U.S. patent application number 10/313852 was filed with the patent office on 2003-06-05 for methods and compositions for lowering the level of tumor necrosis factor (tnf) in tnf-associated disorders.
Invention is credited to Burstein, Haim, Stepan, Anthony M..
Application Number | 20030103942 10/313852 |
Document ID | / |
Family ID | 22535598 |
Filed Date | 2003-06-05 |
United States Patent
Application |
20030103942 |
Kind Code |
A1 |
Burstein, Haim ; et
al. |
June 5, 2003 |
Methods and compositions for lowering the level of tumor necrosis
factor (TNF) in TNF-associated disorders
Abstract
The present invention provides recombinant adeno-associated
virus (rAAV) vectors encoding a tumor necrosis factor (TNF)
antagonist and methods using these vectors to reduce levels of TNF
in a mammal. The invention also provides methods of using these
rAAV vectors in palliating TNF-associated disorders.
Inventors: |
Burstein, Haim; (Redmond,
WA) ; Stepan, Anthony M.; (Seattle, WA) |
Correspondence
Address: |
Catherine M. Polizzi
Morrison & Foerster LLP
755 Page Mill Road
Palo Alto
CA
94304-1018
US
|
Family ID: |
22535598 |
Appl. No.: |
10/313852 |
Filed: |
December 6, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10313852 |
Dec 6, 2002 |
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09579845 |
May 26, 2000 |
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6537540 |
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60150688 |
May 28, 1999 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/320.1; 435/456 |
Current CPC
Class: |
C07K 2319/30 20130101;
A61P 29/00 20180101; C12N 2830/00 20130101; A61K 48/00 20130101;
C12N 2750/14143 20130101; A61K 38/1793 20130101; C07K 2319/00
20130101; A61K 38/1793 20130101; C07K 14/7151 20130101; A61K
48/0075 20130101; A61P 19/02 20180101; A61K 2300/00 20130101; C12N
15/86 20130101; A61K 38/00 20130101 |
Class at
Publication: |
424/93.2 ;
435/456; 435/320.1; 435/235.1 |
International
Class: |
A61K 048/00; C12N
007/00; C12N 015/861 |
Claims
We claim:
1. A recombinant adeno-associated virus (rAAV) vector comprising a
polynucleotide encoding a fusion polypeptide comprising an
extracellular domain of tumor necrosis factor receptor (TNFR) and a
constant domain of an IgG1 molecule.
2. The rAAV vector of claim 1, wherein the TNFR extracellular
domain is from p75 TNFR.
3. The rAAV vector of claim 1, wherein the polynucleotide encoding
the TNFR polypeptide is operably linked to a heterologous
promoter.
4. The rAAV vector of claim 1, wherein the polynucleotide encoding
the TNFR polypeptide is operably linked to a constitutive
promoter.
5. The rAAV vector of claim 1, wherein the polynucleotide encoding
the TNFR polypeptide is operably linked to an inducible
promoter.
6. The rAAV vector of claim 5, wherein the inducible promoter is
from the TNF.alpha. gene.
7. The rAAV vector of claim 1, wherein the polynucleotide further
encodes a polypeptide comprising an interleukin-1 (IL-1)
antagonist.
8. The rAAV vector of claim 7 wherein the IL-1 antagonist is an
IL-1 receptor polypeptide, wherein said IL-1 receptor polypeptide
binds IL-1.
9. The rAAV vector of claim 8 wherein the IL-1 receptor polypeptide
is a IL-1 receptor type II polypeptide.
10. A mammalian cell transfected with the rAAV vector of claim
1.
11. A mammalian cell transfected,with the rAAV vector of claim
7.
12. A composition comprising the rAAV of claim 1.
13. The composition of claim 12, further comprising a
pharmaceutically acceptable excipient.
14. An rAAV virus particle comprising the rAAV vector of claim
1.
15. An rAAV virus particle comprising the rAAV vector of claim
7.
16. A method for reducing TNF levels in a mammal, comprising
administering the rAAV vector of claim 1 to the mammal in an amount
sufficient to reduce TNF levels in the mammal.
17. The method of claim 16, further comprising administering a TNF
antagonist.
18. The method of claim 16, wherein said rAAV vector is
administered by intra-articular injection.
19. The method of claim 16, wherein the rAAV is administered by
injection to connective tissue selected from the group consisting
of a synovium, a cartilage, a ligament, and a tendon of said
mammal.
20. The method of claim 18, wherein said rAAV vector is
administered to synovial cells lining a joint space of said
mammal.
21. The method of claim 16, wherein said rAAV vector is
administered by intramuscular injection.
22. The method of claim 16, wherein said rAAV vector is
administered by intravenous injection.
23. A method for reducing an inflammatory response in a mammal,
comprising administering the rAAV vector of claim 1 to the mammal
in an amount sufficient to reduce the inflammatory response in the
mammal.
24. The method of claim 23, further comprising administering a TNF
antagonist.
25. The method of claim 23, wherein the inflammatory response
occurs in a connective tissue.
26. The method of claim 23, wherein the inflammatory response
occurs in a joint.
27. The method of claim 23, wherein said rAAV vector is
administered by intra-articular injection.
28. The method of claim 23, wherein the rAAV is administered by
injection to connective tissue selected from the group consisting
of a synovium, a cartilage, a ligament, and a tendon of said
mammal.
29. The method of claim 27, wherein said rAAV vector is
administered to synovial cells lining a joint space of said
mammal.
30. The method of claim 23, wherein said rAAV vector is
administered by intramuscular injection.
31. The method of claim 23, wherein said rAAV vector is
administered by intravenous injection.
32. A method for palliating a TNF-associated disorder in a mammal,
comprising administering the rAAV vector of claim 1 to the mammal
in an amount sufficient to palliate the TNF-associated disorder
condition.
33. The method of claim 32, further comprising administering a TNF
antagonist.
34. The method of claim 33, wherein said rAAV vector is
administered by intra-articular injection.
35. The method of claim 32, wherein the rAAV is administered to
connective tissue selected from the group consisting of a synovium,
a cartilage, a ligament, and a tendon of said mammal.
36. The method of claim 32, wherein said rAAV vector is
administered to synovial cells lining a joint space of said
mammal.
37. The method of claim 32, wherein said rAAV vector is
administered by intramuscular injection.
38. The method of claim 30, wherein the TNF-associated disorder in
an inflammatory disorder.
39. The method of claim 32, wherein the inflammatory disorder is an
arthritic condition.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the priority benefit of U.S.
provisional patent application serial No. 60/150,688, filed May 28,
1999, which is incorporated by reference in its entirety.
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
[0002] (Not Applicable)
FIELD OF INVENTION
[0003] This invention relates to the use of adeno-associated virus
(AAV) vectors to lower levels of tumor necrosis factor (TNF). More
specifically, the invention relates to AAV vectors encoding a TNF
antagonist and methods of using the AAV vectors to reduce the
levels of TNF in an individual.
BACKGROUND
[0004] Tumor necrosis factor-.alpha. (TNF.alpha.) and tumor
necrosis factor-.beta. (TNF.beta.) are homologous multifunctional
cytokines; the great similarities in structural and functional
characteristics of which have resulted in their collective
description as tumor necrosis factor or "TNF." Activities generally
ascribed to TNF include: release of other cytokines including IL-1,
IL-6, GM-CSF, and IL-10, induction of chemokines, increase in
adhesion molecules, growth of blood vessels, release of tissue
destructive enzymes and activation of T cells. See, for example,
Feldmann et al., 1997, Adv. Immunol, 64:283-350, Nawroth et al.,
1986, J. Exp. Med., 163:1363-1375; Moser et al., 1989, J. Clin.
Invest., 83:444-455; Shingu et al., 1993, Clin. Exp. Immunol.
94:145-149; MacNaul et al., 1992, Matrix Suppl., 1:198-199; and
Ahmadzadeh et al., 1990, Clin. Exp. Rheumatol. 8:387-391. All of
these activities can serve to enhance an inflammatory response.
[0005] TNF initiates its biological effect through its interaction
with specific, cell surface receptors on TNF-responsive cells.
There are two distinct forms of the cell surface tumor necrosis
factor receptor (TNFR), designated p75 (or Type II) and p55 (or
Type I) (Smith et al., 1990, Science 248:1019-1023; Loetscher et
al., 1990, Cell 61:351-359). TNFR Type I and TNFR Type II each bind
to both TNF.alpha. and TNF.beta.. Soluble, truncated versions of
the TNFRs with a ligand-binding domain are present in body fluids
and joints (Engelmann et al., 1989, J. Biol. Chem. 264:11974-11980;
Roux-Lombard et al., 1993, Arthritis Rheum. 36:485-489).
[0006] A number of disorders are associated with elevated levels of
TNF, many of them of significant medical importance. Among such
TNF-associated disorders are congestive heart failure, inflammatory
bowel diseases (including Crohn's disease), arthritis and
asthma.
[0007] TNF appears to effect the heart and endothelium in
congestive heart failure and has been implicated in the initiation
of an apoptotic process in cardiac myocytes. The role for TNF in
this disease is also supported by a temporal association between
TNF activation and a transition from asymptomatic to symptomatic
congestive heart failure (Ceconi et al., 1998, Prog. Cardiovasc.
Dis. 41:25-30).
[0008] Inflammatory bowel diseases, such as Crohn's disease and
ulcerative colitis, are associated with increased expression of TNF
(Evans et al., 1997, Aliment. Pharmacol. Ther. 11:1031-1035).
Treatment of such disorders have included the widespread use of
immunosuppressive agents, such as azathioprine, methotrexate,
cyclosporine and glucocorticosteroids (Rutgeerts, 1998, Digestion
59:453-469).
[0009] Arthritis is a common crippling condition for which there
are no cures and few effective therapies. Approximately one in
seven people in the United States are affected by one or more forms
of arthritis. Most forms of arthritis are characterized by chronic
inflammation of joints resulting from infection, mechanical injury,
or immunological disturbance. Rheumatoid arthritis (RA) is a
chronic inflammatory disease primarily manifest in the joints by
swelling, pain, stiffness, and tissue destruction (Harris, 1990, N.
Engl. J. Med., 323:994-996). Systemic manifestations can include
elevations in serum levels of acute phase proteins, fever, mild
anemia, thrombocytosis, and granulocytosis. In affected joints,
there is a synovitis characterized by hyperplasia and inflammation
of the synovium with an inflammatory exudate into the joint cavity,
leading to erosion of cartilage and bone.
[0010] Although rheumatoid arthritis is not directly and imminently
life threatening, recent data suggest that RA results in
significantly shorter lifespan, and puts an enormous toll on the
both the health system, the overall economy due to lost
productivity, as well as quality of life resulting from restricted
mobility and activities (Schiff, 1997, Am. J Med.,
102(1A):11S-15S).
[0011] Current commonly employed therapeutics for treatment of RA
fall primarily in three categories: non-steroidal anti-inflammatory
drugs (NSAIDs), disease-modifying anti-rheumatic drugs (DMARDs),
and immunosuppressives. NSAIDs are a large group of drugs often
used as first line therapy for rheumatoid arthritis. The compounds
act primarily through blockade of cyclooxygenase which catalyzes
conversion of arachidonic acid to prostaglandins and thromboxanes.
As a class, DMARDs, including agents such as gold, sulfasalazine,
hydroxychloroquine, and D-penicillamine, are slow acting, quite
toxic and there is little evidence that any of these compounds have
mitigating effects on the underlying disease. NSAIDs can relieve
some of the signs of inflammation and pain associated with
arthritis; however, they appear to be ineffective against the
immune system and in blocking progression of joint destruction and
disease. Immunosuppressive agents, such as corticosteroids and
methotrexate, are commonly used in the treatment of RA for
suppressing the immune system and thus having an anti-inflammatory
effect. However, these agents engender serious systemic toxicity
which limits their use and effectiveness.
[0012] Although it is widely accepted that RA is an immune-based
inflammatory disease, the antigen(s) which trigger the disease
remain unknown. This has led to a large number of approaches to
therapy under pre-clinical or clinical investigation which involve
attempts to modulate the immune response system as a whole.
Examples of several general efforts in this direction are
highlighted below.
[0013] The mechanism of action of NSAIDs has been linked to
blocking of cyclooxygenase, an enzyme with both an inducible and a
constitutive form. As the inducible form of cyclooxygenase appears
to be elevated in inflammatory disease, investigation into
compounds selective for the inducible form are underway. In
addition, attempts to devise vaccines to treat ongoing arthritis
have been made with the use of peptide vaccines directed toward MHC
class II or T cell receptor proteins. Generally, it has been proven
difficult to demonstrate efficacy of vaccines administered to
ongoing disease.
[0014] Much of the tissue destruction in RA appears to be due to
various metalloproteinases. This group of proteases are believed to
be central to the degradation of collagen II and proteoglycan seen
in arthritis. A number of inhibitors of various of these enzymes
are under pre-clinical or clinical investigation.
[0015] A number of broadly immunosuppressive drugs are in clinical
testing for use in rheumatoid arthritis, including cyclosporine A
and mycophenolate mofetil. As a wide range of cytokines are found
in arthritic joints, anti-arthritis therapies have targeted
cytokine pathways including those of IL-1, IL-2, IL-4, IL-10,
IL-11, TGF.beta., and TNF.alpha., as well as, chemokine pathways
(Feldmann et al., 1997). In particular, proinflammatory pathways of
IL-1 have been targeted both by attack of IL-1 directly and via the
naturally occurring interleukin-1 receptor antagonist molecule.
[0016] Methods of administering drug therapy for RA have included,
and have been proposed to include, systemic or local delivery of a
therapeutic drug and, in the case of proposed gene therapies, of a
therapeutic gene. To date, such treatments have fallen short of
delivering effective, safe therapy for arthritis for a variety of
reasons, including: systemic side effects of many drugs, rapid
clearance of therapeutic molecules from injected joints and/or
circulation, inefficiency in DNA integration and expression from
the genome, limited target cell population associated with some
viral delivery vectors, transient gene expression associated with
viral vectors which do not readily integrate and induction of an
immune response associated with the gene delivery virus.
