U.S. patent application number 10/776119 was filed with the patent office on 2004-08-12 for controlling immune response to specific antigens.
Invention is credited to Curiel, David T., Mountz, John D., Zhang, Huang-Ge.
Application Number | 20040157792 10/776119 |
Document ID | / |
Family ID | 30772379 |
Filed Date | 2004-08-12 |
United States Patent
Application |
20040157792 |
Kind Code |
A1 |
Mountz, John D. ; et
al. |
August 12, 2004 |
Controlling immune response to specific antigens
Abstract
One major problem with adenovirus gene therapy has been the
T-cell mediated immune response elicited by inoculation of
adenovirus, which leads to rapid clearance of the virus and loss of
transgene expression. In the instant invention, the immune response
to a virus is prevented by pre-treatment with adenovirus,
adenoassociated virus or herpes virus infected antigen-presenting
cell (APC) expressing Fas ligand with induced T-cell tolerance.
Administration of AdCMVLacZ after tolerance resulted in prolonged
expression of LacZ in tolerized animals compared to control treated
animals. In control, but not tolerized animals, there was
proliferation of CD3.sup.+T-cell in the spleen in response to
AdCMVLacZ treatment. Tolerance induction is also indicated by
decreased production of interferon-.gamma. and IL-2 by peripheral
T-cells isolated from treated animals after stimulation with the
adenovirus infected APCs. T-cell tolerance is specific for the
virus as the T-cell responses to an irrelative virus, mouse
cytomegalovirus (MCMV) remained unimpaired. The instant invention
utilizes virus specific T-cell tolerance, which is induced by APCs
that co-express Fas ligand and virus antigens. The instant
invention involves novel vectors and methods to induce tolerance to
a viral vector gene therapy and prolong expression of a transgene
in a viral host.
Inventors: |
Mountz, John D.;
(Birmingham, AL) ; Curiel, David T.; (Birmingham,
AL) ; Zhang, Huang-Ge; (Birmingham, AL) |
Correspondence
Address: |
GIFFORD, KRASS, GROH, SPRINKLE
ANDERSON & CITKOWSKI, PC
280 N OLD WOODARD AVE
SUITE 400
BIRMINGHAM
MI
48009
US
|
Family ID: |
30772379 |
Appl. No.: |
10/776119 |
Filed: |
February 10, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10776119 |
Feb 10, 2004 |
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09424281 |
Jan 2, 2000 |
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6689605 |
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09424281 |
Jan 2, 2000 |
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PCT/US98/10381 |
May 22, 1998 |
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60047426 |
May 22, 1997 |
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Current U.S.
Class: |
514/44A ;
424/93.21 |
Current CPC
Class: |
A61K 48/005 20130101;
A61K 48/00 20130101; A61K 2039/5154 20130101; C07K 14/70575
20130101; C12N 2799/022 20130101 |
Class at
Publication: |
514/044 ;
424/093.21 |
International
Class: |
A61K 048/00 |
Claims
1. A method for promoting immunotolerance in a host to a gene
therapy vector, comprising the step of: transfecting a host cell
with said vector, such that said vector expresses a transgene, an
antigen and a Fas 2 ligand, wherein expression of said Fas 2 ligand
induces apoptosis in a T-cell raised against said antigen in the
host.
2. The method of claim 1 further comprising the step of: exposing
said host to a second vector following therapeutic gene expression,
said second vector expressing said antigen and a second ligand
wherein expression of said second ligand induces apoptosis in said
T-cell.
3. The method of claim 2 wherein said second ligand induces
apoptosis of said T-cell by the same mechanism as said Fas 2
ligand.
4. The method of claim 3 wherein said Fas 2 ligand interacts with a
death domain region molecule DRX of said T-cell, wherein X is
selected from the group consisting of 3, 4, and 5.
5. The method of claim 1 wherein transfecting said host cell occurs
in vitro.
6. The method of claim 1 wherein transfecting said host cell occurs
in vivo.
7. The method of claim 6 wherein transfecting said host cell occurs
by an intra-nasal pathway.
8. The method of claim 6 wherein transfecting said host cell occurs
by an intravenous pathway.
9. The method of claim 1 wherein said vector is selected from the
group consisting of: a recombinant adenovirus, a recombinant
adeno-associated virus, and a recombinant herpes virus.
10. The method of claim 1 wherein said vector is selected from the
group consisting of: adenovirus, adeno-associated virus and herpes
virus.
11. The method of claim 10 wherein said vector is replication
defective.
12. The method of claim 10 wherein said vector encodes only
nonpathogenic polypeptides.
13. The method of claim 1 wherein said antigen is a polypeptide
encoded for by a vector associated gene.
14. A method for creating an immune privileged site in a tissue of
an organism, said method comprising the steps of: providing a gene
therapy vector encoding and expressing a Fas 2 ligand, a transgene
and an antigen in the tissue of the organism; and infecting cells
of said tissue with said vector, whereby expression of the Fas 2
ligand in said tissue induces apoptosis of T-cells raised against
said antigen to confer specific immunity to infected cells.
15. The method of claim 14 further comprising the step of:
reinfecting said tissue with said vector so as to prolong
expression of said therapeutic gene.
16. The method of claim 14 wherein said transgene is selected from
the group consisting of CFTR, Factor 8, protease inhibitor and
insulin.
17. The method of claim 14 wherein said vector is a recombinant
adenovirus.
18. The method of claim 14 wherein said vector is selected from the
group consisting of: adenovirus, adeno-associated virus and herpes
virus.
19. The method of claim 18 wherein said vector is replication
defective.
20. The method of claim 18 wherein said vector encodes only
nonpathogenic polypeptides.
21. A gene therapy viral vector comprising: a transgene; a viral
vector gene that is expressed as an antigen on an infected host
cell; a Fas 2 ligand gene; and a gene expression control means for
directing product synthesis of said transgene and said Fas 2 ligand
gene in a host.
Description
RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 09/424,281 filed Jan. 2, 2000, now U.S. Pat.
No. 6,689,605, which is a U.S. national phase application under 371
of PCT Application No. PCT/US98/10381 filed May 22, 1998, claiming
priority of U.S. Provisional Patent Application Serial No.
60/047,426 filed May 22, 1997. These applications are incorporated
herein by reference.
FIELD OF THE INVENTION
[0002] This invention relates generally to gene therapy. More
specifically, the invention relates to suppressing immune system
response to antigens expressed on an infected host cell.
BACKGROUND OF THE INVENTION
[0003] The proper function of the immune system of an organism is
to attack and neutralize materials which are perceived as being
foreign to that organism. T-cells are one component of the immune
system. T-cells can become activated to specific antigens, and
function to directly destroy materials which display that antigen,
and they also function to sensitize other components of the immune
system to the presence of that antigen. While a properly
functioning immune system is vital to the health of an organism, in
some instances there is a need for the selective inhibition of an
immune response to particular materials.
[0004] For example, viral vectors, such as adenovirus, are employed
in genetic therapies to introduce genetic material and products
into an organism. One problem encountered with the use of such
viral vectors is that they can provoke an immune response in the
organism. This immune response can destroy the viral vector, and
those host cells which are intentionally infected by the vector, as
well as therapeutic gene products produced by the action of the
vector. Furthermore, immune system "memory" provides a lasting
response to this vector; hence, readministration of the material
will be ineffective. Therefore, there is a need for a method
whereby the immune response to a selected viral vector may be
blocked or destroyed. Suppression of immune response is also
desirable in the instances of autoimmune disease. As is known, such
disease results when the immune system of an organism
inappropriately recognizes an organ or tissue of that organism as
being foreign, and commences an immune response against it. If this
immune response can be blocked, the autoimmune disease can be
controlled. Immune suppression is also needed in those instances
where organs are transplanted. Immune system suppressing drugs are
sometimes employed in the foregoing situations; however, such drugs
produce a generalized suppression of the immune system, which
leaves a patient open to a number of infections. It would therefore
be advantageous if immune response to a specific antigen could be
suppressed and/or an immune suppressed zone of tissue created
within an organism.
[0005] Gene therapy is limited by induction of an immune response
to the virus or the gene-therapy protein product (1-4). A specific
T-cell response to the viral vector usually results in the failure
of re-expression of transgene (5-6). Many efforts have been made to
reduce the T-cell response to the viral vector during gene therapy,
including the blockade of MHC class I and II antigen, reduction of
the antigenicity of the viral vector, and prevention of
co-stimulation of T-cells (1,7-11).
[0006] One important mechanism for maintaining peripheral T-cell
tolerance is clonal deletion of antigen-specific T-cells, which is
mediated by apoptosis (12-15). Cytokine and cytokine ligand
mediated apoptosis has been shown to be an important pathway for
activation-induced cell death in T-cells (16-17). T-cell activation
leads to upregulation of cytokine ligand and cytokine apoptosis
signaling (18,19). Activated macrophages express increased levels
of cytokine ligand and mediate apoptosis in the T-cells during
antigen presentation, which has been thought to be a critical means
of down-modulating T-cell response (20,21).
[0007] In particular, the efficiency of adenovirus-mediated gene
transfer has been found to be far superior to other methods for the
treatment of heart, lung, and liver disease, and is capable of
producing more recombinant protein (22,23). However, the
cell-mediated immune response to E1a-E3-deleted adenoviral (Ad5)
vector and the limited distribution of reporter gene expression
suggest that less immunogenic recombinant vectors and more
homogeneous administration methods are required before Ad5 vectors
can be used successfully for phenotypic modulation. Neonatal
intrathymic injection of the vector was able to induce long-term
LacZ expression for more than 2 months after heart injection,
although neutralizing as well as anti-.beta.-Gal antibodies were
detected in the sera of the animals (24). Pretreatment with the
anti-TCR monoclonal antibody (mAb) H57 resulted in a significant
reduction in lymphocytic infiltration and a prolongation of
transgene expression (25). Studies with adenoviral vectors show
that immune responses limit the efficacy and persistence of gene
expression. HSVtk/ganciclovir therapy was more effective in nude
rats and immunosuppressed Fischer rats than in immunocompetent
Fischer rats (26). The immune response against adenovirally
transduced cells limits the efficacy of the HSVtk/ganciclovir
system and that immunosuppression appears to be a useful adjunct.
Adenoviral transgene expression was transient in the thymus of
immunocompetent mice but persistent in CD8.sup.+T-cell-deficien- t
and severe combined immunodeficiency (SCID) mice, implicating a
role for cytotoxic T lymphocytes in viral clearance (27).
Intrathymic transplantation of syngeneic pancreatic islet cells
infected with adenovirus impaired the normal antiviral cytotoxic
T-lymphocyte response and prolonged hepatic transgene expression
after an intravenous challenge with adenovirus.
[0008] Ad5 vector expressing the lacZ transgene, upon delivery
intra-articularly (5.times.10.sup.8 p.f.u.), lacZ expression was
observed in the articular synovium for at least 14 days.
