leads to extended production of recombinant
sIL-6R protein into the culture media. The kinetics of transgene expression
depends both on the cell type and the species. sIL-6R produced in this
manner is biologically active as it confers responsiveness of human hepatoma
cells (HepG2) to rat IL-6 stimulation. Adenovirus vectors have been shown
to be highly effective for transient delivery of cytokines in vivo. Antibodies
against recombinant rat soluble IL-6R were generated and an ELISA developed
that allowed us to quantify sIL-6R concentrations. The sIL-6R expressing
adenovirus vector has been instilled intratracheally into rats and induced
an increase in lung sIL-6R concentration from Day 1 up to Day 10. We demonstrate
the potency of our system to deliver in vivo or in vitro soluble cytokine
receptors in a prolonged but transient manner.
Gene 1997 Jul 9;193(2):129-140
To explore the utility of the bacteriophage T7 binary system in adenovirus
(Ad) vectors we constructed three Ad5-based vectors containing the T7 RNA
polymerase (T7pol) gene in either early region 1 (E1) or E3. The recombinant
Ad vectors were either deficient (AdT7pol1, AdT7pol2) or competent (AdT7pol3)
for replication in human cells other than Ad5 transformed (293) cells.
To test the ability of the T7 polymerase produced by these vectors to drive
gene expression, a reporter vector was constructed with an E1 substitution
comprising the bacterial beta-galactosidase (betaGal) (lacZ) gene under
the control of the T7 gene 10 promoter (T7pro) and linked to the encephalomyocarditis
virus (EMCV) internal ribosome entry site (IRES) (AdBHG10T7betaGal). Coinfections
were performed with the various AdT7pol vectors and the reporter vector,
and expression was analysed in three different human cell lines: 293, A549
and MRC-5. Depending on the AdT7pol vector used, different levels of expression
were obtained from the reporter gene. In 293 cells, expression was detected
following infection at very low multiplicities of infection (moi) with
all of the T7pol vectors when coinfected with the reporter vector AdBHG10T7betaGal.
In A549 and MRC-5 cells very little expression was detected using AdT7pol1
or pol2 and efficient expression was only obtained when relatively high
moi values of the replication-competent vector were used in the coinfections.
We also constructed a single vector containing both elements of the T7
system (T7pol in E3 and T7 promoter driving expression of the chloramphenicol
acetyl transferase (cat) gene in E1). This vector proved difficult to rescue
but was stable once isolated. Finally, experiments performed to evaluate
the 'leakiness' of the Ad-T7 system detected very little expression from
the T7pro in the absence of T7 polymerase suggesting this system may be
useful for the cloning and expression of genes encoding cytotoxic proteins.
Virology 1997 Jul 7;233(2):423-429
The human-viral junctions of integrated adenovirus type 5 (Ad5) DNA in 293 cells have been cloned and sequenced. The Ad5 sequences extend from nucleotides (nt) 1 to 4344 and are located in the pregnancy-specific beta-1-glycoprotein 4 (PSG 4) gene. This maps the insertion of Ad5 DNA to human chromosome 19 (19q13.2). The Ad5 sequences are represented as a single collinear insertion of viral DNA with no rearrangements. There were 19 bp of PSG4 DNA deleted at the site of insertion and an extra 3 nt (GTC) at the left viral/cellular junction. A short patch of homology between viral DNA and PSG4 DNA (6 nt with 1 mismatch) was present at the right junction.