[0017] Use of TNF antagonists, such as soluble TNFRs and anti-TNF
antibodies, has shown that a blockade of TNF can reverse effects
attributed to TNF including decreases in IL-1, GM-CSF, IL-6, IL-8,
adhesion molecules and tissue destruction (Feldmann et al., 1997).
Such pleiotropic effects apparently due to the blockade of TNF
alone suggests that TNF may lie near the top of the cascade of
cytokine mediated events. Elevated levels of TNF-.alpha. are found
in the synovial fluid of RA patients (Camussi and Lupia, 1998,
Drugs 55:613-620).
[0018] The effect of TNF blockade utilizing a hamster anti-mouse
TNF antibody was tested in a model of collagen type II arthritis in
DBA/1 mice (Williams et al., 1992, Proc. Natl. Acad. Sci. USA,
89:9784-9788). Treatment initiated after the onset of disease
resulted in improvement in footpad swelling, clinical score, and
histopathology of joint destruction. Other studies have obtained
similar results using either antibodies (Thorbecke et al., 1992,
Proc. Natl. Acad. Sci. USA, 89:7375-7379) or TNFR constructs (Husby
et al., 1988, J. Autoimmun. 1:363-71; Tetta et al., 1990, Ann.
Rheum. Dis. 49:665-667; Wooley et al., 1993, J. Immunol.
151:6602-6607; Piguet et al., 1992, Immunology 77:510-514).
[0019] Similar results have also been obtained in other animal
models of ongoing arthritis. In the rabbit, anti-TNF.alpha.
antibody was shown to have an anti-arthritic effect on antigen
induced arthritis (Lewthwaite et al., 1995, Ann. Rheum. Dis.
54:366-374). In the rat, anti-TNF therapy has been demonstrated to
be effective in adjuvant (Mycobacterium) arthritis (Issekutz et
al., 1994, Clin. Exp. Immunol. 97:26-32), in streptococcal cell
wall induced arthritis (Schimmer et al., 1997, J. Immunol.
159:4103-4108) and in collagen induced arthritis (Le et al., 1997,
Arthritis Rheum. 40:1662-1669).
[0020] In the studies described above, the TNF blockade was
achieved by systemic delivery of the blocking agent. In a rat
collagen arthritis model, delivery of a TNFR gene using an
adenoviral vector resulted in transient production of serum levels
of TNFR (up to 8 days) and a significant decrease in disease
progression when the adenovirus was given to animals undergoing
active arthritis (Le et al., 1997). Attempts to deliver the gene
directly to the joint were unsuccessful, however, and resulted in
an inflammatory reaction to the adenovirus.
[0021] A monoclonal antibody directed against TNF.alpha.
(infliximab, REMICADE, Centocor), administered with and without
methotrexate, has demonstrated clinical efficacy in the treatment
of RA (Elliott et al., 1993, Arthritis Rheum. 36:1681-1690; Elliott
et al., 1994, Lancet 344:1105-1110). These data demonstrate
significant reductions in Paulus 20% and 50% criteria at 4, 12 and
26 weeks. This treatment is administered intravenously and the
anti-TNF monoclonal antibody disappears from circulation over a
period of two months. The duration of efficacy appears to decrease
with repeated doses. The patient can generate antibodies against
the anti-TNF antibodies which limit the effectiveness and duration
of this therapy (Kavanaugh et al., 1998, Rheum. Dis. Clin. North
Am. 24:593-614). Administration of methotrexate in combination with
infliximab helps prevent the development of anti-infliximab
antibodies (Maini et al., 1998, Arthritis Rheum. 41:1552-1563).
Infliximab has also demonstrated clinical efficacy in the treatment
of the inflammatory bowel disorder Crohn's disease (Baert et al.,
1999, Gastroenterology 116:22-28).
[0022] Clinical trials of a recombinant version of the soluble
human TNFR (p75) linked to the Fc portion of human IgG1
(sTNFR(p75):Fc, ENBREL, Immunex) have shown that its administration
resulted in significant and rapid reductions in RA disease activity
(Moreland et al., 1997, N. Eng. J. Med., 337:141-147). In addition,
preliminary safety data from an ongoing pediatric clinical trial
for sTNFR(p75):Fc indicates that this drug is generally
well-tolerated by patients with juvenile rheumatoid arthritis (JRA)
(Garrison et al, 1998, Am. College of Rheumatology meeting, Nov. 9,
1998, abstract 584).
[0023] As noted above, ENBREL is a dimeric fusion protein
consisting of the extracellular ligand-binding portion of the human
75 kilodalton (p75) TNFR linked to the Fc portion of human IgG1.
The Fc component of ENBREL contains the CH2 domain, the CH3 domain
and hinge region, but not the CH1 domain of IgG1. ENBREL is
produced in a Chinese hamster ovary (CHO) mammalian cell expression
system. It consists of 934 amino acids and has an apparent
molecular weight of approximately 150 kilodaltons (Smith et al.,
1990, Science 248:1019-1023; Mohler et al., 1993, J. Immunol.
151:1548-1561; U.S. Pat. No. 5,395,760 (Immunex Corporation,
Seattle, Wash.); U.S. Pat. No. 5,605,690 (Immunex Corporation,
Seattle, Wash.).
[0024] Approved by the Food and Drug administration (FDA) (Nov. 2,
1998), ENBREL is currently indicated for reduction in signs and
symptoms of moderately to severely active rheumatoid arthritis in
patients who have had an inadequate response to one or more
disease-modifying antirheumatic drugs (DMARDs). ENBREL can be used
in combination with methotrexate in patients who do not respond
adequately to methotrexate alone. ENBREL is also indicated for
reduction in signs and symptoms of moderately to severely active
polyarticular-course juvenile rheumatoid arthritis in patients who
have had an inadequate response to one or more DMARDs (May 28,
1999). ENBREL is given to RA patients at 25 mg twice weekly as a
subcutaneous injection.
[0025] Currently, treatments using the sTNFR(p75):Fc (ENBREL,
Immunex) preparations, including those described above, are
administered subcutaneously twice weekly, which is costly,
unpleasant and inconvenient for the patient. "Important Drug
Warning" at <http://www.fda.gov/medwa-
tch/safety/1999/enbrel.htm>; "New Warning For Arthritis Drug,
ENBREL" at
<http://www.fda.gov/bbs/topics/ANSWERS/ANS00954.html>;
"ENBREL Injections Difficult for Some Patients" at
<http://dailynews.yahoo.com-
/h/nm/20000516/h1/arthritis_drugs.sub.--1.html>. Further, relief
afforded by this treatment is not sustained. Symptoms associated
with an arthritic condition are reduced during treatment with
sTNFR(p75):Fc but return upon discontinuation of this therapy,
generally within one month. Complications have arisen, including
local reactions at the site of injection. Moreover, long-term
systemic exposure to this TNF-.alpha. antagonist can impose a risk
for increased viral and bacterial infections and possibly cancer.
Since this product was first introduced, serious infections, some
involving death, have been reported in patients using ENBREL.
"Product Information" at <http://www.enbrel.com/patient/html/p-
atpi.htm>; "Proven Tolerability" at
<http://www.enbrel.com/patient/h- tml/patsafety.htm>.
[0026] Additional relevant references include: U.S. Pat. Nos.
5,858,775; 5,858,355; 5,858,351; 5,846,528; 5,843,742; 5,792,751;
5,786,211; 5,780,447; 5,766,585; 5,633,145; International Patent
publications WO 95/16353; WO 94/20517; WO 92/11359; Schwarz, 1998,
Keystone Symp., January 23-29, abstract 412; Song et al. (1998) J.
Clin. Invest. 101:2615-2621; Ghivizzani et al., 1998, Proc. Natl.
Acad. Sci. USA 95:4613-4618; Kang et al., 1997, Biochemical Society
Transactions 25:533-537; Robbins et al., 1997, Drug News &
Perspect. 10:283-292; Firestein et al., 1997, N. Eng. J. Med.
337:195-197; Muller-Ladner et al., 1997, J. Immunol. 158:3492-3498;
and Pelletier et al., 1997, Arthritis Rheum. 40:1012-1019.
[0027] There is a need for new, effective forms of treatment for
TNF-associated disorders such as RA, particularly treatments that
can provide sustained, controlled therapy. The present invention
provides compositions and methods for effective and continuous
treatment of inflammatory processes of arthritis and other
TNF-associated disorders.
[0028] All publications and references cited herein are hereby
incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
[0029] The present invention is directed to compositions and
methods for reducing TNF levels and/or treatment of TNF-associated
disorders of a mammal. The compositions generally comprise a
recombinant adeno-associated virus (rAAV) vector that contains a
polynucleotide encoding a TNF antagonist. The methods generally
employ an rAAV vector to deliver a polynucleotide encoding a TNF
antagonist to the mammal, which in turn reduces the levels of TNF
and results in palliation of a number of TNF-associated disorders,
such as arthritis (including RA), Crohn's disease, asthma and
congestive heart failure. Lowering TNF may in turn reduce levels of
other disease causing or contributing agents, such as other
inflammatory cytokines. Lowering the levels of soluble TNF in
joints exhibiting RA can in turn palliate TNF-associated
conditions, such as arthritis, and can reduce an inflammatory
response in the joints.
[0030] A preferred polynucleotide for the invention in the rAAV
vectors described herein is one encoding a tumor necrosis factor
receptor (TNFR). Since TNFR is capable of binding to soluble TNF,
the introduction of TNFR tends to reduce the levels of TNF in
circulation and/or the affected tissues, such as the joint. In some
embodiments, the invention provides an rAAV vector comprising a
polynucleotide encoding a p75 TNFR polypeptide. In other
embodiments, the rAAV vectors of the invention comprise a
polynucleotide encoding an Fc (constant domain of an immunoglobulin
molecule):p75 fusion polypeptide. In other embodiments, the rAAV
vectors of the invention comprise a polynucleotide encoding a
fusion polypeptide in which the extracellular domain of TNFR is
fused to Fc.
[0031] In some embodiments, the rAAV vectors of the invention
further comprise a polynucleotide encoding an IL-1 antagonist, such
as an IL-1 receptor type II polypeptide.
[0032] In another aspect, the invention provides methods for
reducing TNF levels in a mammal, which comprise administering
(i.e., delivering) any of the rAAV vectors described herein to the
mammal in an amount sufficient to reduce TNF levels. In some
embodiments, the delivery of an rAAV vector is in an arthritic
joint. In some embodiments, these methods further comprise
administering a TNF antagonist.
[0033] In another aspect, the invention provides methods for
reducing an inflammatory response in a mammal, which comprise
administering (i.e., delivering) any of the rAAV vectors described
herein to the mammal in an amount sufficient to reduce the
inflammatory response. In some embodiments, these methods further
comprise administering a TNF antagonist.
[0034] In another aspect, the invention provides methods for
palliating a TNF-associated disoreder, such as an arthritic
condition occurring in a mammal, which comprise administering
(i.e., delivering) any of the rAAV vectors described herein to the
mammal in an amount sufficent to palliate the disorder (such as
arthritic condition). In some embodiments, these methods further
comprise administering a TNF antagonist.
BRIEF DESCRIPTION OF THE FIGURES
[0035] FIG. 1 depicts the amino acid sequence of a TNFR:Fc fusion
polypeptide from U.S. Pat. No. 5,605,690.
[0036] FIG. 2 depicts the polynucleotide and amino acid sequences
of a TNFR:Fc fusion polypeptide from U.S. Pat. No. 5,605,690.
[0037] FIG. 3 depicts the amino acid and polynucleotide sequences
of a human IL-1R type II from GenBank U74649.
[0038] FIG. 4 depicts the nucleotide and amino acid sequences of
rat TNFR (p80) extracellular domain (ECD).
[0039] FIG. 5 depicts the amino acid sequence alignment of rat TNFR
(p80) ECD, murine TNFR (p80) ECD and human TNFR (p75) ECD.
[0040] FIG. 6 depicts a diagram of the rat IgG1 heavy chain cDNA
and the relative location of the PCR primers used to amplify the Fc
portion of the IgG1 cDNA.
[0041] FIG. 7 depicts the nucleotide and amino acid sequences of
rat IgG1Fc.
[0042] FIG. 8 depicts the nucleotide and amino acid sequences of
rat TNFR:Fc fusion construct.
[0043] FIG. 9 depicts a diagram of the pCMVrTNFR-Fc expression
plasmid, including the rat TNFR(p80)ECD-IgG1Fc fusion
polynucleotide and operatively linked control elements.
[0044] FIG. 10 depicts a northern analysis of RNA from cells
tranfected with the pCMVrTNFR-Fc expression plasmid.
[0045] FIG. 11 depicts a diagram of the rAAV vector plasmid
pAAVCMVrTNFRFc, including the rat TNFR(p80)ECD-IgG1Fc fusion
polynucleotide, operatively linked control elements, including AAV
ITRs.
[0046] FIG. 12 is a graph depicting the results of TNF inhibition
bioassays using media collected from cells transfected with
pCMVrTNFR-Fc (--.diamond-solid.--) and from cells transfected with
pCMVGFP (--.diamond.--).
[0047] FIG. 13 is a graph depicting results of TNF inhibition
bioassays using media from cells transduced with AAVCMVrTNFRFc
particles (.diamond-solid.), from cells transduced with AAV-lacZ
particles (.circle-solid.), from mock infected cells
(.tangle-solidup.) and from cells transfected with pCMVrTNFR-Fc
(.box-solid.).
[0048] FIG. 14 is a graph depicting results of TNF inhibition
bioassays using media from cells transduced with AAVCMVrTNFRFc
particles at 100 (.diamond-solid.), 500 (), 1000 (.DELTA.), 5000
(.largecircle.), or 10,000 (.diamond.) particles per cell, as well
as with media from mock infected cells (.sym.) and from cells
transfected with pCMVrTNFR-Fc (.quadrature.).
[0049] FIG. 15 is a graph depicting a time course analysis of
TNFR-Fc polypeptide expression after transduction of cells with
AAVCMVrTNFRFc at 1000 particles per cell. The expression of TNFR-Fc
was determined with TNF inhibition bioassays.