Anti-T-cell mAbs may be useful in inhibiting this immune response.
Improved cell lines allow propagation of Ad with less genetic
material, which decreases the antigenicity (28). The biologic
efficacy and safety profile of second-generation adenovirus for
CFTR gene was evaluated after transfer to baboon lung. This
second-generation virus is deleted of E1 and contains a
temperature-sensitive mutation in the E2a gene, which encodes a
defective DNA-binding protein. Using a second-generation
adenovirus, recombinant gene stability was prolonged and associated
with a diminished level of perivascular inflammation as compared to
first-generation vectors (29). These data suggest that
second-generation adenoviral vectors provide an improved gene
delivery vehicle and are useful in gene therapy for diseases such
as cystic fibrosis.
[0009] Previous attempts to inhibit the immune response to
adenovirus vector or transgenic products have all limited the
utility of transgenic therapies. One technique of pre-toleration of
the adenovirus is to induce neonatal toleration (30). Intratracheal
administration of E1 deleted adenovirus within three days of birth
resulted in transgene expression for over 6 months in cotton rats.
Readministration of virus into 8 to 10 week old animals resulted in
low levels of neutralizing antibodies. Later there was a T-cell
response which correlated with existence of the transgene from the
vector administered at birth, and also the eventual development of
neutralizing antibodies (30). Neonatal administration of E1 deleted
adenovirus to the small intestines also prolonged gene expression
and decreased inflammatory response. Other investigators have used
oral tolerance in rats to prolong gene expression and enable
repeated injections lasting 100 days along with markedly inhibited
lymphocyte response (31). The present invention for tolerance
induction has the advantage that it does not require neonatal
administration of the adenovirus.
[0010] Another mechanism of tolerance is the use of immune
privileged sites. This tolerance makes use of the natural
occurrence of immune privileged sites which has more recently been
thought to be due to production of Fas ligand in subsequent killing
of T-cells that may develop and react with antigens within these
sites. Installation of adenovirus into these sites results in
tolerance to adenovirus and its transgene product. This has been
tested using E1 deleted adenovirus injected into this subretinal
space which resulted in minimal cellular and humeral immune
response (32). The pancreatic islet may also be an immune
privileged site since murine pancreatic islets injected ex-vivo
with Ad5 resulted in high level of beta galactosidase for at least
20 weeks after re-implantation (33). Adenovirus mediated gene
transfer in adult mouse islets does not impair insulin secretion by
the islets (34). Ad lacZ injected subretinally resulted in
prolonged gene expression, which was equivalent to that observed in
either nude mice or after treatment with CTLA4Ig (8). The present
invention is more widely applicable since transgene expression is
not restricted to immune privileged sites.
SUMMARY OF THE INVENTION
[0011] In the instant invention antigen presenting cells (APCs)
that express apoptosis inducing ligands and processed viral vector
antigens are utilized to directly induce apoptosis of T-cells
expressing the ligand receptor resulting in vector-specific T-cell
tolerance. High levels of ligand and vector antigens are induced in
APCs by co-infection. In the case of Fas ligand (FasL) as the
cytokine and adenovirus vector co-infection with
AdLoxpFasL+AxCANCre, pre-treatment of recipient mice with the
adenovirus-infected APCs that express Fas ligand resulted in
induction of T-cell tolerance to the adenovirus. The decreased
T-cell response to the viral vector is demonstrated by decreased
cytokine production, decreased cytotoxic T-cell response,
inhibition of clonal expansion of CD3+ T-cells, and prolonged the
expression of a marker transgene. Induction of T-cell tolerance to
adenovirus requires expression of FasL on the APCs, and does not
occur with adenovirus infected control APCs. T-cell tolerance also
requires expression of Fas on the T-cells of recipient mice, since
lpr/lpr mice are not tolerized. The T-cell tolerance is virus
antigen-specific as there is normal T-cell response to mouse
cytomegalovirus (CMV) in tolerized mice. These results indicate
that pre-tolerization with syngeneic APCs co-infected with
AdLoxpFasL+AxCANCre is a novel immunointervention strategy for
tolerance induction to adenovirus gene therapy.
[0012] The instant invention includes a method for promoting
immunotolerance in a host to a gene therapy vector, including
transfecting a host cell with the vector, such that the vector
expresses a transgene, an antigen and a ligand. Expression of the
ligand induces apoptosis in a T-cell that is raised against the
antigen.
[0013] The instant invention also includes a method for creating an
immune privileged site in a tissue of an organism, the method
including providing a gene therapy vector encoding and capable of
expressing a ligand, a transgene and an antigen and infecting cells
of the tissue with the vector. The expression of the ligand in the
tissue thereby induces apoptosis in T-cells raised against the
ligand so as to confer specific immunity to infected cells.
[0014] The instant invention also teaches a gene therapy viral
vector that includes a transgene, an apoptosis ligand gene and a
gene expression control means for directing product synthesis of
said transgene and said ligand gene. In addition, the use of such a
vector for a gene therapy application is detailed.
[0015] The instant invention also discloses a gene therapy viral
vector including a transgene, a viral vector gene that is expressed
as an antigen on an infected host cell, a functional equivalent of
a Fas ligand gene and a gene expression control means for directing
product synthesis of said transgene and said Fas ligand gene.
BRIEF DESCRIPTION OF THE DRAWINGS
[0016] FIG. 1. A schematic illustrating a production method of gene
therapy viral vector to inhibit an immune response to viral vector
antigens and methods of using the same to produce immune privileged
transduced mammalian host cells.
[0017] FIG. 2. Co-infection of APCs with AdloxpFasL+AxCanCre
(APC-AdFasL) results in high levels of FasL capable of inducing
apoptosis of A20 target cells. The AdLoxpFasL is infected into APCs
from lpr/lpr mice with and without AxCANCre. As a comparison, the
APCs are also electroporation transfected with pcDNA3FasL and
stimulated with lipopolysaccharide (LPS) (1 ug/ml). FasL expression
is determined by ability of the transfected APCs to induce
apoptosis of a 51Cr labeled, Fas sensitive cell line A20.
[0018] FIG. 3. Prolongation of transgene expression by Ad/FasL
expressing APCs. Ten-week-old C57B116-+/+ mice are treated with
1.times.10.sup.6 of the APCs co-infected with AdLoxpFasL plus
AxCANCre (APC-AdFasL) or APCs co-infected with AdLoxpFasL plus
AdCMVGFP (APC-AdControl) or PBS every 3 days for 5 doses. After
induction of T-cell tolerance, mice are intravenously inoculated
with 1010 Ad/LacZ. At the indicated time points, LacZ gene
expression in the liver is analyzed by a quantitative assay and In
situ LacZ histochemical staining. The error bars indicate the
mean.+-.SEM for 3 mice analyzed separately in triplicate assay.
[0019] FIG. 4. Induction of tolerance to adenovirus by APC-AdFasL.
Ten-week-old C57BL/6-+/+ mice are injected intravenously with
1.times.10.sup.6 APC-AdFasL, APC-AdControl or with PBS every 3 days
for 5 doses as described above. On day 7 after the final injection,
mice are challenged with AdCMVlacZ and T-cell cytotoxic response
against APC+adenovirus is determined by killing of the APC cells
infected with AdCMVGFP (5 pfu/cell). The percentages of viable GFP
expressing APC cells are quantitated by FACS analysis. The error
bars indicate the mean.+-.SEM for 3 mice analyzed separately in
triplicate assays.
[0020] FIGS. 5A and 5B. Decreased IFN-gamma and IL-2 induction by
spleen cells from tolerized B6+/+ mice. 10.sup.6 of the APC-AdFasL
or APC-AdControl cells were transferred to B6+/+ mice. The spleen
cells were incubated for 24 hours with APCs that were uninfected,
or infected with adenovirus, and irratiated. Levels of IL-2(A) and
IFN-.gamma. (B) in the supernatant was determined by ELISA.
[0021] FIG. 6. IL-2 induction by spleen cells from tolerized B6+/+
mice. 10.sup.6 of the APC-AdFasL or APC-AdControl cells are
transferred to B6+/+ mice. The spleen cells are incubated for 24
hours with APCs that were uninfected, or infected with adenovirus,
and irratiated. Levels of IL-2 in the supernatant are determined by
ELISA.
[0022] FIG. 7. IFN-gamma induction by spleen cells from B6 lpr/lpr
mice. 10.sup.6 of the lpr APC-AdFasL or APC-AdControl cells are
transferred to B6 lpr/lpr mice. The spleen cells were incubated for
24 hours with APCs that are uninfected, or infected with
adenovirus, and irratiated. Levels of IFN-.gamma. in the
supernatant are determined by ELISA.
[0023] FIG. 8. Ad/FasL APCs induces specific T-cell tolerance to
adenovirus. C57BL/6-+/+ mice (5 mice/group) are treated with either
C57BL/6-+/+ mice (5 mice/group) are treated with APC-AdFasL or
APC-AdControl (M.phi.-CV). Seven days later, mice are challenged in
vivo with either AdCMVLacZ or mouse cytomegalovirus (MCMV). After
an additional 7 days, splenic T-cells are stimulated in vitro with
APCs alone, or APCs infected with MCMV or AdCMVLacZ. IL-2
production in the supernatants was determined by ELISA 48 hours
later.
[0024] FIGS. 9a-9e. Characterization of Fas ligand expressing APCs.
Peritoneal resident macrophages from B6-lpr/lpr mice are isolated
and cultured in RPMI-1640-12% FCS. After short-term culture,
growing macrophages are tested for MHC and B7 expression. (a)-(c)
1.times.10.sup.6 macrophages are stained with biotin-conjugated
anti-H-2 D.sup.b, anti-IA.sup.b (PharMingen) or CTLA4-Ig (Dr.
Linsley: Bristol-Myers Squibb), followed by FITC-conjugated
streptavidin (Southern Biotechnology). 10,000 viable cells are
analyzed by FACScan. (d) Macrophages are transfected with a
pcDNAIII expression vector (Invitrogen) containing a full length
murine Fas ligand cDNA, or empty vector, using a standard
DEAE-Dextran method. Transfected macrophages are selected with 0.5
mg/ml of G418 (Sigma). The selected macrophages are mixed with
[.sup.51C]r-labeled, Fas ligand sensitive A20 cells at the
indicated ratios and, after an 8 h incubation, the specific release
is determined. (e) The splenic T-cells are purified from 4-wk-old
MRL/MpJ-+/+ and MRL/MpJ-lpr/lpr mice (Jackson Laboratory) using a
T-cell enrichment column (R&D Systems). 5.times.10.sup.5
purified T-cells are cultured with 5.times.10.sup.4
.gamma.-irradiated macrophages in round-bottom, 96-well plates for
5 d, and proliferation is determined by adding 1 mCi of
[.sup.3H]-thymidine (Amersham) 16 h prior to harvest.