J Gen Virol 1997 Jul;78( Pt 7):1653-1661
We have developed a number of replication defective adenoviral (Ad) vectors which express transgenes under the control of the human cytomegalovirus (HCMV) immediate early (IE) gene promoter. The orientation of the expression cassette replacing E1 in the vector backbone had a significant effect on the level of transgene expression, with vectors containing expression cassettes directed towards the right end of the Ad genome expressing 7-fold higher levels of beta-galactosidase (beta-gal) than those with inserts in the opposite orientation. Murine cells infected with any of several different Ad vectors in which transgene expression was under the control of the HCMV IE promoter produced 10-100-fold less transgene product (such as beta-gal) than similarly infected human cells. Replacing the HCMV IE promoter with the murine CMV (MCMV) IE promoter resulted in an increase in the levels of beta-gal produced in murine and rat cells by approximately 5-30-fold compared to levels obtained with the HCMV IE promoter, and levels produced in human cells were the same or greater using the MCMV IE promoter compared to the HCMV IE promoter. Similar results were obtained using a luciferase reporter gene. The MCMV IE promoter, therefore, was able to drive high levels of expression without the pronounced species preferences observed for the HCMV IE promoter. The MCMV IE promoter also directed high levels of expression in vivo, suggesting that Ad vectors carrying the MCMV IE promoter may be more effective than those with the HCMV IE promoter for transgene expression in animal models.
Adenoviruses (Ads) are intermediate-sized mammalian DNA viruses with
a double-stranded linear genome of 36 kb. The icosohedral virion has been
shown to accommodate up to 105% of the wild-type genome length, and genomes
larger than this size are either unpackageable or extremely unstable, frequently
undergoing DNA rearrangement. Here we show that the Ad virion also has
a lower packaging limit of approximately 75% of the wild-type genome length.
We have constructed a series of vectors with sizes ranging from 15.1 to
33.6 kb and used these to show that in our Cre/loxP helper-dependent system
(R. J. Parks, L. Chen, M. Anton, U. Sankar, M. A. Rudnicki, and F. L. Graham,
Proc. Natl. Acad. Sci. USA 93:13565-13570, 1996), vectors with genomes
greater than or equal to 27.7 kb are packaged with equal efficiencies,
whereas vectors with smaller genomes are inefficiently packaged. A 15.1-kb
vector, approximately half the size of the wild-type adenovirus genome,
was packaged with an efficiency intermediate between that of the small
(21.3- to 25.7-kb) and large (27.7- to 33.5-kb) vectors. Analysis of vector
DNA after amplification in helper virus-infected cells showed that vectors
below 75% of the Ad genome had undergone DNA rearrangements, whereas larger
vectors were unaltered. The 15.1-kb vector was recovered primarily as a
mix of head-to-tail and tail-to-tail covalent dimers, with a size of 30
kb. We conclude that the Ad virion can efficiently accommodate viral DNA
of greater than 75% of the viral genome but that smaller viral genomes
tend to undergo rearrangement, resulting in a final size of greater than
approximately 27 kb before they can be efficiently packaged. Knowledge
of the lower limit to Ad DNA packaging should allow for the design of better
and more stable vectors.
Interleukin-10 (IL-10) is a potent anti-inflammatory/immune cytokine
and has received growing attention for its therapeutic potential. To aid
therapeutic studies of IL-10 in vivo, a replication-deficient adenoviral
vector expressing mouse IL-10 was constructed and characterized. The transgene
protein IL-10 was shown to markedly inhibit endotoxin-induced tumor necrosis
factor alpha (TNF alpha) production by mouse and rat macrophages in vitro.
Intramuscular injection of this vector in mice resulted in efficient expression
of transgene mRNA in the muscle and active release of IL-10 protein into
the bloodstream. To investigate the therapeutic potential of IL-10 using
this vector, endotoxemia was induced by intraperitoneal injection of a
sublethal dose of endotoxin. Expression of TNF alpha and IL-6 mRNA in the
lung, spleen and heart and the circulating levels of these cytokines markedly
increased in endotoxemia. This endotoxin-induced TNF alpha and IL-6 up-regulation
was however suppressed in mice expressing IL-10 after intramuscular gene
transfer. While cytokine gene expression was inhibited to varying degrees
in different organs, a maximal reduction was seen in the lung, thus also
indicating the efficacy of systemic IL-10 gene product at multiple tissue
sites. Finally, we provided evidence that only when present in abnormally
high concentrations in the circulation following intraperitoneal gene delivery,
IL-10 by itself had some toxic effects of transient nature, primarily manifested
by acute phase reaction and hemostatic disturbance. Thus, our studies demonstrate
the usefulness of adenoviral vectors for therapeutic applications of IL-10
in vivo.