[0050] FIG. 16 is a photograph of joint tissue treated with
rAAV-LacZ and histochemically stained for .beta.-galactosidase
activity.
[0051] FIG. 17 is a photograph of arthritic joint tissue treated
with rAAV-LacZ and histochemically stained for .beta.-galactosidase
activity.
[0052] FIG. 18 is a photograph of arthritic joint tissue treated
with PBS and histochemically stained for .beta.-galactosidase
activity.
[0053] FIG. 19 is a graph depicting suppression of SCW-induced
arthritis by rAAV-ratTNFR:Fc vector. Each point represents the mean
+/- standard error from the mean (SEM) for each group of rats.
[0054] FIG. 20 is a graph depicting suppression of arthritis
symptoms in the contralateral joint by AAV-ratTNFR:Fc vector. The
AI scores for each rear ankle paw was separately recorded and
plotted. Each point represents the mean +/- standard error from the
mean (SEM) for each group of rats.
[0055] FIG. 21 is a graph depicting serum expression of bioactive
rat TNFR:Fc protein in SCW-treated rats. Each point represents the
mean +/- standard deviation (SD) for each group of rats.
[0056] FIG. 22 is a graph depicting serum expression of bioactive
rat TNFR:Fc protein in naive rats. Each point represents the mean
+/- standard deviation (SD) for each group of rats.
DETAILED DESCRIPTION
[0057] We have discovered compositions and methods for reducing
levels of TNF in a tissue, a particular anatomical site and/or the
circulation of an individual and methods for lowering TNF levels
and for palliating TNF-associated disorders. Included are methods
for reducing inflammatory response in a subject by reducing levels
of TNF activity.
[0058] The invention described herein provides materials and
methods for use in the delivery to and expression of a
polynucleotide encoding a TNF antagonist in a mammal. The
polynucleotide encoding a TNF antagonist is delivered to the mammal
through a recombinant adeno-associated virus (rAAV) vector, a
vector which integrates into the genome of the host cell.
Introduction of rAAV DNA into cells generally leads to long-term
persistence and expression of DNA without disturbing the normal
metabolism of the cell. Thus, the invention provides a continuous
source of and ongoing administration of the TNF antagonist to the
mammal. This is a distinct and significant advantage over
previously described treatment modalities (i.e., exogenous
administration of therapeutic agents), which confer only transient
benefits.
[0059] Definitions
[0060] As used in the specification and claims, the singular form
"a", "an" and "the" include plural references unless the context
clearly dictates otherwise. For example, the term "a cell" includes
a plurality of cells, including mixtures thereof.
[0061] A "TNF antagonist" as used herein refers to a polypeptide
that binds TNF and inhibits and/or hinders TNF activity as
reflected in TNF binding to a TNF-receptor including any of the
following: (a) TNFR, preferably endogenous (i.e., native to the
individual or host), cell membrane bound TNFR; (b) the
extracellular domain(s) of TNFR; and/or (c) the TNF binding domains
of TNFR (which may be a portion of the extracellular domain). TNF
antagonists include, but are not limited to, TNF receptors (or
appropriate portions thereof, as described herein) and anti-TNF
antibodies. As used herein, the "biological activity" of a TNF
antagonist is to bind to TNF and inhibit and/or hinder TNF from
binding to any of the following: (a) TNFR, preferably endogenous,
cell membrane bound TNFR; (b) the extracellular domain(s) of TNFR;
and (c) the TNF binding domains of TNFR (which may be a portion of
the extracellular domain). A TNF antagonist can be shown to exhibit
biological activity using assays known in the art to measure TNF
activity and its inhibition, an example of which is provided
herein.
[0062] "TNF-associated disorders" are those disorders or diseases
that are associated with, result from, and/or occur in response to,
elevated levels of TNF. Such disorders may be associated with
episodic or chronic elevated levels of TNF activity and/or with
local or systemic increases in TNF activity. Such disorders
include, but are not limited to, inflammatory diseases, such as
arthritis and inflammatory bowel disease, and congestive heart
failure.
[0063] As used herein, the terms "TNF receptor polypeptide" and
"TNFR polypeptide" refer to polypeptides derived from TNFR (from
any species) which are capable of binding TNF. Two distinct
cell-surface TNFRs have described: Type II TNFR (or p75 TNFR or
TNFRII) and Type I TNFR (or p55 TNFR or TNFRI). The mature
full-length human p75 TNFR is a glycoprotein having a molecular
weight of about 75-80 kilodaltons (kD). The mature full-length
human p55 TNFR is a glycoprotein having a molecular weight of about
55-60 kD. The preferred TNFR polypeptides of this invention are
derived from TNFR Type I and/or TNFR type II.
[0064] TNFR polypeptides, such as "TNFR", "TNFR:Fc" and the like,
when discussed in the context of the present invention and
compositions therefor, refer to the respective intact polypeptide
(such as, TNFR intact), or any fragment or derivative thereof (such
as, an amino acid sequence derivative), that exhibits the desired
biological activity (i.e., binding to TNF). A "TNFR polynucleotide"
is any polynucleotide which encodes a TNFR polypeptide (such as a
TNFR:Fc polypeptide).
[0065] As used herein, an "extracellular domain" of TNFR refers to
a portion of TNFR that is found between the amino-terminus of TNFR
and the amino-terminal end of the TNFR transmembrane region. The
extracellular domain of TNFR binds TNF.
[0066] A "IL-1 antagonist" as used herein refers to a polypeptide
that binds interleukin 1 (IL-1) and inhibits and/or hinders IL-1
activity as reflected in IL-1 binding to an IL-1 receptor including
any of the following: (a) IL-1 receptor (IL-1R), preferably
endogenous (i.e., native to the individual or host), cell membrane
bound IL-1R; (b) the extracellular domain(s) of IL-1R; and/or (c)
the IL-1 binding domains of IL-1R (which may be a portion of the
extracellular domain). IL-1 antagonists include, but are not
limited to, IL-1 receptors (or appropriate portions thereof, as
described herein) and anti-IL-1 antibodies. As used herein, the
"biological activity" of an IL-1 antagonist is to bind-to IL-1 and
inhibit and/or -hinder IL-1 from binding to any of the following:
(a) IL-1R, preferably endogenous, cell membrane bound IL-1R; (b)
the extracellular domain(s) of IL-1R; and/or (c) the IL-1 binding
domains of IL-1R (which may be a portion of the extracellular
domain). An IL-1 antagonist can be shown to exhibit biological
activity using assays known in the art, including IL-1 inhibition
assays, which are described herein as well as in the art.
[0067] As used herein, the term "IL-1 receptor polypeptide" refers
to polypeptides derived from IL-1 receptor (from any species) which
are capable of binding IL-1. IL-1R polypeptides, when discussed in
the context of the present invention and compositions therefor,
refer to the respective intact polypeptide (such as intact IL-1R),
or any fragment or derivative thereof (such as, an amino acid
sequence derivative), that exhibits the desired biological activity
(i.e., binding to IL-1). A "IL-1R polynucleotide" is any
polynucleotide which encodes a IL-1R polypeptide.
[0068] As used herein, an "extracellular domain" of IL-1R refers to
a portion of IL-1R that is found between the amino-terminus of
IL-1R and the amino-terminal end of the IL-1 R transmembrane
region. The extracellular domain of IL-1R binds IL-1.
[0069] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to polymers of amino acids of any
length. The terms also encompass an amino acid polymer that has
been modified; for example, disulfide bond formation,
glycosylation, lipidation, or conjugation with a labeling
component.
[0070] A "chimeric polypeptide" or "fusion polypeptide" is a
polypeptide comprising regions in a different position than occurs
in nature. The regions may normally exist in separate proteins and
are brought together in the chimeric or fusion polypeptide, or they
may normally exist in the same protein but are placed in a new
arrangement in the chimeric or fusion polypeptide. A chimeric or
fusion polypeptide may also arise from polymeric forms, whether
linear or branched, of TNFR polypeptide(s).
[0071] The terms "polynucleotide" and "nucleic acid", used
interchangeably herein, refer to a polymeric form of nucleotides of
any length, including deoxyribonucleotides or ribonucleotides, or
analogs thereof. A polynucleotide may comprise modified
nucleotides, such as methylated nucleotides and nucleotide analogs,
and may be interrupted by non-nucleotide components. If present,
modifications to the nucleotide structure may be imparted before or
after assembly of the polymer. The term polynucleotide, as used
herein, refers interchangeably to double- and single-stranded
molecules. Unless otherwise specified or required, any embodiment
of the invention described herein that is a polynucleotide
encompasses both the double-stranded form and each of two
complementary single-stranded forms known or predicted to make up
the double-stranded form.
[0072] A "chimeric polynucleotide" or "fusion polynucleotide" is a
polynucleotide comprising regions in a different position than
occurs in nature. The regions may normally exist in separate genes
and are brought together in the chimeric or fusion polynucleotide,
or they may normally exist in the same gene but are placed in a new
arrangement in the chimeric or fusion polynucleotide. "AAV" is an
abbreviation for adeno-associated virus, and may be used to refer
to the virus itself or derivatives thereof. The term covers all
subtypes and both naturally occurring and recombinant forms, except
where required otherwise.
[0073] An "rAAV vector" as used herein refers to an AAV vector
comprising a polynucleotide sequence not of AAV origin (i.e., a
polynucleotide heterologous to AAV), typically a sequence of
interest for the genetic transformation of a cell. The heterologous
polynucleotide is flanked by at least one, preferably two, AAV
inverted terminal repeat sequences (ITRs). As described herein, an
rAAV vector can be in any of a number of forms, including, but not
limited to, plasmids, linear artificial chromosomes, complexed with
lipids, encapsulated within liposomes and, most preferably,
encapsidated in a viral particle, particularly an AAV.
[0074] An "rAAV virus" or "rAAV viral particle" refers to a viral
particle composed of at least one AAV capsid protein (preferably by
all of the capsid proteins of a wild-type AAV) and an encapsidated
rAAV.
[0075] "Packaging" refers to a series of intracellular events that
result in the assembly and encapsidation of an AAV particle or rAAV
particle.
[0076] AAV "rep" and "cap" genes refer to polynucleotide sequences
encoding replication and encapsidation proteins of adeno-associated
virus. They have been found in all AAV serotypes examined, and are
described below and in the art. AAV rep and cap are referred to
herein as AAV "packaging genes".
[0077] A "helper virus" for AAV refers to a virus that allows AAV
to be replicated and packaged by a mammalian cell. A variety of
such helper viruses for AAV are known in the art, including
adenoviruses, herpesviruses and poxviruses such as vaccinia. The
adenoviruses encompass a number of different subgroups, although
Adenovirus type 5 of subgroup C is most commonly used. Numerous
adenoviruses of human, non-human mammalian and avian origin are
known and available from depositories such as the ATCC. Viruses of
the herpes family include, for example, herpes simplex viruses
(HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses
(CMV) and pseudorabies viruses (PRV); which are also available from
depositories such as ATCC.
[0078] An "infectious" virus or viral particle is one that
comprises a polynucleotide component which it is capable of
delivering into a cell for which the viral species is trophic. The
term does not necessarily imply any replication capacity of the
virus. Assays for counting infectious viral particles are described
in the art.
[0079] A "replication-competent" virus (e.g., a
replication-competent AAV, sometimes abbreviated as "RCA") refers
to a phenotypically wild-type virus that is infectious, and is also
capable of being replicated in an infected cell (i.e., in the
presence of a helper virus or helper virus functions). In the case
of AAV, replication competence generally requires the presence of
functional AAV packaging genes. Preferred rAAV vectors as described
herein are replication-incompetent in mammalian cells (especially
in human cells) by virtue of the lack of one or more AAV packaging
genes. Preferably, such rAAV vectors lack any AAV packaging gene
sequences in order to minimize the possibility that RCA are
generated by recombination between AAV packaging genes and an rAAV
vector.
[0080] A "gene" refers to a polynucleotide containing at least one
open reading frame that is capable of encoding a particular protein
after being transcribed and translated.
[0081] "Recombinant", as applied to a polynucleotide means that the
polynucleotide is the product of various combinations of cloning,
restriction or ligation steps, and other procedures that result in
a construct that is distinct from a polynucleotide found in nature.
A recombinant virus is a viral particle comprising a recombinant
polynucleotide. The terms respectively include replicates of the
original polynucleotide construct and progeny of the original virus
construct.
[0082] A "control element" or "control sequence" is a nucleotide
sequence involved in an interaction of molecules that contributes
to the functional regulation of a polynucleotide, including
replication, duplication, transcription, splicing, translation, or
degradation of the polynucleotide. The regulation may affect the
frequency, speed, or specificity of the process, and may be
enhancing or inhibitory in nature. Control elements known in the
art include, for example, transcriptional regulatory sequences such
as promoters and enhancers. A promoter is a DNA region capable
under certain conditions of binding RNA polymerase and initiating
transcription of a coding region usually located downstream (in the
3' direction) from the promoter.
[0083] "Operatively linked" or "operably linked" refers to a
juxtaposition of genetic elements, wherein the elements are in a
relationship permitting them to operate in the expected manner. For
instance, a promoter is operatively linked to a coding region if
the promoter helps initiate transcription of the coding sequence.
There may be intervening residues between the promoter and coding
region so long as this functional relationship is maintained.
[0084] "Heterologous" means derived from a genotypically distinct
entity from that of the rest of the entity to which it is being
compared. For example, a polynucleotide introduced by genetic
engineering techniques into a plasmid or vector derived from a
different species is a heterologous polynucleotide. A promoter
removed from its native coding sequence and operatively linked to a
coding sequence with which it is not naturally found linked is a
heterologous promoter.
[0085] "Genetic alteration" refers to a process wherein a genetic
element is introduced into a cell other than by mitosis or meiosis.
The element may be heterologous to the cell, or it may be an
additional copy or improved version of an element already present
in the cell. Genetic alteration may be effected, for example, by
transfecting a cell with a recombinant plasmid or other
polynucleotide through any process known in the art, such as
electroporation, calcium phosphate precipitation, or contacting
with a polynucleotide-liposome complex. Genetic alteration may also
be effected, for example, by transduction or infection with a DNA
or RNA virus or viral vector. Preferably, the genetic element is
introduced into a chromosome or mini-chromosome in the cell; but
any alteration that changes the phenotype and/or genotype of the
cell and its progeny is included in this term.