[0025] FIGS. 10A-10C. Induction of allogeneic T-cell tolerance by
Fas ligand expressing APCs. 4-wk-old of MRL-+/+ and -lpr/lpr mice
are injected i.v. with macrophages (2.times.10.sup.5) transfected
with Fas ligand or control vector every 3 d for 6 times. On d 3 of
the final injection, splenic T-cells are isolated from treated mice
and cultured under various stimulatory conditions. (a)
5.times.10.sup.5 T-cells are cultured with 2.times.10.sup.5
.gamma.-irradiated total spleen cells from B6+/+ mice. (b)
5.times.10.sup.5 T-cells are cultured with 2.times.10.sup.5
.gamma.-irradiated total spleen cells from BALB/c mice. (c)
5.times.10.sup.5 T-cells are cultured with 5 mg/ml of anti-CD3
antibody. T-cell proliferation is determined by incorporation of
[.sup.3H]-thymidine at indicated time points. The error bars
indicate the mean.+-.SEM for 3 mice analyzed separately in
triplicate assays.
[0026] FIGS. 11A-11C. Antigen-specific clonal deletion of the
T-cells induced by Fas ligand expressing APCs in H-2 D.sup.b/HY
reactive TCR transgenic mice. (a) Expression of H-2 D.sup.b is
determined as described above and analyzed by flow cytometric
analysis. (b) Fas ligand activity is assayed by specific lysis of
A20 target cells at the indicated E/T ratio as described in FIG. 9.
(c) The CD4 CD8 T-cells (2.times.10.sup.6) from B6-lpr/lpr female
or male mice are injected every 3 d for 3 times into female, TCR
transgenic D.sup.b/HY -+/+ and -lpr mice. To demonstrate the
requirement for FAS ligand in induction of T-cell tolerance,
identical tolerizing experiments are carried out by co-injection
with 100 mg of purified mouse Fas-Ig fusion protein capable of
neutralizing Fas ligand in vivo. At the end of 12 d,
5.times.10.sup.5 spleen T-cells are stimulated with 5 mg/ml of
anti-CD3 or anti-clonotypic monoclonal antibody (M33), or with
2.times.10.sup.5 irradiated H-2 D.sup.b/HY stimulator cells. The
error bars indicate the mean.+-.SEM for 3 mice analyzed separately
in triplicate assays.
[0027] FIGS. 12A-12C. Tolerance induction due to Fas-mediated
deletion of M33.sup.+CD8.sup.+ T-cells. (a) Expression of M33, CD8,
and Fas on the T-cells in the PLN is determined by 3-color flow
cytometric analysis. 1.times.10.sup.6 total PLN cells are stained
with biotin-conjugated M33, then with FITC-conjugated anti-CD8 and
PE-conjugated anti-Fas (PharMingen). 10,000 viable lymphocytes were
analyzed by FACScan. Two-color contour plots of CD8 and M33 are
shown, and the percentage of M33.sup.+CD8.sup.+ T-cells multiplied
by the total number of spleen cells. The error bars indicate the
mean.+-.SEM for 3 mice analyzed. (b) Fas expression on the
M33.sup.+CD8.sup.+ cells. (c) The M33.sup.+CD8.sup.+ cells are
gated and the histograms of Fas are shown. The percentage of Fas
expression on the gated M33.sup.+CD8.sup.+ T-cells is
indicated.
[0028] FIGS. 13A-13C. Fas ligand expressing p islet cells induce
specific T-cell tolerance. (a) NIT-1 cells are transfected with
pcDNAIII vector containing Fas ligand gene (NIT-i/FL) or empty
vector (NIT-1/Ctl), and selected with G418. Fas ligand activity is
measured by a [.sup.51Cr] release assay. (b) 6-wk-old female NOD
mice are i.p. injected with 5.times.10.sup.5 NMT-1/FL or NIT-1/Ctl
once. Splenic T-cells are isolated 2 wk later and co-cultured with
irradiated NIT-1 cells. Proliferative T-cell response is determined
by [.sup.3H]-thymidine incorporation after 72 h culture. (c) The
splenic T-cells from Example 25 are incubated with
[.sup.51Cr]-labeled NIT-1 cells at indicated E/T ratios, specific
release is determined at 12 h.
[0029] FIGS. 14A and 14B. Histologic Analysis of Insulitis. 6
wk-old female NOD mice are i.p. injected with 5.times.10.sup.5
NIT-1/Ctl (A) or NIT-1/FL (B). Mice are sacrificed at 12 week of
age. H&E stained paraffin sections of pancreas were examined
(400.times.).
[0030] FIG. 15. Prolonged expression of Ad/Luc in muscle
co-transfected with pFasL. Tongue muscles of mice (5 mice/group)
were analyzed at different time points for luciferase production.
There was increased production of luciferase in muscle cells
injected with adenovirus plus FasL compared to muscle injected with
adenovirus and control empty vector.
[0031] FIG. 16. Construction of p.DELTA.E1sp1b/FL and PJM17.
Production of p.DELTA.E1sp1b/FasL. Shown is a 10.5 kb vector that
contains Ad from 0 map units to 1 map unit, the CMV promoter, full
length Fas ligand and a 0.4 kb SV40 polyA tail. This shuttle vector
was combined with the 40.3 kb pJM17 vector containing the
adenovirus genome -.DELTA.E1 and also contains an origin
replication and an ampicillan-resistant site.
[0032] FIG. 17. Production of p.DELTA.E1sp1Bloxp/FasL. A 10.4 kb
shuttle vector containing the fragment of adenovirus from 0 map
unit to 1 map unit is followed by the 0.7 kb CMV promoter. This is
followed by 2 LOXP sites separated by a 2 kb stuffer fragment plus
a 0.3 kb bovine growth hormone polyA tail. The full-length 0.9 kb
Fas ligand is cloned downstream from the stuffer fragment which is
followed by an SV40 PolyA tail and by the 9.8-16.1 map units of
adenovirus.
DETAILED DESCRIPTION OF THE INVENTION
[0033] Vectors and methods are providing for introducing a
transgene into a host using a virus-based delivery system, the
vectors and methods designed to inhibit the host immune system from
interfering with the specific gene therapy vector. The present
invention incorporates the production of apoptosis inducing ligands
into antigen presenting cells through gene therapy. Normally, a
host T-cell directed towards an antigen of a transfected cell
encounters an antigen resulting in elimination of expression of the
transfecting transgene. The present invention promotes
immunotolerance towards transfected host cells. As referred to
herein, the term "gene" or "transgene" is a nucleic acid, either
naturally occurring or synthetic which encodes a polypeptide
product. The term "nucleic acid" is intended to mean natural or
synthetic linear, circular and sequential arrays of nucleotides and
nucleosides, e.g. cDNA, genomic DNA, mRNA, and RNA,
oligonucleotides, oligonucleosides, and derivatives thereof.
[0034] An apoptosis ligand is any polypeptide cytokine that induces
apoptosis or otherwise is lethal to a cell upon complexing the
ligand. Hereafter, the present invention is detailed with the
exemplary naturally occurring ligand, Fas ligand; however, it is
appreciated that other known apoptosis ligands are similarly
operative. Other such ligands illustratively include: Fas ligand 2
which induces apoptosis by acting with death domain region
molecules DR3, DR4 and DR5; TNF which induces apoptosis by acting
with TNFRI; Granzyme B and porferin which are natural killing
molecules associated with T-cells; and antibodies specific to
T-cell apoptosis ligand receptors: anti-Fas, anti-DR3, anti-DR4,
anti-DR5 and anti-TNFR1.
[0035] FIG. 1 is a schematic illustrating a production method of
gene therapy viral vector to inhibit an immune response to viral
vector antigens and methods of using the same to produce immune
privileged transduced mammalian host cells. The method of producing
an immune tolerated gene therapy vector of the present invention
involves a series of steps. In selecting a virus to be modified by
way of the present invention one examines a series of factors
including: viral vector tropism, sites of vector expression within
a host cell, ease of vector gene manipulation, required duration of
expression, pathogenicity and the like. The adenovirus (Ad) affords
many advantages as a vector as evidenced by its popularity. Ad
replicates episomally within a host cell and as such the host cell
genome is unaltered resulting in no transgene expression in host
cell daughters. The adeno-associated virus (AAV) is a smaller virus
than Ad, which is capable of integrating into a host cells
chromosomes, thereby affords the option of long-term expression.
The herpes virus (HV) is trophic for the nervous system of a host
and affords the option of transducing cells of the nervous
system.
[0036] Upon selection of a virus, the upstream regulatory region
(URR) of the virus is excised. A URR contains at least a promoter
which may be regulatable, for example, by TCN or steroids, or
inducible, such as in Loxp/Cre system. The URR is closed into a
shuttle vector plasmid. The shuttle vector contains an origin of
replication, and an apoptosis ligand gene expression cassette.
Optionally, a marker gene, an enhancer, a signal sequence, or a
stuffer fragment are included in the plasmid.
[0037] An apoptosis ligand gene or fragment thereof is excised from
a source cell line and cloned into an apoptosis ligand gene
expression cassette. The cassette contains control elements
necessary for replication within a host cell such as a promoter, a
5' untranslated region and a polyadenylation sequence. The cassette
is incorporated into the shuttle vector plasmid so as to stimulate
apoptosis ligand expression in concert with reading of the viral
URR.
[0038] The shuttle vector plasmid is then combined with a viral
vector replication plasmid from which the pathogenic protein
encoding genes have been deleted or at least inactivated. The
combined plasmids form a recombinant for delivering selected
portions of the viral genome and an apoptosis ligand for
suppressing the immune response to transfected cells presenting
viral antigens thereon. FIG. 1 shows the transgene expression
cassette as being incorporated into the vector replication
cassette. Alternatively the transgene expression cassette is
incorporated into the shuttle vector plasmid.
[0039] The recombinant is then alternatively introduced into a cell
culture or into a mammalian host. The transfection of a cell
culture is carried out by a prior art method (35). The transfected
cells expressing viral antigens and an apoptosis ligand are
identified by methods illustratively including indirect immune
fluorescent assay and .sup.51Cr release assay. Preferably, the
transiently transfected antigen presenting cell lines are
macrophages or NIT-1.beta. islet pancreas cells. It is appreciated
that the present invention is readily extended to be mediated by a
cell-cell interaction where the apoptosis ligand is expressed on
cell type one which also expresses a different ligand, the
different ligand being able to activate a receptor on a second cell
type. The preferred situation is where the different ligand of cell
type one--receptor of the second cell type interaction up-regulates
a death domain molecule in the second cell type.
[0040] Cultured cells expressing both the ligand and viral vector
antigens are then exposed to T-cells that have been sensitized to
the viral vector. The immune system challenged transfected cells
are then assayed as to proliferation, or cytotoxicity or as to the
inducement anti-viral vector antibody production. These assays are
designed to, alone or in combination assess the extent of
immuno-tolerance sensitized immune system components have towards
the gene therapy vector.