Adv Pharmacol 1997;40:137-206
J Immunol 1996 Dec 1;157(11):5076-5084
RANTES (regulated on activation, normal T cell expressed and secreted) is a member of the C-C superfamily of chemokines and is reported to function as a potent chemoattractant for monocytes, eosinophils, and a subpopulation of CD4+ T cells. Using a recombinant human type 5 adenovirus containing the murine RANTES cDNA (Ad5E3 mRANTES), which is capable of expressing biologically active cytokine upon infection, we initiated a study to characterize the biologic functions of RANTES cytokine in vivo. Intratracheal administration of Ad5E3 mRANTES targeted transient RANTES expression to the bronchial epithelium of the lung in Sprague-Dawley rats. Bronchoalveolar lavage fluids (BAL) collected at 24 h had increased chemotactic activity vs controls as measured in a murine CD4+ T cell Boyden chamber microchemotaxis assay. There was a dramatic increase in the number of cells (macrophage, monocytes, and neutrophils) recovered from BAL samples taken from Ad5E3 mRANTES-treated animals at 24 h, with a >50-fold increase in monocytes, indicating a proinflammatory effect for this cytokine in vivo. This effect on monocytes was transient, decreasing by 7 days, with evidence of increased eosinophils and lymphocytes at this time. Histologic examination of lung sections at 24 h revealed greatly increased numbers of mononuclear cells, primarily monocytes, within the lungs of Ad5E3 mRANTES-treated animals, with increased extravasation of monocytes around blood vessels, indicating an ongoing process of peripheral blood monocyte recruitment. This study provides further evidence for RANTES to be a monocyte chemoattractant in vivo.
Adenoviruses are attractive vectors for the delivery of foreign genes into mammalian cells for gene therapy. However, current vectors retain many viral genes that, when expressed at low levels, contribute to the induction of a host immune response against transduced cells. We have developed a helper-dependent packaging system for production of vectors that have large regions of the genome deleted. Helper viruses were constructed with packaging signals flanked by loxP sites so that in 293 cells that stably express the Cre recombinase (293Cre), the packaging signal was efficiently excised, rendering the helper virus genome unpackageable. However, the helper virus DNA was replicated at normal levels and could thus express all of the functions necessary in trans for replication and packaging of a vector genome containing the appropriate cis-acting elements. Serial passage of the vector in helper virus-infected 293Cre cells resulted in an approximately 10-fold increase in vector titer per passage. The vector could be partially separated from residual helper virus by cesium chloride buoyant density centrifugation. Large scale preparations of vector yielded semi-purified stocks of approximately 10(10) transducing virions/ml, with < 0.01% contamination by the E1-deleted helper virus. This system should have great utility for the generation of adenovirus-based vectors with increased cloning capacity, increased safety and reduced immunogenicity.
Somat Cell Mol Genet 1996 Nov;22(6):477-488
We have constructed 293 cell lines expressing the site-specific Cre
recombinase from bacteriophage P1, that acts on a 34 bp target sequence
called loxP. Stably transformed cells were obtained by transfection with
a plasmid containing Cre and a selectable marker under the control of viral
promoters. The resulting 293Cre cell lines could be used to induce expression
from adenovirus vectors containing reporter genes under the control of
a Cre responsive "molecular switch." High efficiency recombination was
observed for Ad viral DNA containing loxP sites. The Cre expressing cell
lines described here are likely to be useful for several purposes: For
expression of toxic gene products from Cre inducible viral vectors, to
induce recombination between loxP sites in transfected plasmids, and to
induce deletions or rearrangements of genes defined by loxP sites in viral
genomes.
Hum Gene Ther 1996 Oct 20;7(16):1995-2002
Mice bearing breast tumors were treated with a single dose of an adenovirus
expressing interleukin-12 (AdmIL-12.1) injected intratumorally, which produced
regressions in greater than 75% of the treated tumors; approximately one-third
of the animals remained tumor free. Complete regression was associated
with immunity to secondary challenge with fresh tumor cells. Analysis of
local cytokine expression demonstrated maximum expression of IL-12 within
the tumor between 24 and 72 hr post-injection, reaching 600-800 ng per
tumor, with elevated local levels of IL-12 detectable for at least 9 days.