[0086] A cell is said to be "stably" altered, transduced, or
transformed with a genetic sequence if the sequence is available to
perform its function during extended culture of the cell in vitro.
In preferred examples, such a cell is "inheritably" altered in that
a genetic alteration is introduced which is also inheritable by
progeny of the altered cell.
[0087] "Stable integration" of a polynucleotide into a cell means
that the polynucleotide has been integrated into a replicon that
tends to be stably maintained in the cell. Although episomes such
as plasmids can sometimes be maintained for many generations,
genetic material carried episomally is generally more susceptible
to loss than chromosomally-integrated material. However,
maintenance of a polynucleotide can often be effected by
incorporating a selectable marker into or adjacent to a
polynucleotide, and then maintaining cells carrying the
polynucleotide under selective pressure. In some cases, sequences
cannot be effectively maintained stably unless they have become
integrated into a chromosome; and, therefore, selection for
retention of a sequence comprising a selectable marker can result
in the selection of cells in which the marker has become
stably-integrated into a chromosome. Antibiotic resistance genes
can be conveniently employed as such selectable markers, as is well
known in the art. Typically, stably-integrated polynucleotides
would be expected to be maintained on average for at least about
twenty generations, preferably at least about one hundred
generations, still more preferably they would be maintained
permanently. The chromatin structure of eukaryotic chromosomes can
also influence the level of expression of an integrated
polynucleotide. Having the genes carried on stably-maintained
episomes can be particularly useful where it is desired to have
multiple stably-maintained copies of a particular gene. The
selection of stable cell lines having properties that are
particularly desirable in the context of the present invention are
described and illustrated below.
[0088] An "isolated" plasmid, virus, or other substance refers to a
preparation of the substance devoid of at least some of the other
components that may also be present where the substance or a
similar substance naturally occurs or is initially prepared from.
Thus, for example, an isolated substance may be prepared by using a
purification technique to enrich it from a source mixture.
Enrichment can be measured on an absolute basis, such as weight per
volume of solution, or it can be measured in relation to a second,
potentially interfering substance present in the source mixture.
Increasing enrichments of the embodiments of this invention are
increasingly more preferred. Thus, for example, a 2-fold enrichment
is preferred, 10-fold enrichment is more preferred, 100-fold
enrichment is more preferred, 1000-fold enrichment is even more
preferred.
[0089] A preparation of rAAV is said to be "substantially free" of
helper virus if the ratio of infectious rAAV particles to
infectious helper virus particles is at least about 10.sup.2:1;
preferably at least about 10.sup.4:1, more preferably at least
about 10.sup.6:1; still more preferably at least about 10.sup.8:1.
Preparations are also preferably free of equivalent amounts of
helper virus proteins (i.e., proteins as would be present as a
result of such a level of helper virus if the helper virus particle
impurities noted above were present in disrupted form). Viral
and/or cellular protein contamination can generally be observed as
the presence of Coomassie staining bands on SDS gels (e.g. the
appearance of bands other than those corresponding to the AAV
capsid proteins VP1, VP2 and VP3).
[0090] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient for vector(s) or for
incorporation of polynucleotides and/or proteins. Host cells
include progeny of a single host cell, and the progeny may not
necessarily be completely identical (in morphology or in genomic of
total DNA complement) to the original parent cell due to natural,
accidental, or deliberate mutation. A host cell includes cells
transfected in vivo with a polynucleotide(s) of this invention.
[0091] "Transformation" or "transfection" refers to the insertion
of an exogenous polynucleotide into a host cell, irrespective of
the method used for the insertion, for example, lipofection,
transduction, infection or electroporation. The exogenous
polynucleotide may be maintained as a non-integrated vector, for
example, a plasmid, or alternatively, may be integrated into the
host cell genome.
[0092] An "individual" or "subject" refers to vertebrates,
particularly members of a mammalian species, and includes, but is
not limited to, domestic animals, sports animals, rodents and
primates, including humans.
[0093] An "effective amount" is an amount sufficient to effect
beneficial or desired clinical results. An effective amount can be
administered in one or more administrations. For purposes of this
invention, an "effective amount" is an amount that achieves any of
the following: reduction of TNF levels; reduction of an
inflammatory response; and/or palliation, amelioration,
stabilization, reversal, slowing or delay in the progression of the
disease state.
[0094] As used herein, "in conjunction with" refers to
administration of one treatment modality in addition to another
treatment modality, such as adminstration of a TNF antagonist to a
subject in addition to the delivery of an rAAV to the same subject,
or administration of two different rAAV vectors to the same
subject. As such, "in conjunction with" refers to administration of
one treatment modality before, during or after delivery of the
other treatment modality to the subject.
[0095] An "arthritic condition" is a term well-understood in the
art refers to a state characterized by inflammation of a joint or
joints.
[0096] As used herein, "treatment" is an approach for obtaining
beneficial or desired clinical results. For purposes of this
invention, beneficial or desired clinical results include, but are
not limited to, alleviation of symptoms, diminishment of extent of
disease, stabilized (i.e., not worsening) state of disease,
preventing spread of disease, delay or slowing of disease
progression, amelioration or palliation of the disease state, and
remission (whether partial or total), whether detectable or
undetectable. "Treatment" can also mean prolonging survival as
compared to expected survival if not receiving treatment. For
example, treatment of an individual may be undertaken to decrease
or limit the pathology associated with elevated levels of TNF,
including, but not limited to, an inherited or induced genetic
deficiency, infection by a viral, bacterial, or parasitic organism,
a neoplastic or aplastic condition, or an immune system dysfunction
such as autoimmunity. Treatment may be performed either
prophylactically or therapeutically; that is, either prior or
subsequent to the initiation of a pathologic event or contact with
an etiologic agent.
[0097] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom, and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples.
[0098] "Palliating" a disease means that the extent and/or
undesirable clinical manifestations of a disease state are lessened
and/or time course of the progression is slowed or lengthened, as
compared to not administering rAAV vectors of the present
invention.
[0099] General Techniques
[0100] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of molecular biology,
virology, animal cell culture and biochemistry which are within the
skill of the art. Such techniques are explained fully in the
literature. See, for example, "Molecular Cloning: A Laboratory
Manual", Second Edition (Sambrook, Fritsch & Maniatis, 1989);
"Animal Cell Culture" (R. I. Freshney, ed., 1987); "Gene Transfer
Vectors for Mammalian Cells" (J. M. Miller & M. P. Calos, eds.,
1987); "Current Protocols in Molecular Biology" (F. M. Ausubel et
al., eds., 1987); "Current Protocols in Protein Science" (John E
Coligan, et al. eds. Wiley and Sons, 1995); and "Protein
Purification: Principles and Practice" (Robert K. Scopes,
Springer-Verlag, 1994).
[0101] rAAV Vectors for Delivery of TNF Antagonist
[0102] This invention provides recombinant AAV (rAAV) vectors for
reducing levels of TNF in a subject. This reduction may occur
anywhere in the body, such as in a tissue(s), a particular
anatomical site and/or circulation. Generally, these rAAV vectors
comprise a polynucleotide encoding a TNF antagonist. Preferably the
TNF antagonist is a TNFR, or a TNFR polypeptide (including
biologically active derivative(s) thereof). In the present
invention, a preferred TNFR is derived from the p75 TNFR.
[0103] An rAAV vector of this invention comprises a heterologous
(i.e. non-AAV) polynucleotide of interest in place of the AAV rep
and/or cap genes that normally make up the bulk of the AAV genome.
As in the wild-type AAV-genome, however, the heterologous
polynucleotide is preferably flanked by at least one, more
preferably two, AAV inverted terminal repeats (ITRs). Variations in
which an rAAV construct is flanked by a only a single (typically
modified) ITR have been described in the art and can be employed in
connection with the present invention.
[0104] TNF Antagonists
[0105] In the present invention, a TNF antagonist is supplied to an
individual, preferably a mammal, most preferably a human, as an
expressed product of a polynucleotide which encodes a TNF
antagonist. The polynucleotide encoding the TNF antagonist is
delivered to the mammal in the form of an rAAV vector. As defined,
such a TNF antagonist may be any polypeptide which binds to TNF
including, but not limited to, a TNFR polypeptide and an anti-TNF
antibody.
[0106] The TNF antagonist is secreted by the cell that receives the
rAAV vector; preferably the TNF antagonist is soluble (i.e., not
attached to the cell). For example, soluble TNF antagonists are
devoid of a transmembrane region and are secreted from the cell.
Techniques to identify and remove polynucleotide sequences which
encode transmembrane domains are known in the art.
[0107] Preferably, the TNF antagonist is a TNFR polypeptide. TNFR
polypeptide may be an intact TNFR (preferably from the same species
that receives the rAAV) or a suitable fragment of TNFR. U.S. Pat.
No. 5,605,690 provides examples of TNFR polypeptides, including
soluble TNFR polypeptides, appropriate for use in the present
invention. Preferably, the TNFR polypeptide comprises an
extracelluar domain of TNFR. More preferably, the TNFR polypeptide
is a fusion polypeptide comprising an extracellular domain of TNFR
linked to a constant domain of an immunoglobulin molecule; still
more preferably, the TNFR polypeptide is a fusion polypeptide
comprising an extracellular domain of the p75 TNFR linked to a
constant domain of an IgG1 molecule. Preferably when administration
to humans is contemplated, an Ig used for fusion proteins is human,
preferably human IgG1.
[0108] Monovalent and multivalent forms of TNFR polypeptides may be
used in the present invention. Multivalent forms of TNFR
polypeptides possess more than one TNF binding site. Multivalent
forms of TNFR polypeptides may be encoded in an rAAV vector, for
example, through the repeated ligation of polynucleotides encoding
TNF binding domains, each repeat being separated by a linker
region. Preferably, the TNFR of the present invention is a
bivalent, or dimeric, form of TNFR. For example, as described in
U.S. Pat. No. 5,605,690 and in Mohler et al., 1993, J. Immunol.,
151:1548-1561, a chimeric antibody polypeptide with TNFR
extracellular domains substituted for the variable domains of
either or both of the immunoglobulin heavy or light chains Would
provide a TNFR polypeptide for the present invention. Generally,
when such a chimeric TNFR:antibody polypeptide is produced by
cells, it forms a bivalent molecule through disulfide linkages
between the immunoglobulin domains. Such a chimeric
TNFR:antibody-polypeptide is referred to as TNFR:Fc.
[0109] The TNFR polypeptide construct sTNFR(p75):Fc is a preferred
embodiment of a TNF antagonist of the present invention. The
polypeptide sequence of sTNFR(p75):Fc is depicted in FIG. 1. The
coding sequence for this TNF antagonist is found in plasmid
pCAVDHFRhuTNFRFc as described in U.S. Pat. No. 5,605,690. Any
polynucleotide which encodes this sTNFR(p75):Fc polypeptide is
suitable for use in the present invention. A polynucleotide
sequence encoding sTNFR(p75):Fc is depicted in FIG. 2.
[0110] In the present invention, additional TNFR polypeptide
sequences include, but are not limited to, those indicated in FIGS.
2 and 3 of U.S. Pat. No. 5,395,760.
[0111] Polynucleotides which encode TNFR polypeptides can be
generated using methods known in the art from TNFR polynucleotide
sequences known in the art. In the present invention, preferable
polynucleotide sequences which encode TNFR polypeptides include,
but are not limited to, TNFR polynucleotide sequences found in U.S.
Pat. Nos. 5,395,760 and 5,605,690 and GenBank entries M32315 (human
TNFR) and M59378 (murine TNFRI). Suitable polynucleotides for use
in the present invention can be synthesized using standard
synthesis and recombinant methods.
[0112] Methods to assess TNF antagonist activity are known in the
art and exemplified herein. For example, TNF antagonist activity
may be assessed with a cell-based competitive binding assay. In
such an assay, radiolabelled TNF is mixed with serially diluted TNF
antagonist and cells expressing cell membrane bound TNFR. Portions
of the suspension are centrifuged to separate free and bound TNF
and the amount of radioactivity in the free and bound fractions
determined. TNF antagonist activity is assessed by inhibition of
TNF binding to the cells in the presence of the TNF antagonist.
[0113] As another example, TNF antagonists may be analyzed for the
ability to neutralize TNF activity in vitro in a bioassay using
cells susceptible to the cytotoxic activity of TNF as target cells,
such as L929 cells (see, for example, Example 3). In such an assay,
target cells, cultured with TNF, are treated with varying amounts
of TNF antagonist and subsequently are examined for cytolysis. TNF
antagonist activity is assessed by a decrease in TNF-induced target
cell cytolysis in the presence of the TNF antagonist.
[0114] The invention also provides rAAV vectors comprising a
polynucleotide encoding an interleukin 1 (IL-1) antagonist. The
cytokine IL-1 has been implicated as a pivotal mediator in both the
early and late disease stages of RA (Joosten et al., 1996,
Arthritis Rheum. 39:797-809). In RA, IL-1 appears to be involved in
infiltration of inflammatory cells and cartilage destruction in the
affected joint. A clinical trial with an IL-1 antagonist in
patients with RA indicated that blocking IL-1 activity may result
in amelioration of RA symptoms (Campion et al., 1996, Arthritis
Rheum. 39:1092-1101; Bresnihan et al., 1996, Arthritis Rheum.
39:S73). In a murine arthritis model, a combined
anti-TNF.alpha./anti-IL-- 1 treatment led to both diminished
inflammation and to diminished joint cartilage damage (Kuiper et
al., 1998, Cytokine 10:690-702).
[0115] As IL-1 and TNF appear to mediate different aspects of RA,
the present invention provides rAAV vectors comprising a
polynucleotide encoding a TNF antagonist (such as sTNFR(p75):Fc)
and an IL-1 antagonist (or, the rAAV vector comprises a
polynucleotide which encodes a TNF antagonist and an IL-1
antagonist). The present invention also provides rAAV vectors
comprising a polynucleotide encoding an IL-1 antagonist.