[0041] The recombinant is introduced into a mammalian host by a
route dictated by the targeted host cells. For instance, lung
tissue to be transfected with CFTR or protease inhibitor so as to
treat cystic fibrosis is preferably administered intra-nasally as
an aerosol suspension; blood to be transfected with Factor 8 so as
to treat hemophilia is preferably administered intravenously, intra
peritoneally to transfect organ specific diseases of the liver,
pancreas, etc., intra marrow for marrow and intra-myocardial for
heart tissue It is recognized that adjuvants are readily added to a
gene therapy vector of the present invention to facilitate
administration. The host transfected tissues are biopsied or
excreted gene product markers associated with the gene therapy are
assayed to monitor the efficacy of the therapy. In vitro assays are
also applicable to in vivo therapy monitoring.
[0042] The present invention provides for a gene therapy vector
capable of delivering a complete apoptosis ligand, as well as
smaller functional components of these ligands. Certain truncations
of these ligands interact with death domain molecules and thereby
induce T-cell apoptosis. For example, the nucleic acid sequences
coding for Fas ligand, Fas ligand 2, Granzyme B and porferin can be
altered by substitutions, additions, deletions or multimeric
expression that provide for functionally equivalent ligands. Due to
the degeneracy of nucleic acid coding sequences, other sequences
which encode substantially the same amino acid sequences as those
of the naturally occurring ligands may be used in the practice of
the present invention. These include, but are not limited to,
nucleic acid sequences comprising all or portions of the nucleic
acid sequences encoding the above ligands, which are altered by the
substitution of different codons that encode a functionally
equivalent amino acid residue within the sequence, thus producing a
silent change. For example, one or more amino acid residues within
a sequence can be substituted by another amino acid of a similar
polarity which acts as a functional equivalent, resulting in a
silent alteration. Substitutes for an amino acid within the
sequence may be selected from other members of the class to which
the amino acid belongs. For example, the nonpolar (hydrophobic)
amino acids include alanine, leucine, isoleucine, valine, proline,
phenylalanine, tryptophan and methionine. The polar neutral amino
acids include glycine, serine, threonine, cysteine, tyrosine,
asparagine, and glutamine. The positively charged (basic) amino
acids include arginine, lysine and histidine. The negatively
charged (acidic) amino acids include aspartic acid and glutamic
acid. Also included within the scope of the present invention are
ligands or fragments or derivatives thereof which are
differentially modified during or after translation, e.g., by
glycosolation, protolytic cleavage, linkage to an antibody molecule
or other cellular ligands, etc. In addition, the recombinant ligand
encoding nucleic acid sequences of the present invention may be
engineered so as to modify processing or expression of a ligand.
For example, a signal sequence may be inserted upstream of a ligand
encoding sequence to permit secretion of the ligand and thereby
facilitate apoptosis.
[0043] Additionally, a ligand encoding nucleic acid sequence can be
mutated in vitro or in vivo to create and/or destroy translation,
initiation, and/or termination sequences or to create variations in
coding regions and/or form new restriction endonuclease sites or
destroy pre-existing ones, to facilitate further in vitro
modification. Any technique for mutagenesis known in the art can be
used, including but not limited to in vitro site directed
mutagenesis (36), use of Tab linkers (Pharmacea), etc.
[0044] In the case of the Fas ligand, polymorphisms in the
intracellular domain modify the hydrophilic regions of the ligand
but do not greatly affect Fas ligand function in inducing
apoptosis. Thus, mutations of Fas ligand that do not affect the
apoptosis inducing potential of the ligand including additions,
substitutions, truncations and the like are recognized to be usable
in the present invention. Indeed, a polynucleotide modification of
Fas ligand to produce multimers of the Fas ligand is a means of
increasing apoptosis potential of the Fas ligand. By extension, the
same holds true for other ligands. It is known to the art that
soluble Fas ligand binds to Fas and may impede apoptosis by
endocytosis of Fas without inducing apoptosis. Therefore, larger
conglomerates of Fas ligand such as surface Fas ligand or Fas
ligand that has been engineered to be cross-lined and produced by
cells is more affective in the induction of apoptosis than the
naturally occurring Fas ligand.
[0045] In accord with the present invention, T-cells which are
activated against a specific antigen, are selectively eliminated
thereby preventing or reducing immune response to that antigen.
T-cells are eliminated by activation-induced cell death of T-cells,
which is caused by Fas-mediated apoptosis of those activated
T-cells that express Fas and Fas ligand.
[0046] In the immune system, macrophages and other specialized
cells, collectively referred to as antigen presenting cells (APCs),
ingest antigen materials and break them into smaller peptide
fragments which are presented on the surface of the APCs as a
complex with major histocompatibility complex (MHC) molecules. A
particular peptide/MHC complex is recognized by the specialized
receptor on the surface of a T-cell thereby activating that T-cell.
Activated T-cells then reproduce, and the offspring proceed to
initiate an immune response by attacking those materials displaying
the foreign antigen, and by further activating B cells and other
components of the immune system. In addition, some of the activated
T-cells persist so as to provide an enhanced response to further
infection. In accord with the present invention, it has been found
that immune response to a specific antigen can be very effectively
blocked, if the activated T-cells, responsive to that antigen, are
eliminated.
[0047] It has further been found in accord with the present
invention that the Fas ligand can be employed to produce apoptosis
of T-cells that express Fas. More specifically, it has been found
that introduction of APCs, that express a Fas ligand, into an
organism will induce apoptosis of T-cells that express Fas, thereby
resulting in antigen specific T-cell tolerance. It has been found
that an adenovirus capable of expressing the Fas ligand can be used
to transfect macrophages and other APCs. This results in a highly
efficient presentation of adenovirus antigens and Fas ligand on the
APCs. Such APCs will then confer immune tolerance to the adenovirus
vector by selectively eliminating those T-cells which are capable
of reacting with antigens from the adenovirus vector. This novel
therapeutic approach greatly enhances the utility of adenoviral
based gene therapies by producing specific tolerance to the
therapeutic materials. Since the therapy of the present invention
is highly selective, adverse affects heretofore encountered with
broad immunosuppressive approaches are eliminated.
[0048] It has further been found that adenovirus, expressing the
Fas ligand, can be targeted to APCs via the mannose receptors on
the APCs. APCs can be transfected with Fas, in vitro, and the
transfected cells introduced into the organism; or, transfection
may occur in vivo, by administration of the adenovirus vector to
the organism.
[0049] Principles of the present invention are also be employed to
inhibit autoimmune responses. As is known, autoimmune disease
occurs when an organism's immune system becomes activated toward
tissue of the organism itself. Selective apoptosis of those
activated T-cells which cause the autoimmune response will control
autoimmune disease. Transfection of those cells which elicit the
autoimmune response with the Fas gene will produce syngeneic cells
which will induce tolerance to an autoimmune antigen, in T-cells
via Fas mediated apoptosis. The syngeneic cells may comprise APCs
or they may comprise cells of the tissue provoking the autoimmune
disease, in which instance these cells will then cause the APCs to
present the Fas ligand and the autoimmune antigen.
[0050] These principles are also employed to produce immune
privileged sites within an organism. Provision of an immune
privileged site facilitates organ transplant and other such tissue
graft procedures. An immune privileged site also prolongs
expression of an adenovirus gene product at that site. Creation of
the immune privileged site is accomplished by causing cells at the
site to produce the Fas ligand, and the presence of this ligand
will protect an adenovirus from immune system attack. Production of
Fas ligand is accomplished by the virus used for the therapy
itself, or by genes introduced into the tissue via another
carrier.
[0051] Fas ligand expression induces specific tolerance by
apoptosis. Fas ligand expression is also induced by clonal
deletion. Peripheral T-cell tolerance is maintained by
activation-induced cell death of the T-cells, which is mediated by
Fas-mediated apoptosis of the activated T-cells that express Fas
and Fas ligand (37-41). Thus, Fas ligand expression is used to
create immune-privileged sites and prevent graft rejection by
inducing apoptosis in the T-cells (42-44). Transplantation of APCs
expressing Fas ligand induces apoptosis of T-cells that express
Fas, resulting in antigen-specific T-cell tolerance. The instant
invention includes a novel immunointervention strategy for
adenovirus gene therapy in which Fas ligand gene therapy is used to
confer immune privilege. This response is mediated at the cell
level and an immune response to cells is prevented by Fas ligand
production by these cells. In one embodiment of the instant
invention, the mouse FasL is introduced into the E1A site of Ad to
produce a recombinant virus which is both replicative defective and
expresses high levels of Fas ligand. Such a transgene vector
inhibits the immune response of the host thereto, resulting in
highly efficient presentation of adenovirus antigens and Fas ligand
on the macrophages. This confers immune tolerance to the adenovirus
gene therapy by selectively eliminating T-cells capable of reacting
with adenovirus vector antigens.
[0052] The current results demonstrate that AdLoxpFasL co-infection
with AxCANCre results in very high levels of FasL in a majority of
infected APCs. These APCs can express high levels of Fas ligand
without undergoing autocrine suicide. This is in contrast to low
efficiency transfection of DNA into APCs using lipofectin (1%-5%)
or electroporation (8%). The present invention utilizes several
unique technologies to allow high expressions of Fas ligand plus
high expression of process adenovirus antigen on an antigen
presenting cell to induce apoptosis of T-cells that react with this
antigen.
[0053] The present invention demonstrates extremely efficient
inhibition of CD3.sup.+ T-cell expansion that are potentially
reactive with APC processed adenovirus antigens leading to
prolongation of gene expression by challenge after tolerance with
AdCMVLacZ. High efficiency inhibition of adenovirus-reactive
T-cells is achieved by first treatment of mice with 5 dosages of
APC-AdasL using APCs from B6-lpr/lpr mice. After administration
every three days with 5 dosages, these APCs toleralize to antigens
for up to four weeks by inhibition of APC/antigen reactive T-cells.
Therefore, administration of AdCMVLacZ (10.sup.10 pfu.)
intravenously one week after tolerance does not lead to a
significant T-cell response since there is deletion or inhibition
of all potentially reactive T-cells. One week after challenge with
intravenous AdCMVLacZ, there was no visible expansion of CD3.sup.+
T-cells in the spleen. The absence of cytotoxic T-cells at 7 days
post-infection with AdCMVLacZ correlates with a prolonged
expression of LacZ in toleralized mice compared to non-toleralized
mice. The present invention shows that adenovirus expression of Fas
ligand within an antigen presenting cell used as pretreatment can
be utilized to toleralize against second administration of
adenovirus/gene therapy product.
[0054] Mice are toleralized with APC-AdFas-L. There are several
independently novel features to the inventive tolerance procedure.