This expression was highly localized as serum IL-12 peaked at 40-60 ng/ml
at 24 hr and was less than 10 ng/ml from day 3 onward. Interferon-gamma
(IFN-gamma) concentrations were markedly increased within the tumor following
AdmIL-12.1 administration, demonstrating that IL-12 was acting locally.
Tumor-draining lymph node cells spontaneously produced IFN-gamma following
AdmIL-12.1 treatment, suggesting these cells were activated by IL-12. These
data demonstrate that AdmIL-12.1 can be used to deliver very high levels
of localized cytokine production. Moreover, we have confirmed that the
IL-12 produced from our vector actually affects the local cytokine environment
of the tumor and activates responder cells present within the tumor.
The prevalence of wildlife rabies throughout the world and the continued
spread of this disease in North America highlights the need for oral vaccines
which may be used safely and effectively to vaccinate a number of species
that are reservoirs or vectors of rabies. We have previously shown that
AdRG1, a replication competent recombinant human adenovirus type 5 (Ad5)
expressing a rabies glycoprotein (RG), can induce immunity to rabies in
rodent, canine, and skunk model systems. To improve the Ad5 vector system
as a potential oral vaccine, we have constructed additional Ad5 recombinant
vectors and compared RG expression in cell culture and immunogenicity in
animals. Two new replication competent vectors are compared. AdRG1.3, which
carries RG with accompanying SV40 poly A addition sequences within an E3
deletion, and AdRG4, which has RG in the E3 deletion but under the control
of an exogenous Ad2 major late promoter, both express higher levels of
RG in permissive cell culture than did AdRG1 and both elicit high levels
of serum anti-rabies antibodies by parenteral or oral routes in animals.
AdRG1.3 may be a more effective vaccine vector in species which are non-permissive
for the replication of human Ad5.
Oncogene 1996 Jul 18;13(2):445-446
Cell extracts from knockout mice can provide definitive proof of antibody
specificity. Two NF-kappa B1-p50 antibodies, sc-114 (a commercial antibody)
and NR1157, were observed to recognize proteins having distinct electrophoretic
mobilities of 52-55 kD and 50 kD, respectively, by Western blot analysis.
In order to discriminate the specificity of these antibodies for NF-kappa
B1-p50, whole cell extracts derived from NF-kappa B1-p105 knockout mice
were employed. While the NR1157 antibody completely failed to recognize
its 50 kD product in p105-/- knockout extracts, the sc-114 antibody still
strongly recognized its 52-55 kD product. These data demonstrate that NR1157,
but not sc-114, is highly specific for NF-kappa B1-p50 by Western blot
analysis. In addition, these results highlight the utility of knockout
cell extracts for discerning antibody specificity.
J Leukoc Biol 1996 Apr;59(4):481-488
Using adenoviral-mediated gene transfer techniques, the murine granulocyte-macrophage
colony-stimulating factor (GM-CSF) transgene is efficiently targeted to
and highly expressed by the respiratory epithelium of rat lung. This lung
tissue-directed expression of GM-CSF induces accumulation of both eosinophils
and macrophages at early stages and an irreversible fibrotic reaction at
later stages. These tissue responses to GM-CSF appear to be distinct from
those induced by other proinflammatory cytokines, interleukin (IL)-5, IL-6,
macrophage inflammatory protein-2 (MIP-2), or RANTES overexpressed in the
lung. These findings clearly demonstrate that GM-CSF is more than a hematopoietic
cytokine in the lung and may play a pivotal role in the multiple pathological
processes underlying numerous respiratory illnesses, including asthma.
In this overview, the differences in tissue responses induced by GM-CSF
and other individual cytokines are highlighted. In addition, the mechanisms
by which GM-CSF and other individual cytokines are highlighted. In addition,
the mechanisms by which GM-CSF contributes to the development of eosinophilia,
macrophage granuloma, and fibrosis are discussed in conjunction with the
recent findings from us and others.