Preferably, the IL-1 antagonist is an IL-1 receptor (IL-1R), or an
IL-1R polypeptide (including biologically active derivatives(s)
thereof), that exhibits the desired biological activity (ie.,
binding to IL-1). Preferably, the IL-1R is derived from IL-1R type
II. In the present invention, preferable IL-1R polypeptide
sequences include, but are not limited to, that depicted in FIG. 3
and those found in IL-1R GenBank entry U74649 and U.S. Pat. No.
5,350,683, Any polynucleotide which encodes an IL-1R polypeptide is
suitable for use in the present invention. A polynucleotide
sequence encoding a preferred IL-1R polypeptide is depicted in FIG.
3. Suitable polynucleotides for use in the present invention can be
synthesized using standard synthesis and recombinant methods.
[0116] Methods to assess IL-1 antagonist activity are known in the
art. For example, IL-1 antagonist activity may be assessed with a
cell-based competitive binding assay as described herein for TNF
antagonists. As another example, IL-1 antagonist activity may be
assessed for the ability to neutralize IL-1 activity in vitro in a
bioassay for IL-1. In such an assay, a cell line (for example, EL-4
NOB-1) is used that produces interleukin 2 (IL-2) in response to
treatment with IL-1. This IL-1 responsive cell line is used in
combination with a IL-2 sensitive cell line (for example, CTLL-2).
Proliferation of the IL-2 sensitive cell line is dependent on the
IL-1 responsive cell line producing IL-2 and thus, is used as a
measure of I1-1 stimulation of the IL-1 responsive cell line. IL-1
antagonist activity would be assessed by its ability to neutralize
IL-1 activity in such a IL-1 bioassay (Gearing et al., 1991, J.
Immunol. Methods 99:7-11; Kuiper et al., 1998).
[0117] In preferred embodiments, the vector(s) of the invention are
encapsidated into an rAAV virus particle. Accordingly, the
invention includes an rAAV virus particle (recombinant because it
contains a recombinant polynucleotide) comprising any of the
vectors described herein. Methods of producing such particles are
described below.
[0118] The present invention also provides compositions containing
any of the rAAV vectors (and/or rAAV virus particles comprising the
rAAV vectors) described herein. These compositions are especially
useful for administration to individuals who may benefit from a
reduction in the level of TNF.
[0119] Generally, the compositions of the invention for use in
reducing TNF levels comprise an effective amount of an rAAV vector
encoding a TNF antagonist, preferably in a pharmaceutically
acceptable excipient. As is well known in the art, pharmaceutically
acceptable excipients are relatively inert substances that
facilitate administration of a pharmacologically effective
substance and can be supplied as liquid solutions or suspensions,
as emulsions, or as solid forms suitable for dissolution or
suspension in liquid prior to use. For example, an excipient can
give form or consistency, or act as a diluent. Suitable excipients
include but are not limited to stabilizing agents, wetting and
emulsifying agents, salts for varying osmolarity, encapsulating
agents, and buffers. Excipients as well as formulations for
parenteral and nonparenteral drug delivery are set forth in
Remington's Pharmaceutical Sciences 19th Ed. Mack Publishing
(1995).
[0120] Generally, these rAAV compositions are formulated for
administration by injection (e.g., intra-articularly,
intravenously, intramuscularly, etc.). Accordingly, these
compositions are preferably combined with pharmaceutically
acceptable vehicles such as saline, Ringer's balanced salt solution
(pH 7.4), dextrose solution, and the like. Although not required,
pharmaceutical compositions may optionally be supplied in unit
dosage form suitable for administration of a precise amount.
[0121] The invention also includes any of the above vectors (or
compositions comprising the vectors) for use in treatment of
TNF-associated disorders, such as inflammatory conditions
(including arthritis). The invention also includes any of the above
vectors (or compositions comprising the vectors) for use in
reducing TNF levels in an individual. The invention further
provides use of any of the above vectors (or compositions
comprising the vectors) in the manufacture of a medicament for
treatment of TNF-associated disorders, such as inflammatory
condiitons (including arthritis). The invention also provides use
of any of the above vectors (or compositions comprising the
vectors) in the manufacture of a medicament for reducing TNF
activity levels in an individual.
[0122] Host Cells Comprising an rAAV of the Invention
[0123] The present invention also provides host cells comprising
rAAV vectors described herein. Among eukaryotic host cells are
yeast, insect, avian, plant and mammalian cells. Preferably, the
host cells are mammalian. For example, host cells include, but are
not limited to, HeLa and 293 cells, both of human origin and both
readily avaliable.
[0124] The development of host cells able to express the rAAV
vector sequence provides an established source of the material that
is expressed at a reliable level. Methods and compositions for
introducing the rAAV vector into the host cell and then for
determining whether a host cell contains the rAAV vector are
discussed in a later section, have been described art and are
widely available.
[0125] Included in these embodiments, and discussed in a later
section are so called "producer cells" used as a basis for
producing packaged rAAV vectors.
[0126] Preparation of the rAAV of the Invention
[0127] The rAAV vectors of this invention may be prepared using
standard methods in the art. Adeno-associated viruses of any
serotype are suitable, since the various serotypes are functionally
and structurally related, even at the genetic level (see, e.g.,
Blacklow, pp. 165-174 of "Parvoviruses and Human Disease" J. R.
Pattison, ed. (1988); and Rose, Comprehensive Virology 3:1, 1974).
All AAV serotypes apparently exhibit similar replication properties
mediated by homologous rep genes; and all generally bear three
related capsid proteins such as those expressed in AAV2. The degree
of relatedness is further suggested by heteroduplex analysis which
reveals extensive cross-hybridization between serotypes along the
length of the genome; and the presence of analogous self-annealing
segments at the termini that correspond to ITRs. The similar
infectivity patterns also suggest that the replication functions in
each serotype are under similar regulatory control. Among the
various AAV serotypes, AAV2 is most commonly employed. For a
general review of AAV biology and genetics, see, e.g., Carter,
"Handbook of Parvoviruses", Vol. I, pp. 169-228 (1989), and Berns,
"Virology", pp. 1743-1764, Raven Press, (1990). General principles
of rAAV vector construction are known in the art. See, e.g.,
Carter, 1992, Current Opinion in Biotechnology, 3:533-539; and
Muzyczka, 1992, Curr. Top. Microbiol Immunol., 158:97-129.
[0128] As described above, the rAAV vectors of this invention
comprise a heterologous polynucleotide that encodes a TNF
antagonist. The rAAV vectors may also encode additional
polypeptides, such as an IL-1 receptor type II. Alternatively, the
rAAV vectors may comprise a heterologous polynucleotide that
encodes an IL-1 antagonist, such as an IL-1R. Such a heterologous
polynucleotide will generally be of sufficient length to provide
the encoding sequence and desired function. For encapisdation
within AAV2 particles, the heterologous polynucleotide will
preferably be less than about 5 kb although other serotypes and/or
modifications may be employed to allow larger sequences to packaged
into the AAV viral particles. For example, a preferred
polynucleotide encodes a TNFR:Fc as represented in SEQ ID NO: 1, is
about 1.5 kb in length.
[0129] Since transcription of the heterologous polynucleotide is
desired in the intended target cell, it can be operably linked to
its own or to a heterologous promoter and/or enhancer, depending
for example on the desired level and/or specificity of
transcription within the target cell, as is known in the art.
Various types of promoters and enhancers are suitable for use in
this context. For example, Feldhaus (U.S. patent application Ser.
No. 09/171,759, filed Oct. 20, 1998) describes a modified ITR
comprising a promoter to regulate expression from an rAAV.
Constitutive promoters provide an ongoing level of gene
transcription, and are preferred when it is desired that the
therapeutic polynucleotide be expressed on an ongoing basis.
Inducible or regulatable promoters generally exhibit low activity
in the absence of the inducer, and are up-regulated in the presence
of the inducer. They may be preferred when expression is desired
only at certain times or at certain locations, or when it is
desirable to titrate the level of expression using an inducing
agent. Promoters and enhancers may also be tissue-specific, that
is, they exhibit their activity only in certain cell types,
presumably due to gene regulatory elements found uniquely in those
cells. Such tissue-specific promoters and enhancers are known in
the art. By way of illustration, an example of tissue-specific
promoters includes various myosin promoters for expression in
muscle. Another example of tissue-specific promoters and enhancers
are of regulatory elements for cell and/or tissue types that are in
a joint.
[0130] Preferred inducible or regulated promoters and/or enhancers
include those that are physiologically responsive, such as those
that are responsive to inflammatory signals and/or conditions. For
example, use of promoters and/or enhancers that are activated in
response to mediators that drive inflammatory flares, including,
but not limited to, those from proinflammatory cytokine genes
(e.g., TNF.alpha., IL-1.beta. and IFN.gamma.), would result in the
expression of a TNF antagonist during the period of inflammatory
flare (Varley et al., 1998, Mol. Med. Today 4:445-451). The
TNF.alpha. promoter region is approximately 1.2 kb, and the
sequence has been reported by Takashiba et al., 1993, Gene,
131:307-308.
[0131] Further illustrative examples of promoters are the SV40 late
promoter from simian virus 40, the Baculovirus polyhedron
enhancer/promoter element, Herpes Simplex Virus thymidine kinase
(HSV tk), the immediate early promoter from cytomegalovirus (CMV)
and various retroviral promoters including LTR elements. Additional
inducible promoters include heavy metal ion inducible promoters
(such as the mouse mammary tumor virus (mMTV) promoter or various
growth hormone promoters), and the promoters from T7 phage which
are active in the presence of T7 RNA polymerase. A large variety of
other promoters are known and generally available in the art, and
the sequences for many such promoters are available in sequence
databases such as the GenBank database.
[0132] As translation is also desired in the intended target cell,
the heterologous polynucleotide encoding a TNF antagonist will
preferably also comprise control elements that facilitate
translation (such as a ribosome binding site or "RBS" and a
polyadenylation signal). Accordingly, the heterologous
polynucleotide will generally comprise at least one coding region
operatively linked to a suitable promoter, and can also comprise,
for example, an operatively linked enhancer, ribosome binding site
and poly-A signal. The heterologous polynucleotide can comprise one
encoding region, or more than one encoding region under the control
of the same or different promoters. The entire unit, containing a
combination of control elements and encoding region, is often
referred to as an expression cassette.
[0133] A heterologous polynucleotide encoding a TNF antagonist is
integrated by recombinant techniques into or preferably in place of
the AAV genomic coding region (i.e., in place of the AAV rep and
cap genes), but is generally flanked on either side by AAV ITRs.
This means that an ITR appears both upstream and downstream from
the coding sequence, either in direct juxtaposition, preferably
(although not necessarily) without any intervening sequence of AAV
origin in order to reduce the likelihood of recombination that
might regenerate a replication-competent AAV ("RCA") genome. Recent
evidence suggests that a single ITR can be sufficient to carry out
the functions normally associated with configurations comprising
two ITRs (U.S. Pat. No. 5,478745), and vector constructs with only
one ITR can thus be employed in conjunction with the packaging and
production methods described herein. The resultant rAAV vector is
referred to as being "defective" in AAV functions when specific AAV
coding sequences are deleted from the vector.
[0134] Given the relative encapsidation size limits of various AAV
genomes, insertion of a large heterologous polynucleotide into the
genome necessitates removal of a portion of the AAV genome, in
particular, one or more of the packaging genes may be removed.
Removal of one or more AAV genes is in any case desirable, to
reduce the likelihood of generating RCA. Accordingly, encoding or
promoter sequences for rep, cap, or both, are preferably removed,
since the functions provided by these genes can be provided in
trans.
[0135] The rAAV vectors are provided in a variety of forms, such as
in the form of bacterial plasmids, AAV particles, liposomes or any
combination thereof. In other embodiments, the rAAV vector sequence
is provided in the eukaryotic cells transfected with the rAAV
vector.
[0136] If the rAAV is to be used in the form of a packaged rAAV
particle, there are at least three desirable features of an rAAV
virus preparation for use in gene transfer. First, it is preferred
that the rAAV virus should be generated at titers sufficiently high
to transduce an effective proportion of cells in the target tissue.
High number of rAAV viral particles are typically required for gene
transfer in vivo. For example, some treatments may require in
excess of 10.sup.8 particles. Second, it is preferred that the rAAV
virus preparations should be essentially free of
replication-competent AAV (i.e., phenotypically wild-type AAV which
can be replicated in the presence of helper virus or helper virus
functions). Third, it is preferred that the rAAV virus preparation
as a whole be essentially free of other viruses (such as a helper
virus used in AAV production) as well as helper virus and cellular
proteins, and other components such as lipids and carbohydrates, so
as to minimize or eliminate any risk of generating an immune
response in the context of gene transfer. This latter point is
especially significant in the context of AAV because AAV is a
"helper-dependent" virus that requires co-infection with a helper
virus (typically adenovirus) or other provision of helper virus
functions in order to be effectively replicated and packaged during
the process of AAV production; and, moreover, as described above,
adenovirus has been observed to generate a host immune response in
the context of gene transfer applications (see, e.g., Le et al.,
1997; Byrnes et al., 1995, Neuroscience, 66:1015; McCoy et al.,
1995, Human Gene Therapy, 6:1553; and Barr et al., 1995, Gene
Therapy, 2:151).
[0137] If an rAAV vector is to be packaged in an AAV particle, in
order to replicate and package the rAAV vector, the missing
functions are complemented with a packaging gene, or a plurality
thereof, which together encode the necessary functions for the
various missing rep and/or cap gene products. The packaging genes
or gene cassettes are preferably not flanked by AAV ITRs and
preferably do not share any substantial homology with the rAAV
genome. Thus, in order to minimize homologous recombination during
replication between the vector sequence and separately provided
packaging genes, it is desirable to avoid overlap of the two
polynucleotide sequences. The level of homology and corresponding
frequency of recombination increase with increasing length of the
homologous sequences and with their level of shared identity. The
level of homology that will pose a concern in a given system can be
determined theoretically and confirmed experimentally, as is known
in the art. Generally, however, recombination can be substantially
reduced or eliminated if the overlapping sequence is less than
about a 25 nucleotide sequence if it is at least 80% identical over
its entire length, or less than about a 50 nucleotide sequence if
it is at least 70% identical over its entire length. Of course,
even lower levels of homology are preferable since they will
further reduce the likelihood of recombination. It appears that,
even without any overlapping homology, there is some residual
frequency of generating RCA. Even further reductions in the
frequency of generating RCA (e.g., by nonhomologous recombination)
can be obtained by "splitting" the replication and encapsidation
functions of AAV, as described by Allen et al. in U.S. patent
application Ser. No. 08/769,728, filed Dec. 18, 1996.