First, although direct intravenous injection of AdLoxpFasL+AxCANCre
results in high co-infection of liver cells and extensive liver
necrosis (45), there was no liver toxicity due to APC-Fas ligand
cell therapy. Therefore, the use of APCs cell therapy results in
high migration of APCs to lymphoid organs, such as the spleen, and
not the liver. Second, AdCMVLacZ is used to challenge mice, but the
LacZ gene is not encoded in the AdLoxpFasL+AxCANCre viruses
infecting the toleralizing APCs, since this would require a triple
adenovirus infection, with potentially lower infection efficiency.
Nevertheless, there is tolerance to readministration of AdCMVLacZ
during challenge. AdCMVLacZ elicits an immune response to LacZ as
well as adenovirus (46-48). These results indicate that tolerance
to adenovirus alone can prolong gene therapy even in the absence of
tolerance to one of the more immunogenic transgenes, LacZ.
[0055] Tolerance induction by APCs infected with a viral vector
expressing high levels of FasL is specific for the viral vector,
but not with an irrelevant virus. These results are demonstrated by
tolerizing the mice with APC-AdFasL, and then challenging one week
later with either AdCMVLacZ or murine cytomegalovirus (MCMV), and
determining the cytotoxic response one week after challenge. There
is no stimulatory response, determined by IL-2 production, after
stimulation of splenic T-cells in vitro with APCs infected with
AdCMVLacZ, whereas there was normal IL-2 production by T-cells from
identically toleralized mice, after challenge in vivo, and
stimulation in vitro with MCMV. This is significant since other
methods for induction of tolerance, or immunosuppression to a viral
vector gene therapy are associated with a more generalized
immunosuppressed state, which would be undesirable for long-term
gene therapy use. However, the present toleralizing technique
completely abrogates the ability of T-cells responding to the
toleralizing virus used to infect the APC, but not to APC infected
with an irrelevant virus. Therefore, the present invention for
toleralizing to a viral vector gene therapy is widely applicable,
does not result in generalizing immune-suppression and is amenable
to readministration for repeated treatment without inducing an
immune-suppressed state.
[0056] Specific targeting of adenovirus to macrophages is
accomplished by either of two methods. The first approach uses a
method to couple the adenovirus fiber/knob to a mannosylated
polylysine peptide. The modified receptor is targeted to
macrophages. This technique is used to attach mannosylated
polylysine to a modified, replicative defective adenovirus to
determine adenovirus redirection to combine with high efficiency to
APCs in vivo. These experiments show that modified adenovirus is
directed to macrophages in vivo and macrophage expression of Fas
ligand combined with presentation of adenovirus gene products and
the desired new gene product is efficacious in prolonged
expression. The result is a decrease in the initial inflammatory
response to the adenovirus, along with induction of long-term
T-cell tolerance, allowing for prolonged survival of cells
expressing the adenovirus gene product, as well as decreased
immunogenicity to the adenovirus and to the adenovirus gene
product. Another method for APC infection with Ad involves using
the adenovirus-polylysine infection technique to deliver
adenovirus-polylysine-DNA complexes to accompany gene therapy to
targeted cells for cell lines that did not already express Fas
ligand. This is advantageous in the creation of immunoprivileged
sites in cells that do not express Fas ligand or do not undergo
apoptosis after expressing Fas ligand. This may be especially
advantageous for creating immune privileged cells in vitro or for
delivering to sites where low Fas expression occurs such as in the
lung.
[0057] A more stringent test of tolerance induction involves later
challenges of the mice in vivo with either the Ad-APC-FL or Ad-APC,
as well as control Ad without APC. This subsequent reaction elicits
a strong secondary immune response in the mice that were previously
immunized with adenovirus, but there is little or no response in
mice that have been tolerized with Ad-APC-FL. The use of the
Ad-APC-FL and Ad-APC, or Ad in the subsequent administration
determines if Ad-APC-FL is required with each administration of
adenovirus for a specific APC, or if the initial induction of
tolerance confers long-term tolerance to adenovirus. This technique
is used to induce tolerance to alloantigens, and that systemic
administration of APC-FL does not induce significant toxicity to
the liver or long and has no other apparent toxic effect on the
mouse. Thus, it may be advantageous to have continued expression of
FasL by the Ad infected cell to create immune privilege sites.
[0058] Fas-ligand gene therapy is useful as a strategy to prevent
immune response to viral vector antigens and in this embodiment of
the invention, adenovirus. The ability to exploit this strategy is
supported by the finding that Fas ligand expression can be targeted
to APC in vitro using the polylysine method for targeting Fas
ligand and adenovirus. This method promotes targeted gene delivery
via the receptor mediator endocytosis pathway (49-53). It is
necessary in this approach to link the vector, such as adenovirus
to molecular conjugates and, at the same time, preserve both the
binding and endosome disruption capabilities of the virus. Since
fiber and penton proteins are believed to be primary responsible
for binding and internalization, respectively, and hexon protein is
thought to be a "scaffolding protein," the conjugates are
preferably linked through the hexon protein. The linkage is
accomplished by an antibody bridge through a molecular conjugate
and the viral vector. This is accomplished by conjugating a
monoclonal antibody against a foreign epitope on the viral vector
hexon protein to the polylysine.
[0059] Preferably, the normal viral tropism of the vector is
ablated. In the case of an adenoviral vector, redirection to
macrophages optionally involves the mannosylated fiber-knob
(53-57). Regulation of the macrophage mannose receptor expression
and cloning of the mannose receptor has been carried out (58-60).
The first three exons of the mannose receptor gene encode: a signal
sequence, the NH.sub.2-terminal cysteine rich domain, and the
fibronectin type II repeat, while the final exons encode the
transmembrane anchor and the cytoplasmic tail. The intervening 26
exons encode the 8 carbohydrate-recognition domains and intervening
spacer elements. The mannose receptor is expressed on alveolar
macrophages and a highly homologous receptor DEC-205 is expressed
on dendritic cells and thymic epithelial cells (58). DEC-205 is
able to bind carbohydrates and mediate endocytosis. It is rapidly
taken up into the coated pits forming vesicles and delivered to a
multi-vesicular endosomal compartment that resembles the MHC class
II-containing vesicles. Thus, the mannose receptor on macrophages
and APCs provides an excellent target for modified adenovirus
tropism and delivery of genes to APCs. The present invention
preferably utilizes adenovirus expressing Fas ligand under the
regulation of a well characterized target cell lysozyme promoter or
a similar target specific promoter to transfect into a target cell
(61-63) efficiently present of viral vector antigens and a cytokine
ligand on the target cells.
EXAMPLES
Example 1
Animals
[0060] Four to six week-old, female C57BL/6-+/+ and C57BL/6-lpr/lpr
mice were obtained from the Jackson Laboratory (Bar Harbor, Mass.).
Mice were maintained in pathogen free condition.
Example 2
Construct Fas Ligand Expression Adenovirus Vector
[0061] This is carried out as previously described (45). Briefly, a
10.4 kb shuttle vector containing the fragment of adenovirus from 0
map unit to 1 map unit followed by the 1.6 kb chicken .beta.-actin
promoter plus CMV enhancer. This is followed by 2 Loxp sites
separated by a Neo resistant gene plus a 0.3 kb bovine growth
hormone poly A tail. The full-length 0.9 kb FasL is cloned
down-stream from the bovine growth hormone poly A tail which is
followed by an SV40 polyA tail and by the 9.8-16.1 map units of
adenovirus.
Example 3
MCMV Virus
[0062] MCMV Virus Smith strain is obtained from the American Type
Culture Collection (Rockville, Md.). The virus are titrated as
duplicates in log.sub.10 dilutions on subconfluent primary murine
embryo fibroblasts in 12-well plates. Seven days later, monolayers
are stained with neutral red and the number of plaques counted. The
supernatant is dispensed into aliquots, which are stored at
-80.degree. C. and used as the MCMV stock virus pool
(3.times.10.sup.7 PFU/ml).
Example 4
Infection of Antigen Presenting Cells for Fas Ligand Expression
[0063] This is carried out as previously described (45). Murine
B6-lpr/lpr APCs are infected with either AdLoxpFasL plus AxCANCre
(APC-AdFasL) or AdLoxpFasL plus AdCMVGFP (APC-AdControl) at 5
pfu/cell of each viruses for 1 hour at 37.degree. C., and then
infected cells continue to incubated at 37.degree. C. for
additional 24 hrs. Expressed murine FasL and adenoviral antigens on
the surface of B6-lpr/lpr APCs are identified using indirect immune
fluorescent assay (64) and the killing activity is evaluated by
.sup.51Cr release assay (65).
Example 5
Analysis of FasL by APCs Infected with AdLoxpFasL plus AxCANCre
[0064] Fas ligand (FasL) cytotoxicity is assayed as previously
described (65). FasL expression is determined by ability of the
transfected APCs to induce apoptosis of a .sup.51Cr labeled, Fas
sensitive cell line A20. Target cells (1.times.10.sup.6), which are
sensitive to cytotoxic lysis, are incubated with 20 .mu.Ci of
[.sup.51Cr]-sodium chromate in 100 .mu.l of RPMI-1640 containing
10% FCS at 37.degree. C. for 1 h. After washing with medium, these
cells are used as target cells. Effector cells are prepared from
B6-lpr/lpr APCs infected with AdLoxpFasL plus AxCANCre as described
above. These effector cells are then incubated with
[.sup.51Cr]-labeled target cells (1.times.10.sup.4) at different
effector/target (E/T) ratios in a total volume of 200 .mu.l of the
medium. Release of .sup.51Cr into the supernatant is assessed 6 h
later using a .beta.-counter.
[0065] The percentage of specific .sup.51Cr release is calculated
as follows: 1 % specific lysis = ( experimental 51 Cr release -
spontaneous 51 Cr release ) ( maximum 51 Cr release - spontaneous
51 Cr release )
[0066] The spontaneous release of .sup.51Cr using these assays has
routinely been 8%-12% of the maximum release.
Example 6
Administration of APC-AdFasL for Induction of Tolerance
[0067] Ten-week-old C57BL/6-+/+ mice are injected intravenously
with 1.times.10.sup.6 of the APCs co-infected with AdLoxpFasL plus
AxCANCre (APC-AdFasL) or APCs co-infected with AdLoxpFasL plus
AdCMVGFP (APC-AdControl) or with PBS every 3 days for 5 doses. On
day 7 after the final injection, mice are challenged with AdCMVlacZ
and T-cell cytotoxic response against APC+adenovirus is determined
one week after challenge
Example 7
Analysis of Immune Response to Adenovirus and MCMV after
Tolerance
[0068] One week after tolerance, mice are treated with AdCMVlacZ
(1.times.10.sup.10 pfu i.v.) or MCMV (1.times.10.sup.5 pfu i.v.).
After an additional 7 days, purified splenic T-cells are stimulated
in vitro with APCs alone, or APCs that are incubated either with
MCMV or AdCMVlacZ. After 48 hours the supernate is collected and
analyzed for IL-2 and Ifn-.gamma. expression.
Example 8
Quantitation of .beta.-galactosidase Expression in Liver
[0069] .beta.-galactosidase activity is determined as previously
described (66). Freshly isolated liver tissue is homogenization for
10 s in a tissumizer in 1 ml of .beta.-gal buffer (Tropix, Inc.,
Bedford Mass.). The homogenate is centrifuged at 12,500.times.g for
10 min at 4.degree. C., and the supernatant is heated for 60 min at
48.degree. C. to inactivate the endogenous eukaryotic
.beta.-galactosidase activity. The sample is then centrifuged at
12,500.times.g for 5 min, and 10 .mu.l of the supernatant is
assayed for .beta.-galactosidase activity using the
Galacto-light.TM. (Tropix, Inc., Bedford Mass.) chemi-luminescent
reporter assay. The reaction is carried out for 10 min at room
temperature (RT) and .beta.-galactosidase activity is assayed using
a luminomiter (Monolight 500). The protein concentration is
determined by the Bradford assay (Bio-Rad). The activity is
expressed as the relative light units/min/mg of total protein in
the liver.
Example 9
Analysis of Adenovirus Specific Cytotoxic T-Cell Analysis Using
AdCMVGFP infected Target Cells
[0070] The adenovirus shuttle vector construct is produced by
cloning the enhanced GFP gene from pCA13 (Clonetech) into the
HindIII-XbaI site. This is cotransfected with pJM17 to produce
recombinant AdCMVGFP. AdCMVGFP is plaque purified by 3 rounds of
selection. These are used to infect APC to be used as target cells
for analysis of cytotoxic effector T-cells from mice treated with
APC (AdLoxpFasL+AdCMVGFP) and APC (AdLoxpFasL+AxCANCre). Effector
cells are prepared from spleen, and peripheral lymph nodes of
Ad-immunized and non-immunized mice. These effector cells are then
incubated with AdCMVGFP-infected target cells (1.times.10.sup.5) at
different effector/target (E/T) ratios in round-bottom microtiter
plates in a total volume of 200 .mu.l of the medium for 48 hours,
and Green fluorescent positive APC are sorted using FACS analysis.
The percentage of specific cytotoxicity was calculated as follows:
2 % specific lysis = ( 100 % - experimental % GFP + - spontaneous %
GFP + ) ( 100 % - maximum % GFP + - spontaneous % GFP + )
Example 10
Cytokine Production In Vitro in Response to APC Infected With
Adenovirus
[0071] B6 lpr/lpr APCs are infected with AdCMVLacZ (10 pfu/cell)
for 1 hour in 1 ml of media and then diluted through addition of 10
ml of RPMI1640 supplemented with 10% fetal bovine serum. The cells
continue to culture at 37.degree. C. for 24 hours. Before serving
as a targeting cells, the APC is .gamma.-irradiated, and
1.times.10.sup.5 APC are mixed at different ratios of T-cells
isolated from the spleen of tolerized mice. The mixed cells are
incubated at 96 well plate for 2 days at 37.degree. C. The
supernatants are collected and induction of IL2 and interferon
gamma are determined using ELISA assay kit (R & D systems Inc.,
MN).
Example 11
Histopathological Examination of Tissue Sections
[0072] Animals are sacrificed by cervical dislocation. Organs were
removed and fixed in neutral 10% formalin/phosphate-buffered saline
for 24 hr, followed by fixation in 70% ethanol for 24 hr. Tissues
are then embedded in paraffin blocks, sectioned into 10 .mu.m
thickness, and stained with hematoxylin and eosin (H&E).
Example 12
Immunohistochemistry
[0073] Paraffin-embedded tissue sections are deparaffinated and
treated with 3% H.sub.2O.sub.2 at RT for 15 min. After washing 3
times with neutral phosphate buffered saline, tissues are stained
with an antibody against anti-CD3 (Dako Corporation, Carpinteria,
Calif.) following standard avidin-biotin conjugate (ABC)
immunohistochemical techniques according to manufacturer's manual
(Dako Corporation, Carpinteria, Calif.). A peroxidase-conjugated
secondary antibody is then applied to the sections at RT for 2 h.
Positive staining is visualized using diaminobenzidine (DAB)
substrate (Dako Corporation).
Example 13
Statistical Analysis
[0074] The two-tailed Student's t-test is used for statistical
analysis when two different groups of samples are compared. The one
factor analysis of variance (ANOVA) test is used when more than two
groups of samples were compared. A p value of less than 0.05 was
considered significant.
Example 14
Co-infection of AdLoxpFasL+AxCANCre Results in High Levels of FasL
Capable of Inducing Apoptosis of A20 Target Cells
[0075] The instant invention includes an AdLoxpFasL modified
adenovirus to yield high titer production of the virus in 293 cells
(45). This technique also facilitates control of FasL expression
since FasL is not expressed in the absence of co-infection with
AxCANCre. This technique is used to induce high FasL expression by
a APC cell from Fas-mutant B6-lpr/lpr mice which could induce
apoptosis of A20 target cells (FIG. 2). There are very high lyses
of the A20 target cells by APC infected with AdLoxpFasL+AxCANCre
(APC-AdFasL) as FasL activity in APC-AdFasL is 10-fold higher
compared with that of APCs transfected by electroporation with a
pcDNAIII-FasL expression vector, and 100-fold higher compared to
LPS-activated APCs. High levels of FasL expression by the APCs is
sustained for at least 7 days of in vitro culture (FIG. 3). There
is no cytotoxicity using APC+AdLoxpFasL (APC-AdControl) alone (not
shown).
Example 15
Prolonged Lac Z Expression in the Liver After Tolerance With
APCs/AdFasL Therapy
[0076] Expression of adenovirus gene therapy in the liver is
limited due to an acute inflammatory response and a chronic
cytotoxic T-cell response (67). To determine if induction of
adenoviral vector specific T-cell tolerance by AdFasL expressing
APCs leads to prolongation of transgene expression delivered by
adenoviral vector, the APC-AdFasL tolerized and APC-AdControl
treated mice are inoculated with AdCMVlacZ (1.times.10.sup.10 pfu).
LacZ gene expression in the liver is kinetically analyzed by
quantitative measurement of LacZ protein and histochemistry
staining. The levels of LacZ gene expression in the liver rapidly
decreased in mice treated with APC-AdControl (FIG. 3). In contrast,
in mice treated with APC-AdFasL, the levels of LacZ gene expression
is not decreased and is sustained for at least 50 days after gene
delivery (FIG. 3). Histochemistry staining shows that LacZ positive
cells are detectable in the liver of mice which received Ad/FasL
expressing APCs at day 30 after delivery, whereas there were few
LacZ positive cells in the liver which received control treatment
(FIG. 3).
Example 16
Decreased Cytotoxic Response After FasL Toleration
[0077] LacZ expression peaked at day 7 after expression of
AdCMVLacZ in both toleralized and non-toleralized mice, and rapidly
decreased in non-toleralized mice compared to toleralized after day
7. To determine if this prolonged expression of LacZ after day 7 in
the liver is associated with a decreased cytotoxic response to
adenovirus, mice are toleralized in vivo as described above and
challenged with AdCMVLacZ. Seven days after challenge splenic
T-cells are purified and used as effect cells at different E/T
ratios to kill AdCMVGFP infected APCs. There is a high cytotoxic
response by T-cells from mice treated with APC-AdControl after
challenged with AdCMVLacZ (FIG. 4). This is indicated by the
increased killing of APC infected with AdCMVGFP. In contrast there
was low cytotoxicity of mice toleralized with APC-AdFasL or PBS and
challenged with AdCMVLacZ to the AdCMVGFP infected APCs.
Example 17
T-Cell Tolerance Demonstrated by Decreased IFN-.gamma. and IL-2
Production In Vivo
[0078] Mice are tolerized as above with either APC-AdFasL or
APC-AdControl as a control. Thirty days after tolerance induction,
mice are sacrificed and spleen cells are stimulated with APC or APC
infected with AdCMVlacZ. Non-infected APCs did not stimulate
T-cells as determined by low IL-2 (FIG. 5A) and IFN-.gamma. (FIG.
5B) in the supernate at 24 or 48 hours (FIG. 4). In contrast, there
is high production of IL-2 and IFN-.gamma. from spleen cells from
C57BL/6 which are tolerized with APC-AdControl, which do not
express FasL. B6+/+ mice that are tolerized with APC-AdFasL are
tolerized as indicated by low IL-2 (FIG. 5A) and IFN-.gamma. (FIG.
5B) in the supernate at 24 or 48 hours.
Example 18
Fas Expression by Recipient T-Cells is Required for Tolerance
Induction
[0079] Fas expression in recipient C57BL/6 mice is required for
tolerance induction since spleen cells from B6-lpr/lpr mice
produced high levels of IFN-.gamma. and IL-2 despite being
tolerized with APC-AdFasL (FIGS. 6, 7).
Example 19
Decreased T-Cell Expansion in APC-AdFasL Treated Mice
[0080] B6+/+ mice were treated with APC-AdFasL or APC-AdControl
every 3 days for 5 doses, and then all treated mice were i.v.
challenged with AdCMVlacZ (1.times.10.sup.10 pfu). Three days
later, frozen sections of spleen from naive mice, control APC
treated mice, FasL APC treated mice and were stained with anti-CD3
antibody using a standard ABC technique. There was no expansion of
CD3.sup.+ T-cells in tolerized mice spleen, whereas mice treated
with control APCs resulted in clonal expansion in spleen after
challenge.
Example 20
APC-AdFasL Induces Specific Tolerance to Adenovirus
[0081] To determine if T-cell tolerance induced by Ad/FasL
expressing APCs is specific for adenoviral vector rather than a
general immune suppression to viral infection, the T-cell response
by APC-AdFasL and APC-AdControl tolerized mice to an irrelevant
viral infection is measured. B6+/+ mice are treated with APC-AdFasL
as described above for induction of tolerance, and then challenged
7 days later with either adenovirus or mouse cytomegalovirus
(MCMV). Although there is a reduction of T-cell response to
adenoviral vector, the T-cell response to MCMV is not impaired as
demonstrated by the comparable levels of IL-2 produced by the
T-cells from both control and FasL APC treated mice (FIG. 8).
Example 21
Fas Ligand Expressing Adenovirus (Ad/FasL-.beta.Gal) Provides Both
Systemic Immune Tolerance to Ad Transfected APCs and Confers
Privilege on Cells That are Transfected With the
Ad/FasL-.beta.Gal.
[0082] APCs transfected with Fas ligand induce specific apoptosis
and specific T-cell tolerance to antigens both in vitro and in
vivo. This is observed using a macrophage cell line derived from
Fas-deficient C57BL/6(B6)-lpr/lpr mice that are transiently
transfected with Fas ligand, and then injected into mice of a
different MHC. In addition, macrophages co-infected with Fas ligand
and viral vector are highly efficient presenters of viral vector
antigens and Fas ligand. This results in antigen-specific apoptosis
of vector-reactive T-cells. Transfection of Fas ligand into a
.beta.-islet cell line also confers immune privilege on the host
.beta.-islet-reactive T-cells and prevention of diabetes where the
vector is adenovirus. These results show that muscle cells infected
with Ad and co-transfected with Fas ligand created an immune
privileged site in which the adenovirus is not capable of inducing
an immune response.