[0138] The rAAV vector construct, and the complementary packaging
gene constructs can be implemented in this invention in a number of
different forms. Viral particles, plasmids, and stably transformed
host cells can all be used to introduce such constructs into the
packaging cell, either transiently or stably.
[0139] A variety of different genetically altered cells can thus be
used in the context of this invention. By way of illustration, a
mammalian host cell may be used with at least one intact copy of a
stably integrated rAAV vector. An AAV packaging plasmid comprising
at least an AAV rep gene operably linked to a promoter can be used
to supply replication functions (as described in U.S. Pat. No.
5,658,776). Alternatively, a stable mammalian cell line with an AAV
rep gene operably linked to a promoter can be used to supply
replication functions (see, e.g., Trempe et al., U.S. Pat. No.
5,837,484; Burstein et al., WO 98/27207; and Johnson et al., U.S.
Pat. No. 5,658,785). The AAV cap gene, providing the encapsidation
proteins as described above, can be provided together with an AAV
rep gene or separately (see, e.g., the above-referenced
applications and patents as well as Allen et al. (WO 96/17947).
Other combinations are possible.
[0140] As is described in the art, and illustrated in the
references cited above and in Examples below, genetic material can
be introduced into cells (such as mammalian "producer" cells for
the production of rAAV) using any of a variety of means to
transform or transduce such cells. By way of illustration, such
techniques include, but are not limited to, transfection with
bacterial plasmids, infection with viral vectors, electroporation,
calcium phosphate precipitation, and introduction using any of a
variety of lipid-based compositions (a process often referred to as
"lipofection"). Methods and compositions for performing these
techniques have been described in the art and are widely
available.
[0141] Selection of suitably altered cells may be conducted by any
technique in the art. For example, the polynucleotide sequences
used to alter the cell may be introduced simultaneously with or
operably linked to one or more detectable or selectable markers as
is known in the art. By way of illustration, one can employ a drug
resistance gene as a selectable marker. Drug resistant cells can
then be picked and grown, and then tested for expression of the
desired sequence (i.e., a product of the heterologous
polynucleotide). Testing for acquisition, localization and/or
maintenance of an introduced polynucleotide can be performed using
DNA hybridization-based techniques (such as Southern blotting and
other procedures as known in the art). Testing for expression can
be readily performed by Northern analysis of RNA extracted from the
genetically altered cells, or by indirect immunofluorescence for
the corresponding gene product. Testing and confirmation of
packaging capabilities and efficiencies can be obtained by
introducing to the cell the remaining functional components of AAV
and a helper virus, to test for production of AAV particles. Where
a cell is inheritably altered with a plurality of polynucleotide
constructs, it is generally more convenient (though not essential)
to introduce them to the cell separately, and validate each step
seriatim. References describing such techniques include those cited
herein.
[0142] In one approach to packaging rAAV vectors in an AAV
particle, the rAAV vector sequence (i.e., the sequence flanked by
AAV ITRs), and the AAV packaging genes to be provided in trans, are
introduced into the host cell in separate bacterial plasmids.
Examples of this approach are described in Ratschin et al., 1984,
Mol. Cell. Biol., 4:2072; Hermonat et al., 1984, Proc. Natl. Acad.
Sci. USA, 81:6466; Tratschin et al., 1985, Mol. Cell. Biol.,
5:3251; McLaughlin et al., 1988, J. Virol., 62:1963; Lebkowski et
al., 1988, Mol. Cell. Biol., 7:349; Samulski et al., 1989, J.
Virol., 63:3822-3828; and Flotte et al., 1992, Am. J. Respir. Cell.
Mol. Biol., 7:349.
[0143] A second approach is to provide either the rAAV vector
sequence, or the AAV packaging genes, in the form of an episomal
plasmid in a mammalian cell used for AAV replication. See, for
example, U.S. Pat. No. 5,173,414.
[0144] A third approach is to provide either the rAAV vector
sequence or the AAV packaging genes, or both, stably integrated
into the genome of the mammalian cell used for replication, as
exemplified in Example 2 below.
[0145] One exemplary technique of this third approach is outlined
in international patent application WO 95/13365 (Targeted Genetics
Corporation and Johns Hopkins University) and corresponding U.S.
Pat. No. 5,658,776 (by Flotte et al.). This example uses a
mammalian cell with at least one intact copy of a stably integrated
rAAV vector, wherein the vector comprises an AAV ITR and a
transcription promoter operably linked to a target polynucleotide,
but wherein the expression of rep is limiting in the cell. In a
preferred embodiment, an AAV packaging plasmid comprising the rep
gene operably linked to a heterologous promoter is introduced into
the cell, and then the cell is incubated under conditions that
allow replication and packaging of the rAAV vector sequence into
particles.
[0146] Another approach is outlined in Trempe et al., U.S. Pat. No.
5,837,484. This example uses a stable mammalian cell line with an
AAV rep gene operably linked to a heterologous promoter so as to be
capable of expressing functional Rep protein. In various preferred
embodiments, the AAV cap gene can be provided stably as well or can
be introduced transiently (e.g. on a plasmid). An rAAV vector can
also be introduced stably or transiently.
[0147] Another approach is outlined in patent application WO
96/17947 (Targeted Genetics Corporation). This example uses a
mammalian cell which comprises a stably integrated AAV cap gene,
and a stably integrated AAV rep gene operably linked to a helper
virus-inducible heterologous promoter. A plasmid comprising the
rAAV vector sequence is also introduced into the cells (either
stably or transiently). The packaging of rAAV vector into particles
is then initiated by introduction of the helper virus.
[0148] Methods for achieving high titers of rAAV virus preparations
that are substantially free of contaminating virus and/or viral or
cellular proteins are outlined by Atkinson et al. in WO 99/11764.
Techniques described therein can be employed for the large-scale
production of rAAV viral particle preparations. Other methods for
preparing rAAV described in WO 00/14205, WO 99/20773, and WO
99/20779.
[0149] These various examples address the issue of producing rAAV
viral particles at sufficiently high titer, minimizing
recombination between rAAV vector and sequences encoding packaging
components, reducing or avoiding the potential difficulties
associated with the expression of the AAV rep gene in mammalian
cell line (since the Rep proteins can not only limit their own
expression but can also affect cellular metabolism) and producing
rAAV virus preparations that are substantially free of
contaminating virus and/or viral or cellular protein.
[0150] Packaging of an AAV vector into viral particles relies on
the presence of a suitable helper virus for AAV or the provision of
helper virus functions. Helper viruses capable of supporting AAV
replication are exemplified by adenovirus, but include other
viruses such, as herpes viruses (including, but not limited to,
HSV1, cytomegalovirus and HHV-6) and pox virus (particularly
vaccinia). Any such virus may be used.
[0151] Frequently, the helper virus will be an adenovirus of a type
and subgroup that can infect the intended host cell. Human
adenovirus of subgroup C, particularly serotypes 1, 2, 4, 6, and 7,
are commonly used. Serotype 5 is generally preferred.
[0152] The features and growth patterns of adenovirus are known in
the art. See, for example, Horowitz, "Adenoviridae and their
replication", pp 771-816 in "Fundamental Virology", Fields et al.,
eds. The packaged adenovirus genome is a linear DNA molecule,
linked through adenovirus ITRs at the left- and right-hand termini
through a terminal protein complex to form a circle. Control and
encoding regions for early, intermediate, and late components
overlap within the genome. Early region genes are implicated in
replication of the adenovirus genome, and are grouped depending on
their location into the E1, E2, E3, and E4 regions.
[0153] Although not essential, in principle it is desirable that
the helper virus strain be defective for replication in the subject
ultimately to receive the genetic therapy. Thus, any residual
helper virus present in an rAAV virus preparation will be
replication-incompetent. Adenoviruses from which the E1A or both
the E1A and the E3 region have been removed are not infectious for
most human cells. They can be replicated in a permissive cell line
(e.g., the human 293 cell line) which is capable of complementing
the missing activity. Regions of adenovirus that appear to be
associated with helper function, as well as regions that do not,
have been identified and described in the art (see, e.g., P. Colosi
et al., WO97/17458, and references cited therein).
[0154] For example, as described in Atkinson et al. (WO 99/11764),
a "conditionally-sensitive" helper virus can also be employed to
provide helper virus activity. Such a helper virus strain must
minimally have the property of being able to support AAV
replication in a host cell under at least one set of conditions
where it itself does not undergo efficient genomic replication.
Where helper virus activity is supplied as intact virus particles,
it is also generally necessary that the virus be capable of
replication in a host cell under a second set of conditions. The
first set of conditions will differ from the second set of
conditions by a readily controllable feature, such as the presence
or absence of a required cofactor (such as a cation), the presence
or absence of an inhibitory drug, or a shift in an environmental
condition such as temperature. Most conveniently, the difference
between the two conditions is temperature, and such a
conditionally-sensitive virus is thus referred to as a
temperature-sensitive helper virus.
[0155] Helper virus may be prepared in any cell that is permissive
for viral replication. For adenovirus, preferred cells include 293
cells and HeLa cells. It is preferable to employ culture techniques
that permit an increase in seeding density. 293 cells and HeLa cell
variants are available that have been adapted to suspension
culture. HeLa is preferable for reasons of cell growth, viability
and morphology in suspension. These cells can be grown at
sufficient density (2.times.10.sup.6 per ml) to make up for the
lower replication rate of the temperature-sensitive adenovirus
strain. Once established, cells are infected with the virus and
cultured at the permissive temperature for a sufficient period;
generally 3-7 days and typically about 5 days.
[0156] Examples of methods useful for helper virus preparation,
isolation and concentration can be found in Atkinson et al. (WO
99/11764).
[0157] Several criteria influence selection of cells for use in
producing rAAV particles as described herein. As an initial matter,
the cell must be permissive for replication and packaging of the
rAAV vector when using the selected helper virus. However, since
most mammalian cells can be productively infected by AAV, and many
can also be infected by helper viruses such as adenovirus, it is
clear that a large variety of mammalian cells and cell lines
effectively satisfy these criteria. Among these, the more preferred
cells and cell lines are those that can be easily grown in culture
so as to facilitate large-scale production of rAAV virus
preparations. Again, however, many such cells effectively satisfy
this criterion. Where large-scale production is desired, the choice
of production method will also influence the selection of the host
cell. For example, as described in more detail in Atkinson et al.
(WO 99/11764) and in the art, some production techniques and
culture vessels or chambers are designed for growth of adherent or
attached cells, whereas others are designed for growth of cells in
suspension. In the latter case, the host cell would thus preferably
be adapted or adaptable to growth in suspension. However, even in
the case of cells and cell lines that are regarded as adherent or
anchorage-dependent, it is possible to derive suspension-adapted
variants of an anchorage-dependent parental line by serially
selecting for cells capable of growth in suspension. See, for
example, Atkinson et al. (WO 99/11764).
[0158] Ultimately, the helper virus, the rAAV vector sequence, and
all AAV sequences needed for replication and packaging must be
present in the same cell. Where one or more AAV packaging genes are
provided separately from the vector, a host cell is provided that
comprises: (i) one or more AAV packaging genes, wherein each said
AAV packaging gene encodes an AAV replication or encapsidation
protein; (ii) a heterologous polynucleotide introduced into said
host cell using an rAAV vector, wherein said rAAV vector comprises
said heterologous polynucleotide flanked by at least one AAV ITR
and is deficient in said AAV packaging gene(s); and (iii) a helper
virus or sequences encoding the requisite helper virus functions.
It should be noted, however, that one or more of these elements may
be combined on a single replicon.
[0159] The helper virus is preferably introduced into the cell
culture at a level sufficient to infect most of the cells in
culture, but can otherwise be kept to a minimum in order to limit
the amount of helper virus present in the resulting preparation. A
multiplicity of infection or "MOI" of 1-100 may be used, but an MOI
of 5-10 is typically adequate.
[0160] Similarly, if the rAAV vector and/or packaging genes are
transiently introduced into the packaging cell (as opposed to being
stably introduced), they are preferably introduced at a level
sufficient to genetically alter most of the cells in culture.
Amounts generally required are of the order of 10 .mu.g per
10.sup.6 cells, if supplied as a bacterial plasmid; or 10.sup.8
particles per 10.sup.5 cells, if supplied as an AAV particle.
Determination of an optimal amount is an exercise of routine
titration that is within the ordinary skill of the artisan.
[0161] These elements can be introduced into the cell, either
simultaneously, or sequentially in any order. Where the cell is
inheritably altered by any of the elements, the cell can be
selected and allowed to proliferate before introducing the next
element.
[0162] In one preferred example, the helper virus is introduced
last into the cell to rescue and package a resident rAAV vector.
The cell will generally already be supplemented to the extent
necessary with AAV packaging genes. Preferably, either the rAAV
vector or the packaging genes, and more preferably both are stably
integrated into the cell. It is readily appreciated that other
combinations are possible. Such combinations are included within
the scope of the invention.
[0163] Once the host cell is provided with the requisite elements,
the cell is cultured under conditions that are permissive for the
replication AAV, to allow replication and packaging of the rAAV
vector. Culture time is preferably adjusted to correspond to peak
production levels, and is typically 3-6 days. rAAV particles are
then collected, and isolated from the cells used to prepare
them.
[0164] Optionally, rAAV virus preparations can be further processed
to enrich for rAAV particles, deplete helper virus particles, or
otherwise render them suitable for administration to a subject. See
Atkinson et al. for exemplary techniques (WO 99/11764).