Example 22
APCs Transfected With Fas Ligand Induce Apoptosis and Specific
T-Cell Tolerance to Antigens In Vitro and In Vivo
[0083] An APC line derived by short-term culture of peritoneal
macrophages from Fas mutant B6-lpr/lpr mice does not express Fas,
but expressed MHC class II IA.sup.b, MHC class I H-2D.sup.b
antigens (FIG. 9a, 9b), Mac-1, and Fc-.gamma. receptor (data not
shown). Significant levels of the B7 costimulatory molecule are
expressed on 50% of the cells (FIG. 9c). This cell line is
transfected with a eukaryotic expression vector (pcDNAIII)
containing the full-length murine Fas ligand and selected using
G418. APCs transfected with Fas ligand (APC-FL), but not control
vector (APC-CV), exhibit high Fas ligand activity (FIG. 9d). APC-CV
cells are capable of presenting alloantigen as the
.gamma.-irradiated cells induced a proliferative responses in
co-cultured splenic H-2.sup.k T-cells (MRL-+/+ or MRL-lpr/lpr)
(FIG. 9e). APC-FasL cells are capable of presenting alloantigen and
induce a proliferative response if the responding T-cells are
obtained from MRL-lpr/lpr mice, which do not express Fas. However,
presentation of antigen by APCs that express Fas ligand to T-cells
that express Fas antigen, obtained from MRL-+/+ mice, abrogated the
proliferative response. Thus, in the present invention,
presentation of antigen by APCs that express Fas ligand induces
tolerance of the Fas-positive responding T-cells.
Example 23
Induction of Allogeneic T-Cell Tolerance by Fas Ligand Expressing
APCs
[0084] 4-wk-old of MRL-+/+ and -lpr/lpr mice are injected i.v. with
macrophages (2.times.10.sup.5) transfected with Fas ligand or
control vector every 3 d for 6 times. On d 3 of the final
injection, splenic T-cells are isolated from treated mice and
cultured under various stimulatory conditions. 5.times.10.sup.5
T-cells are cultured with 2.times.10.sup.5 .gamma.-irradiated total
spleen cells from B6+/+ mice (FIG. 10a). 5.times.10.sup.5 T-cells
are cultured with 2.times.10.sup.5 .gamma.-irradiated total spleen
cells from BALB/c mice (FIG. 10b). 5.times.10.sup.5 T-cells are
cultured with 5 mg/ml of anti-CD3 antibody (FIG. 10c). T-cell
proliferation is determined by incorporation of [.sup.3H]-thymidine
at 24, 48, 72 and 96 hours.
Example 24
Antigen-Specific Clonal Deletion of the T-Cells Induced by Fas
Ligand Expressing APCs in H-2 D.sup.b/HY Reactive TCR Transgenic
Mice
[0085] The ability of APCs that express Fas-ligand to mediate
clonal deletion of antigen-specific T-cells is directly tested in
female, T-cell receptor (TCR) transgenic, H-2 D.sup.b HY-reactive
mice. In these mice, the majority of peripheral CD8.sup.+ T-cells
bear the transgenic TCR and are reactive with the male HY antigen
presented in the context of the H-2 D.sup.b antigen. To obtain
cells that bear H-2 D.sup.b, HY antigen and high levels of Fas
ligand but not Fas, CD4.sup.-CD8.sup.-T-cells are isolated from the
peripheral lymph nodes of 5-month-old, male, B6-lpr/lpr mice.
CD4.sup.-CD8.sup.-T-cells isolated from 5-month-old, female,
B6-lpr/lpr mice are used as controls in which the HY antigen is not
expressed. The CD4.sup.-CD8.sup.-T-cells obtained from both male
and female B6-lpr/pr mice expressed high levels of H-2 D.sup.b
antigen (FIG. 11a). The Fas ligand activity of the
CD4.sup.-CD8.sup.-T-cells is high and results in specific
inhibition of this release by soluble Fas-Ig fusion protein (FIG.
11b). Alloantigen-specific T-cell tolerance was analyzed after i.v.
injection of 1.times.10.sup.6 CD4.sup.-CD8.sup.-T-cel- ls from
either male or female mice into 4-wk-old, female, TCR transgenic
+/+ or lpr/lpr mice. T-cells from female, TCR transgenic +/+ mice
treated with Fas ligand .sup.+HY.sup.+ and male cells exhibited a
decreased proliferative response upon stimulation with either H-2
D.sup.b/HY spleen cells or crosslinking with the M33
anti-clonotypic TCR antibody, but not anti-CD3. Fas ligand-positive
cells derived from H-2 D.sup.b female mice had no effect on the H-2
D.sup.b/HY reactivity of recipient T-cells in TCR transgenic female
mice. Comparable levels of T-cell proliferation were observed in
response to stimulation with anti-CD3, M33 antibody, or H-2
D.sup.b/HY cells when the TCR transgenic female mice were treated
with CD4.sup.-CD8.sup.-T-cells of female mice (FIG. 11c). These
results indicate that the decreased response requires the presence
of the H-2 D.sup.b/HY antigen on the APCs and is specific for H-2
D.sup.b/HY reactive T-cells as there was a normal response to
crosslinking with anti-CD3.
Example 25
Tolerance Induction Due to Fas-Mediated Deletion of H-2 D.sup.b/HY
Reactive CD8.sup.+ T-Cells
[0086] Clonal deletion of H-2 D.sup.b/HY cells is examined by
analyzing the numbers of H-2 D.sup.b/HY reactive CD8.sup.+ T-cells
in the female TCR transgenic mice using the anti-clonotypic mAb
M33. Tolerance induction is carried out as described above and the
numbers of M33.sup.+CD8.sup.+ T-cells in the peripheral lymph node
(FIG. 12a) and spleen (FIG. 12b) cells are determined. In
untreated, female, transgenic +/+ and lpr/lpr mice, approx. 30% of
the PLN cells were M33.sup.+CD8.sup.+ T-cells and this percentage
is not altered by treatment with female H-2 D.sup.b cells lacking
HY antigen (FIG. 12a). After tolerance induction in female, TCR
transgenic, +/+ mice by Fas ligand-positive H-2 D.sup.b/HY cells,
however, only 4% of PLN cells are M33.sup.+ and CD8.sup.+. This
depletion of M33.sup.+CD8.sup.+ T-cells is inhibited significantly
by Fas-Ig treatment in that 19% of the cells are
M33.sup.+CD8.sup.+. Thus, induction of tolerance by Fas ligand
expressing APCs is associated with Fas ligand-mediated clonal
deletion of antigen-specific T-cells that recognize the antigen
presented by the APCs. Time-course analysis of the deletion of
M33.sup.+CD8.sup.+ T-cells in the spleen showed that the depletion
commenced as early as 24 h after treatment in the female TCR
transgenic +/+ mice that received Fas ligand-positive H-2
D.sup.b/HY cells and continued during the 10-d period of the
treatment. Fas-Ig effectively inhibited the deletion in the TCR
transgenic +/+ mice, which further supports the role of Fas ligand
expression on the APCs in clonal deletion. Fas expression also is
analyzed in M33.sup.+CD8.sup.+ PLN T-cells in the female TCR
transgenic lpr/lpr mice did not express Fas antigen regardless of
treatment. Fas expression on M33.sup.+CD8.sup.+ T-cells expressed
low levels of Fas (14%), whereas additional treatment with Fas-Ig
led to the majority of M33.sup.+CD8.sup.+ T-cells being deleted by
Fas ligand expressing APCs.
Example 26
Inhibition of Insulitis in NOD Mice Using a Synegeic .beta.-Islet
Cell Line That Expresses Fas Ligand to Induce T-Cell Tolerance
[0087] NOD mice develop spontaneous insulitis and diabetes due to a
T-cell-mediated autoimmune response to self-.beta. cells. The
syngeneic .beta. cell line, NIT-1, is used as the APC for Fas
ligand expression. NIT-1 cells do not express Fas antigen and do
not undergo either anti-Fas antibody or Fas ligand mediated
apoptosis (data not shown). This cell line is transfected with an
expression vector containing Fas ligand mediated apoptosis (data
not shown). This cell line is transfected with an expression vector
containing Fas ligand (pcDNAIII) as described in Example 9. Fas
ligand transfected, but not control, cells expressed functional Fas
ligand (FIG. 13a). 6-wk-old, female, NOD mice are injected once
with 5.times.10.sup.5 Fas ligand expressing, or control, NIT-1
cells. Seven d later, the splenic T-cells are isolated from treated
NOD mice and co-cultured with irradiated NIT-1 cells. There are
increased T-cell proliferative and cytotoxic responses in NOD mice
treated with control NIT-1 cells (FIG. 13b,c). In contrast, NOD
mice treated with Fas ligand expressing NIT-1 cells only exhibit a
minimal increase in response compared with the untreated control.
100% of NOD mice that received no treatment or treatment with
NIT-1/CV developed insulitis, and 100% of islets from each
individual mouse are involved. In contrast, only 1 of 3 mice
receiving NIT-1/FasL developed minor insulitis, with only 10% of
islets involved (FIG. 14).
Example 27
Inhibition of Insulitis in Nod Mice Using NIT-1--AdFasL as a
Syngenic .beta. Islet Cell to Induce T-Cell Tolerance to an Ad
Vector
[0088] The procedure of Example 25 is repeated with the expression
vector of Example 14 substituted therein. NIT-1--Ad control treated
mice develop insulitis involving 100% of islet cells of individual
mice. NIT-1--AdFasL treated mice did not develop insulitis.
Example 28
Transfection With Fas Ligand and Adenovirus Results in High
Expression of .beta.-Gal in Macrophages
[0089] The polylysine method is used for targeting Fas ligand and
Ad to APC via the receptor-mediated endocytosis pathway (49-51, 68,
69). It is important to link Ad to molecular conjugates, and at the
same time preserve both the binding and endosome disruption
capabilities of the virus. The linkage is accomplished by
conjugating a molecular antibody against a foreign epitope on the
adenovirus hexon protein to the polylysine-protein complex. For
this purpose a chimeric adenovirus containing a foreign epitope in
the surface region of its hexon protein is constructed. The loop
region of the hexon protein is a useful foreign epitope expression
region.
Example 29
Creation of an Immune-Privileged Site for Prolonged Expression of
the Adenovirus Gene Product Using Co-Expression of FasL and
Adenovirus in Muscle
[0090] 10.sup.9 adenovirus is co-injected into mouse muscle tissue
with 5 .mu.g of purified FasL DNA under the regulation of the CMV
promoter (pFasL), or with identical control plasmid DNA which does
not express Fas ligand. FasL production by adenovirus confers a
high level of specific immunity to the adenovirus, prevent immune
elimination of cells expressing the adenovirus, and result in
prolonged expression of the adenovirus gene product. These results
are consistent with previous studies showing that FasL production
in muscle cells created an immune privileged site (42).
Example 30
Modification of Viral Tropism to Allow High Efficiency Targeting to
Macrophages
[0091] In addition to the in vitro infection and tolerance
induction by Ad/FasL, in vivo infection by an Ad/FasL virus is
operative. A FasL Tg mouse which overexpresses FasL specifically in
T-cells without cytotoxicity is used (70). Similar techniques
direct Ad/FasL for high transfection of APCs in vivo (macrophages)
by targeting adenovirus to the macrophage mannose receptor. This is
accomplished using a synthetic molecular conjugate consisting of a
mannosylated polylysine protein combined with the adenovirus
fiber/knob protein. A mannosylated polylysine has been demonstrated
to bind to the macrophage mannose receptor and lead to high
efficiency transfection of DNA complexes into islet cells (71, 72).
Modification of adenovirus tropism uses the methods detailed in
U.S. Provisional Patent Application 60/054,112 for modification of
the adenovirus knob/fiber protein to include a 10 amino acid
polypeptide capable of binding E-selectin and targeting adenovirus
to inflamed sites in the synovium and also using an anti-adenovirus
sFv/IL-2 fusion protein to direct adenovirus tropism to
T-cells.
Example 31
Production of an Adenovirus-Infected, Fas Ligand Expressing
Macrophage for Induction of Tolerance to Adenovirus
[0092] The APC line of Example 22 expresses high level of MHC class
I and II antigen, B7 and Fas ligand. This macrophage cell line
express high levels of H-2 D.sup.b and I-A.sup.b as well as B7 upon
the stimulation with LPS or IFN-.gamma.. This cell lines does not
express Fas, exhibits low levels of Fas ligand activity, and has
been transfected with a CMV promoter/FasL construct to produce a
stable transfected macrophage cell line which expresses FasL. This
cell line can also be infected with Ad by known techniques to allow
expression of adenovirus antigens and gene products.
Example 32
Analysis of Tolerance to Ad/Fas Ligand
[0093] Tolerance to adenovirus is analyzed using a macrophage cell
line that stably expresses Fas ligand (APC-FL), such as that of
Example 22, and are infected with the adenovirus by intravenous
(i.v.) or intranasal (i.n.) injection to induce tolerance.
Tolerance is analyzed at d 2, 7, 14, 28, and 56 after injection of
5.times.10.sup.6 Ad-APC-FL. Mice are bled by retro-orbital sinus
puncture for analysis of antibody titer to adenovirus.
Example 33
Determination of Tolerance to Ad
[0094] Single cell suspensions of spleen and lung are prepared for
determination of the proliferative response upon co-culture with
normal, irradiated H-2b Ad-APC. T-cells tolerance are evaluated by
[.sup.3H]-thymidine incorporation to measure the T-cell
proliferative response, BrdU incorporation, and flow cytometric
analysis of BrdU-positive T-cells to determine the frequency of
proliferative T-cells, and 7AAD three color flow cytometric
analysis to determine apoptosis of the T-cells. The level of IL-2
in the culture supernatants is also measured to determine T-cell
activation. A similar technique is used to test to determine if
cytotoxic CD8.sup.+ T-cells are toleralized or deleted from the
spleen in vivo. The CD8.sup.+ T-cells are tested for their ability
to lyse chromium-labeled Ad-APC. Purified T-cells are isolated as
described in reference to FIG. 10. A suitable effector: target
(E:T) ratio of CD8.sup.+ T-cells to chromium-labeled,
adenovirus-pulsed macrophage target cells is thereby obtained.
Example 34
Construction of an Adenovirus Producing Fas Ligand
[0095] First, a full length 1114 bp murine Fas ligand cDNA clone is
obtained by conventional methods (73-75). Second, this Fas ligand
clone is used to produce the Ad/FasL vector (FIG. 16). Third, this
clone has undergone recombination with the adenovirus genome in 293
cells. This construct and variations of this construct are used in
the present invention. The Fas ligand cDNA clone is introduced into
the p.DELTA.E1sp1b shuttle vector. To produce recombinant
adenovirus, this DNA is co-transfected into weakly Fas.sup.+ 293
cells. A total of 6 transfections are carried out using 3 different
transfection methods including: lipofectin, dotap, and the calcium
chloride precipitation method. Under all conditions, the majority
of the transfected 293 cells undergo apoptosis within 24 h, whereas
minimal apoptosis occurs after transfection of 293 cells with the
control shuttle vector.
Example 35
Production of p.DELTA.E1sp1Bloxp/FasL
[0096] A Fas ligand expressing recombinant adenovirus, denoted as
AdLOXP/FasL recombinant virus is shown in FIG. 17. The
p.DELTA.E1sp1Bloxp/Fas shuttle vector is co-transfected with pJM17
to produce the AdLOXP/FasL. The AdLOXP/FasL is co-infected with the
Ad/CRE recombinant adenovirus. The CRE excises the LOXP sites
placing FasL under the control of the CMV promoter resulting in
high levels of expression of FasL. As outlined in the detailed
description of the invention, AdLOXP/FasL does not induce toxicity
in the 293 cells. The AdLOXP/FasL adenovirus is combined with the
Ad/CRE recombinant adenovirus. The CRE protein has been well
studied and is demonstrated to be able to excise the LOXP sites
which in the present invention construct results in the production
of FasL under the CMV promoter. This system was first heavily
utilized for production of transgenic mice. It has applied by
several investigators for adenovirus recombination (73-75). These
viruses can be co-infected into any cell, such as macrophages used
herein with high efficiency.
Example 36
Confirmation That the Macrophage Cell Line Transfected With the
Adenovirus Expresses lacZ and Fas Ligand
[0097] Macrophages are transfected with the recombinant adenovirus.
Lac z expression is confirmed by .beta.-galactosidase staining as
described in Example 8. After gene therapy, mice are analyzed at
different time courses for expression of the lacZ marker gene in
the lung and liver. Fas ligand expression is confirmed by ability
of the transfected macrophages to induce apoptosis of .sup.51Cr
labeled and Fas sensitive cell line A20 as per Example 5. These
experiments are carried out with and without the presence of a
soluble Fas (sFas) capable of neutralizing Fas ligand activity to
demonstrate that cytotoxicity is specific for Fas ligand.
Example 37
Treatment of a Lung Disease With AdCF/FasL Transfected into
APCs
[0098] The CF gene is ligated into the EcoRV site of the Ad shuttle
vector of FIG. 16 so as to be under the control of the regulatory
element. The CF modified vector, Ad Shuttle CF is co-transfected
with pJM17 to produce recombinant AdCF. To produce FasL, this is
co-infected with the AdLOXP FasL and AxCanCre. These three viruses
will be co-administered intra-nasally into the airways of 6 week
old, female bleomycin--IPF mice. On day 7 after the injection, the
mice are challenged with AdCF. The mice so treated are tolerant of
the CF gene therapy vector 7 days after challenge.
Example 38
Treatment of a Lung Disease With AdPI/FasL Transfected into
APCs
[0099] The protease inhibitor (PI) gene is ligated into the EcoRV
site of the Ad shuttle vector of FIG. 16 so as to be under the
control of the regulatory element. The PI modified vector, Ad
Shuttle PI is co-transfected with pJM17 to produce recombinant
AdPI. To produce FasL, this is co-infected with the AdLOXP FasL and
AxCanCre. These three viruses will be co-administered intra-nasally
into the airways of 6 week old, female bleomycin--IPF mice. On day
7 after the injection, the mice are challenged with AdPI. The mice
so treated are tolerant of the PI gene therapy vector 7 days after
challenge.
Example 39
Treatment of Hemophilia with AdF8/FasL Transfected in APCs
[0100] The factor VIII gene is ligated into the EcoRV site of the
pJM 17 vector of FIG. 16 so as to be under the control of the
regulatory element. The PI modified vector, Ad Shuttle Factor VIII
is co-transfected with pJM 17 to produce recombinant Ad Factor
VIII. To produce FasL, this is co-infected with the Ad LOXP FasL
and AxCanCre. These three viruses will be co-administered
intra-nasally into the airways of 6 week old, female bleomycin--IPF
mice. On day 7 after the injection, the mice are challenged with Ad
Factor VIII. The mice so treated are tolerant of the Factor VIII
gene therapy vector 7 days after challenge.
Example 40
Determine the Expression of Fas Ligand and Ad/.beta.-gal In Vivo at
Different Time Points After Infection In Vivo in Tolerized and
Non-Tolerized Mice
[0101] Detailed analysis of expression of Fas ligand RNA and
protein, viral RNA and protein, and .beta.-gal is carried out at
different time point and under different conditions of tolerance
induction involves analysis of tissue sections using
immunohistochemical staining for Fas, .beta.-Gal. Tissue sections
are also analyzed for in-situ expression by RT-PCR and for
apoptosis by the tunel method. The phenotype of T-cell and
macrophages in lymphoid organs and lung is determined by flow
cytometry analysis. Fas ligand expression by single cell suspension
is determined by 1) Cr release assay of Fas apoptosis sensitive
target cells, 2) frequency analysis by single cell Fas ligand
PCR.
Example 41
Mechanism to Abolish Fas Expression of Fas Apoptosis Signaling by
the Cell That is Infected With the Ad/FasL-Gene Therapy Vector
[0102] To abolish cell surface Fas expression, it is sufficient to
prevent Fas apoptosis signaling, since it is well established that
Fas expression does not necessarily correlate with Fas apoptosis
signaling (76-81). The analysis of Fas-apoptosis signaling and
inhibition of this by IL-1.beta. converting enzyme family members
and also inhibitors of HCP are useful in testing abolition. The
will be accomplished by incorporating both Fas and known inhibitory
proteins of Fas apoptosis into modified Ad virus. A specific
construct capable of expressing Fas ligand safely and at the same
time protect the Fas ligand expressing cell from autocrine-mediated
apoptosis includes both FasL and fragments of IL-1.beta. or repeats
of the peptide sequence the CPP32/Yama inhibitor DEVD-CHO, the ICE
inhibitor YVAD-CHO which inhibit ICE and CPP32 and prevent
Fas-mediated apoptosis in different cells, and Crm A, which block
the cas pase pathway (81). These experiments show the ablation of
the endogenous tropism of the adenovirus and the addition novel
tropism of the adenovirus to antigen presenting cells. Highly
efficient ablation of endogenous tropism is important for using the
immune modulating strategies proposed of the present invention.
[0103] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. These patents and publications are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
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[0185] Various modifications of the present invention, in addition
to those shown and described herein, will be apparent to those
skilled in the art of the above description. Such modifications are
also intended to fall within the scope of the appended claims.
* * * * *