Purification techniques can include isopynic gradient
centrifugation, and chromatographic techniques. Reduction of
infectious helper virus activity can include inactivation by heat
treatment or by pH treatment as is known in the art. Other
processes can include concentration, filtration, diafiltration, or
mixing with a suitable buffer or pharmaceutical excipient.
Preparations can be divided into unit dose and multi dose aliquots
for distribution, which will retain the essential characteristics
of the batch, such as the homogeneity of antigenic and genetic
content, and the relative proportion of contaminating helper
virus.
[0165] Various methods for the determination of the infectious
titer of a viral preparation are known in the art. For example, one
method for titer determination is a high-throughput titering assay
as provided by Atkinson et al. (WO 99/11764). Virus titers
determined by this rapid and quantitative method closely correspond
to the titers determined by more classical techniques. In addition,
however, this high-throughput method allows for the concurrent
processing and analysis of many viral replication reactions and
thus has many others uses, including for example the screening of
cell lines permissive or non-permissive for viral replication and
infectivity.
[0166] Methods of Using rAAV of the Invention
[0167] The invention also provides methods in which administration
of rAAV vectors described herein is used to reduce levels of TNF in
a subject. Such methods may be particularly beneficial to
individuals with a TNF-associated disorder. Disorders suitable for
these methods are those associated with elevated levels of TNF and
include, but are not limited to, arthritis (including RA),
psoriatic arthritis, inflammatory bowel diseases (including Crohn's
disease and ulcerative colitis), asthma and congestive heart
failure.
[0168] The level of TNF may be circulating levels of TNF and/or
levels of TNF in a tissue and/or at a particular anatomical site.
It is understood that TNF levels are reduced when compared to TNF
levels of a subject prior to receiving rAAV encoding a TNF
antagonist or when compared to TNF levels of an individual that
does not receive rAAV encoding a TNF antagonist. It is understood
that TNF levels refers to levels of free (uncomplexed or unbound)
or active TNF. Methods to detect TNF levels are described
below.
[0169] In one embodiment, methods provided herein for reducing
levels of TNF include administration (delivery) of rAAV vectors (or
compositions comprising the vectors) described herein. In another
embodiment, rAAV vectors are administered in conjunction with
administration of a TNF antagonist, such as TNFR or anti-TNF
antibody. The TNF antagonist, preferably in composition with
physiologically acceptable carriers, exicipients or diluents, may
be administered by suitable techniques including, but not limited
to, intra-articular, intraperitoneal or subcutaneous routes by
bolus injection, continuous infusion or sustained release from
implants. As discussed below, the TNF antagonist may also be
administered directly to the connective tissue, particularly the
joint.
[0170] The invention also provides methods in which administration
of rAAV vectors described herein (or compositions comprising an
rAAV vector(s)) is used to reduce an inflammatory response in a
subject. Preferably, an inflammatory response is reduced in a
connective tissue, including, but not limited to, synovium,
cartilage, ligament and tendon. A preferred anatomical site for
reduction of an inflammatory response is an affected joint in a
subject with arthritis, such as RA. It is understood that an
inflammatory response is reduced when compared to an inflammatory
response in a subject prior to receiving rAAV encoding a TNF
antagonist or when compared to an inflammatory response in an
individual that does not receive rAAV encoding TNF antagonist.
[0171] The invention also provides methods in which administration
of rAAV vectors described herein (or compositions comprising an
rAAV vector(s)) is used to palliate a TNF-associated disorder,
including inflammatory diseases such as arthritis (i.e., an
arthritic condition) occuring in a subject. Preferably, an
arthritic condition is palliated in a joint, preferably connective
tissue which includes, but is not limited to, synovium, cartilage,
ligament and tendon. It is understood that an arthritic condition
is palliated when compared to an arthritic condition in a subject
prior to receiving rAAV encoding a TNF antagonist or when compared
to an arthritic condition in an individual that does not receive
rAAV encoding TNF antagonist.
[0172] In a preferred embodiment, the rAAV vector (or compositions
comprising an rAAV vector(s)) is delivered to an arthritic joint of
a mammal thus providing a source of the TNF antagonist at the site
of inflammation. Even more preferably, the rAAV vector comprises a
polynucleotide encoding sTNFR(p75):Fc.
[0173] In another preferred embodiment, the rAAV vector(s) (or
compositions comprising an rAAV vector(s)) is delivered to an
arthritic joint of a mammal providing a source of the TNF
antagonist and a source of IL-1 antagonist at the site of
inflammation. Preferably, the rAAV vector comprises a
polynucleotide encoding sTNFR(p75):Fc and a polynucleotide encoding
IL-1R.
[0174] In another preferred embodiment, a source of the TNF
antagonist and a source of IL-1 antagonist are delivered to an
arthritic joint of a mammal at the site of inflammation through the
administration of at least two different rAAV vectors (or
compositions comprising at least two different rAAV vectors).
Preferably, one of the rAAV vectors comprises a polynucleotide
encoding a TNFR and another one of the rAAV vectors comprises a
polynucleotide encoding an IL-1R. In these two different rAAV
vectors, the heterologous polynucleotides may be operably linked to
transcriptional promoters and/or enhancers which are active under
similar conditions or to transcriptional promoters and/or enhancers
which are active under different conditions, e.g., independently
regulated. In various refinements of administration, the two
different rAAV vectors (i.e., one comprising a polynucleotide
encoding a TNFR and one comprising a polynucleotide encoding IL-1R)
may be administered to the mammal at the same time or at different
times, at the same or at different frequencies and/or in the same
or at differing amounts.
[0175] For any of the above methods, it is understood that one or
more rAAV vectors may be administered. For example, as discussed
above, a vector may be administered that encodes a TNF antagonist,
such as TNF receptor (most preferably sTNFR(p75):Fc).
Alternatively, an additional vector may be administered that
encodes an IL-1 antagonist, such as an IL-1 receptor polypeptide.
Alternatively, a single vector encoding both a TNF antagonist and
an IL-1 antagonist may be administered. This single vector may have
the coding sequences under control of the same or different
transcriptional regulatory elements. If more than one vector is
used, it is understood that they may be administered at the same or
at different times and/or frequencies.
[0176] Further, it is understood that, for any of the above
methods, in preferred embodiments, the individual receiving rAAV
vector(s) will have cells which contain the rAAV vector (after
administration), and most preferably will have cells in which the
rAAV vector(s) is integrated into the cellular genome. Stable
integration of rAAV is a distinct advantage, as it allows more
persistent expression than episomal vectors. Accordingly, in
preferred embodiments, cells (i.e., at least one cell) in the
individual will comprise stably integrated rAAV. Stated
alternatively, for any of the above methods, administration of
rAAV(s) results in integration of the rAAV(s) into cellular genomes
(although, as is understood by those in the art, not all rAAV
vectors need be integrated). Methods of determining and/or
distinguishing integrated vs. non-integrated forms, such as
Southern detection methods, are well known in art.
[0177] Administration of rAAV vectors (preferably packaged as AAV
particles) may be through any of a number of routes. A preferred
mode of administration is through intramuscular delivery.
Intramuscular delivery of the rAAV vectors can reduce TNF levels
both in tissue and inter-tissue spaces near the site of injection
and also in circulation. Another preferred mode of administration
of the rAAV compositions is through intravenous delivery. Another
preferred mode of administration of rAAV compositions of the
invention is through injection of the composition(s) directly to
the tissue or anatomical site. A preferred mode of such an
administration is by intra-articular injection of the composition.
Preferably, the rAAV composition is delivered to the synovium of
the affected joint; more preferably, to synovial cells lining the
joint space. Administration to the joint can be single or repeated
administrations. Repeated administration would be at suitable
intervals, such as about any of the following: once a month, once
every 6 weeks, once every two months, once every three months, once
every four months, once every five months, once very six months.
Repeated administrations may also occur at varying intervals.
[0178] Another preferred mode of administration of rAAV
compositions of the invention is through naso-pharyngeal and
pulmonary routes of administration including, but not limited to,
by-inhalation, transbronchial and transalveolar routes. The
invention includes rAAV compositions suitable for by-inhalation
administration including, but not limited to, various types of
aerosols for inhalations, as well as powder forms for delivery
systems. Devices suitable for by-inhalation administration of rAAV
compositions include, but are not limited to, atomizers and
vaporizers.
[0179] An effective amount of rAAV (preferably in the form of AAV
particles) is administered, depending on the objectives of
treatment. An effective amount may be given in single or divided
doses. Where a low percentage of transduction can achieve a
therapeutic effect, then the objective of treatment is generally to
meet or exceed this level of transduction. In some instances, this
level of transduction can be achieved by transduction of only about
1 to 5% of the target cells, but is more typically about 20% of the
cells of the desired tissue type, usually at least about 50%,
preferably at least about 80%, more preferably at least about 95%,
and even more preferably at least about 99% of the cells of the
desired tissue type.
[0180] As an guide, the number of rAAV particles administered per
injection will generally be between about 1.times.10.sup.6 and
about 1.times.10.sup.14 particles, preferably, between about
1.times.10.sup.7 and 1.times.10.sup.13 particles, more preferably
about 1.times.10.sup.9 and 1.times.10.sup.12 particles and even
more preferably about 1.times.10.sup.11 particles.
[0181] The number of rAAV particles administered per joint by
intra-articular injection, for example, will generally be at least
about 1.times.10.sup.8, and is more typically about
5.times.10.sup.8, about 1.times.10.sup.10, and on some occasions
about 1.times.10.sup.11 particles, including both DNAse resistant
and DNAse susceptible particles. In terms of DNAse resistant
particles, the dose will generally be between about
1.times.10.sup.6 and about 1.times.10.sup.14 particles, more
generally between about 1.times.10.sup.8 and about
1.times.10.sup.12 particles.
[0182] The number of rAAV particles administered per intramuscular
injection and per intravenous administration, for example, will
generally be at least about 1.times.10.sup.10, and is more
typically about any of the following: 5.times.10.sup.10,
1.times.10.sup.11, 5.times.10.sup.11, 1.times.10.sup.12,
5.times.10.sup.12 and on some occasions about 1.times.10.sup.13
particles, including both DNAse resistant and DNAse susceptible
particles. In terms of DNAse resistant particles, the dose will
generally be between about 1.times.10.sup.6 and about
1.times.10.sup.14 particles, more generally between about
1.times.10.sup.10 and 1.times.10.sup.13 particles.
[0183] The effectiveness of rAAV delivery can be monitored by
several criteria. For example, samples removed by biopsy or
surgical excision may be analyzed by in situ hybridization, PCR
amplification using vector-specific probes and/or RNAse protection
to detect rAAV DNA and/or rAAV mRNA. Also, for example, harvested
tissue, joint fluid and/or serum samples can be monitored for the
presence of TNF antagonist encoded by the rAAV with immunoassays,
including, but not limited to, immunoblotting, immunoprecipitation,
immunohistology and/or immunofluorescent cell counting, or with
function-based bioassays dependent on TNF antagonist-mediated
inhibition of TNF activity. For example, when the rAAV encoded TNF
antagonist is a TNFR polypeptide, the presence of the encoded TNFR
in harvested samples can be monitored with a TNFR immunoassay or a
function-based bioassay dependent on TNFR-mediated inhibition of
TNF killing of mouse L929 cells. Examples of such assays are known
in the art and described herein.
[0184] The invention also provides methods in which administration
of rAAV vectors described herein use ex vivo strategies for
delivery of polynucleotides to the mammal. Such methods and
techniques are known in the art. See, for example, U.S. Pat. No.
5,399,346. Generally, cells are transduced by the rAAV vectors in
vitro and then the transduced cells are introduced into the mammal,
for example, into an arthritic joint. Suitable cells are known to
those skilled in the art and include autologous cells, such as stem
cells.
[0185] The effectiveness of the methods provided herein may, for
example, be monitored by assessment of the relative levels of TNF
in harvested tissue, joint fluid and/or serum subsequent to
delivery of the rAAV vectors described herein. Assays for assessing
TNF levels are known in the art and include, but are not limited
to, immunoassays for TNF, including, but not limited to, immunoblot
and/or immunoprecipitation assays, and cytotoxicity assays with
cells sensitive to the cytotoxic activity of TNF. See, for example,
Khabar et al., 1995, Immunol. Lett. 46:107-110.
[0186] The treated subject may also be monitored for clinical
features which accompany the TNF-associated disorder. For example,
subjects may be monitored for reduction is symptoms associated with
inflammation. For example, after treatment of RA in a subject using
methods of the present invention, the subject may be assessed for
improvements in a number of clinical parameters including, but not
limited to, joint swelling, joint tenderness, morning stiffness,
pain, erythrocyte sedimentation rate, and c-reactive protein.
[0187] The selection of a particular composition, dosage regimen
(i.e., dose, timing and repetition) and route of administration
will depend on a number of different factors, including, but not
limited to, the subject's medical history and features of the
condition and the subject being treated. The assessment of such
features and the design of an appropriate therapeutic regimen is
ultimately the responsibility of the prescribing physician. The
particular dosage regimen may be determined empirically.
[0188] The foregoing description provides, inter alia, compositions
and methods for reducing the levels of TNF in a mammal. It is
understood that variations may be applied to these methods by those
of skill in this art without departing from the spirit of this
invention.
[0189] The examples presented below are provided as a further guide
to a practitioner of ordinary skill in the art, and are not meant
to be limiting in any way.
EXAMPLES
Example 1
[0190] Rat (P80) TNFR:Fc Fusion Constructs and Expression of
Same
[0191] Cloning the Rat (p80) TNFR Extracellular Domain (ECD)
[0192] cDNA encoding the extracellular domain (EDC) of the rat p80
TNFR (Type II) was isolated from MARATHON-READY rat spleen cDNA
(Clontech) using 5' RACE PCR (Clontech) with a gene-specific PCR
primer (5'-CTAACGACGTTAACGATGCAGGTGAC-3') (Frohman et al., 1988,
Proc. Natl. Acad. Sci. USA 85:8998-9002). This primer was selected
from the 259 bp sequence of the cytoplasmic region of the rat TNFR
(p80) gene (Bader et al., 1996, J. Immunol. 157:3089-3096). Five
separate 5' RACE PCR reactions were performed. The products from
each PCR reaction were ligated into pCR 2.1 plasmid (Invitrogen
Corporation) followed by transformation into TOP10F' competent
cells, using the TOPO TA Cloning.RTM. Kit (Invitrogen Corporation).
A representative panel of clones were completely sequenced and a
full consensus sequence of the rat TNFR (p80) ECD was generated.
The cDNA sequence and the amino acid sequence are depicted in FIG.
1. DNA and protein sequence alignments were carried out using the
murine p80 TNFR and the human p75 TNFR as reference sequences. FIG.
2 depicts a protein alignment of the rat p80 TNFR ECD, the murine
p80 TNFR ECD and the human p75 TNFR ECD. The rat TNFR (p80) ECD
plasmid was denoted pCRrTNFR.ECD.
[0193] Cloning the Rat IgG1 Fc Region
[0194] Rat spleen poly(A) RNA was reverse transcribed with Oligo
d(T).sub.16 as a primer and the IgG1 Fc cDNA (encompassing the
hinge, CH2 and CH3 domains) was subsequently amplified using the
GeneAmp/E RNA PCR Kit (Perkin Elmer) (FIG. 3). PCR primers were
designed based on the rat IgG1 sequence (GenBank RAT IGG1Z,
Accesion # M28670) (Bruggemann, 1988, Gene 74:473-82). The forward
(hinge region) primer: 5'-cggaattcGTGCCCAGAAACTGTGGAG-3' included
an EcoRI site (lower case). The reverse (CH3 region) primer:
5'-gctctagaTCATTTACCCGGAGAGTGG-3' included an XbaI site. The PCR
product was ligated to pCR 2.1 plasmid DNA followed by
transformation into TOP10F' competent cells, using the TOPO TA
Cloning.RTM. Kit (Invitrogen Corporation). A panel of clones were
analyzed by restriction enzyme and sequence analyses. The cDNA
sequence of the rat IgG1Fc and the corresponding amino acid
sequence is depicted in FIG. 4. One clone was used for further
manipulations (see below) and was denoted pCRrIgG1Fc.
[0195] Generation of Rat (p80) TNFR-Fc Fusion Construct and
Expression Vector
[0196] To facilitate the fusion of the rat TNFR ECD with the IgG1Fc
region (at the hinge region), PCR was used to engineer a NotI
restriction site at the 5' end and a KpnI restriction site at the
3' end of the TNFR ECD. For the PCR reactions, plasmid pCRrTNFR.ECD
was used as a template, the forward primer (p80-5 NotI) was
5'-CATAAGGGCCCGCAAGAGCGG GAGCTACCGCCG-3' and the reverse primer
(p80-3 KpnI) was 5'-GGTACCCCACCCGTGATGCTTGGTTCAATG- -3'. Similarly,
PCR was used to engineer a KpnI restriction site at the 5' end of
the IgG1Fc (at the hinge region). For this, pCRrIgG1Fc was used as
a template, the forward primer (5r IgG1 Fe) was
5'-GGGTACCCAGAAACTGTGGAGG- TGATTGC-3' and the reverse primer
(HBRATG1/3') was 5'-GCTCTAGATCATTTACCCGG- AGAGTGG-3'.
[0197] The site of the sequence fusion was modeled after the human
(p75)TNFR:Fc fusion protein (Mohler et al., 1993, J. Immunol.
151:1548-1561). The TNFR ECD and IgG1Fc PCR products were ligated
via their KpnI restriction sites and subcloned into pCR 2.1. A
panel of clones were analyzed by restriction enzyme and sequence
analyses. One plasmid with the fusion polynucleotide (pCRrTNFR-Fc)
was used for further manipulations. The nucleotide sequence of the
rat TNFR:Fc fusion polynucleotide and the encoded amino acid
sequence are depicted in FIG. 5.
[0198] To construct a mammalian expression vector, the plasmid
pCRrTNFR-Fc was digested with NotI restriction enzyme and a 1.6 kb
DNA fragment containing the rTNFR-Fc fusion gene was isolated and
purified. The mammalian expression plasmid pCMV.beta. (Clontech)
was digested with NotI to remove the .beta.-galactosidase gene and
the 3.6 kb plasmid DNA backbone fragment was isolated and purified.
The 1.6 kb rTNFR-Fc gene fragment was ligated to the 3.6 kb plasmid
backbone and the resulting expression plasmid was designated
pCMVrTNFR-Fc (diagrammed in FIG. 6).
[0199] Analysis of Expression from pCMVrTNFR-Fc
[0200] The expression plasmid pCMVrTNFR-Fc (10 .mu.g) was
transfected into 293A cells using LIPOFECTAMINE (Life
Technologies). A mock-transfection was included as a negative
control. At 48 hours post-transfection, cells were harvested and
total cellular RNA was extracted using the Rneasy Mini Kit
(Qiagen). RNA samples (10 .mu.g) were subjected to northern blot
analysis using a rat TNFR-specific .sup.32P-labeled probe. A 1.6 kb
band corresponding to the rat TNFR-Fc RNA was present only in the
RNA sample from pCMVrTNFR-Fc-transfected cells (FIG. 7).
[0201] To assess protein expression from the rat TNFR-Fc expression
vector, 293 cells in 60 mm dishes were transfected with 10 .mu.g of
either pCMVrTNFR-Fc or a control plasmid (pCMVGFP) using
LIPOFECTAMINE (Life Technologies). A mock-transfection was also
included. At 48 hours post-transfection, cells were washed with PBS
and fixed for 10 min in methanol/acetone at room temperature. The
cells were then washed with PBS, incubated with blocking buffer for
1 hour at room temperature, washed again with PBS and then
incubated with alkaline phosphatase-conjugated anti-rat IgG1
(diluted 1:5000 in PBS) for 1 hour at 37.degree. C. The cells were
washed with PBS and were incubated with the alkaline phosphatase
detection system 1-STEP NBT/BCIP plus Suppressor (PIERCE) for 2 to
4 hours at room temperature.
Example 2
[0202] Generation of rAAV Vectors and Producer Cell Lines
[0203] rAAV Vectors
[0204] The principles of rAAV vector construction follow from the
genetics of the virus. Generally, the AAV rep and cap genes are
deleted and the cis-acting ITR sequences are retained in the
construction of an rAAV vector. Rep and cap functions can be
provided by a variety approaches including, but not limited to,
those based on transient transfections (see, for example, Samulski
et al., 1989; Flotte et al., 1995, Gene Ther. 2:29-37) and those
based on stable cell lines (see, for example, Clark et al., 1995,
Hum. Gene Ther. 6:1329-1341; Tamayose et al., 1996, Hum. Gene Ther.
7:507-513) to allow for rAAV virus generation.
[0205] Construction of the AAV Vector Plasmid pAAVCMVrTNFR-Fc
[0206] The expression plasmid pCMVrTNFR-Fc DNA was digested with
NotI and XbaI restriction enzymes and the 1.6 kb DNA fragment
containing the rat TNFR-Fc fusion gene was isolated and purified.
An rAAV vector plasmid, pAAVflagLUC, was digested with NotI and
XbaI restriction enzymes to remove the flagLUC DNA fragment and the
rAAV vector backbone was isolated and purified. The 1.6 kb rat
TNFR-Fc gene fragment was then subcloned into the NotI and XbaI
restriction sites of the rAAV vector plasmid. The diagram in FIG. 8
depicts the resulting rAAV vector in which the rat TNFR-Fc fusion
polynucleotide is located between, and operably linked to, the
human immediate early CMV enhancer promoter and a synthetic polyA
addition signal. The transcription unit containing the TNFR-Fc
fusion gene is enclosed between the AAV-2 ITRs. This rAAV vector
plasmid was denoted pAAVCMVrTNFR-Fc.
[0207] Generation of a Stable Producer Cell Line for
AAVCMVrTNFR-Fc
[0208] Generally, rAAV producer cell lines are generated by
transfection of cells with vector plasmid, followed by selection
with antibiotics (typically G418, hygromycin, or histidinol) and
cloning of individual colonies. Colonies are first screened for
vector replication. Clones showing high level replication of vector
following adenovirus infection are then tested for production of
infectious vector.
[0209] Plasmid pAAVCMVrTNFR-Fc (30 .mu.g) was transfected into the
Hela C12 packaging cell line by electroporation (Potter et al.,
1984, Proc. Natl. Acad. Sci. USA 79:7161-7165). The C12 cell line
contains the AAV2 rep and cap genes that are transcriptionally
quiescent until induction upon infection with adenovirus helper
(Clark et al., 1995; Clark et al., 1996, Gene Therapy 3:1124-1132).
Twenty four hours post-transfection, the cells were trypsinized and
replated in 100 mm plates at densities ranging from
5.times.10.sup.3 to 5.times.10.sup.4 cells per plate. The cells
were subjected to selection in DMEM containing 10% fetal bovine
serum and 300 .mu.g/ml hygromycin B. Drug-resistant cell clones
were isolated, expanded and their ability to produce infectious
AAVCMVrTNFR-Fc vectors was tested and compared in an infectivity
assay as described in Atkinson et al., 1998, Nucleic Acid Res.
26:2821-2823. One such producer cell clone (C12-55) was further
used for production of AAVCMVrTNFR-Fc vector. Production,
purification and titration were carried out essentially as
described herein and as generally described in Atkinson et al. (WO
99/11764).
Example 3
[0210] Rat TNFR-FC as a TNF Antagonist
[0211] Expression of Rat TNFR-Fc Activity After Transfection with
pCMVrTNFR-Fc
[0212] Cells were transfected with the rat TNFR-Fc expression
vector to determine (1) whether rat TNFR-Fc would be secreted from
cells and (2) whether rat TNFR-Fc had the ability to neutralize
TNF-.alpha. activity.
[0213] 293 cells (2.times.10.sup.6) in T-75 flasks were transfected
with either 10 .mu.g of pCMVrTNFR-Fc or pCMVGFP using LIPOFECTAMINE
(Life Technologies, Inc.). After 48 hours, the medium was collected
and tested in a TNF-.alpha. inhibition bioassay as follows. Mouse
fibrosarcoma WEHI-13var cells (ATCC, CRL-2148) were seeded in
96-well microplate at 4.times.10.sup.4 cells per well in 100 .mu.l
RPMI 1640 medium containing 10% fetal bovine serum. After overnight
incubation, actinomycin D (1 .mu.g/ml) and recombinant rat
TNF-.alpha. (0.75 ng/ml; BioSource International, PRC 3014) were
added to each well in a total volume of 100 .mu.l. Samples of
medium from the transfected 293 cells were added to the first row
of wells and serially diluted 2-fold, in triplicate. The cells were
incubated overnight at 37.degree. C. supplemented with 5% CO.sub.2.
The next day, 50 .mu.l of XTT labeling mixture (Cell proliferation
kit, Boehringer Mannheim, #1-465-015) was added to each well, and
the cells were incubated at 37.degree. C. for 4 hours. Finally, the
plate was placed in Spectra MAX 250 plate reader (Molecular
Devices) and the absorbance at 490 nm was recorded using Delta Soft
analysis software. The absorbance measured directly correlates to
the cell number and thus, to cell proliferation in the assay. If
not inhibited, TNF-.alpha. induces cell death in this assay.
[0214] Results from such a TNF inhibition bioassay are depicted in
FIG. 9 and demonstrate that pCMVrTNFR-Fc-transfected 293 cells
expressed and secreted the rat TNFR-Fc fusion protein into the
medium and that this TNFR-Fc protein inhibited killing of
WEHI-13var cells by TNF-.alpha. in a dose-dependent manner. Medium
from pCMVGFP-transfected 293 cells appeared to have no effect on
TNF-.alpha. activity.
[0215] Rat TNFR-Fc Activity After Transduction With
AAVCMVrTNFR-Fc
[0216] Cells were infected with the rAAV virus particles to
determine whether transduced cells could express and secrete rat
TNFR-Fc. The rat TNFR-Fc produced from the transduced cells was
also tested for the ability to act as a TNF antagonist.
[0217] 293 cells in a 24-well plate were mock-infected, infected
with a LacZ gene-containing AAV vector (Clark et al., 1995; Clark
et al., 1996) or with AAVCMVrTNFR-Fc at 10.sup.4 particles per
cell. The infected cells were maintained in DMEM containing 10%
fetal bovine serum (1 ml per well). Forty eight hours
post-infection, the media was collected and samples ranging from
0.3125 .mu.l to 20 .mu.l were analyzed in a TNF-.alpha. inhibition
assay, as described above. 293 cells transduced with
AAVCMVrTNFR-Fc, but not cells transduced with the LacZ
gene-containing vector (D6) nor mock-infected cells, expressed and
secreted a TNFR-Fc polypeptide with TNF-.alpha. neutralizing
activity (FIG. 10).
[0218] In another experiment, 293 cells were either mock-infected
or infected with AAVCMVrTNFR-Fc vector at 10.sup.2,
5.times.10.sup.2, 10.sup.3, 5.times.10.sup.3 and 10.sup.4 particles
per cell. At 48 hours post-infection, the media were collected and
subjected to a TNF-.alpha. inhibition assay as described above. The
rat TNFR-Fc protein was secreted from transduced cells in a
dose-dependent manner (FIG. 11). Time-course analysis of TNFR-Fc
protein expression after transduction of 293 cells with
AAVCMVrTNFR-Fc at 10.sup.3 particles per cell showed a steady
increase in secretion of a TNFR-FC protein with TNF-.alpha.
antagonist activity over 120 hours (FIG. 12).
Example 4
[0219] rAAV Vector Delivery to Joints
[0220] AAV vectors have been shown to mediate efficient and
persistent gene delivery to a variety of tissue targets in vivo.
These targets have included airway epithelium, vasculature, muscle,
liver, and central nervous system. See, for example, Flotte et al.,
1993, Proc. Natl. Acad. Sci. USA 90:10613-10617; Lynch, et al.,
1997, Circ. Res. 80:497-505; Kessler et al., 1996, Proc. Natl.
Acad. Sci. USA 93:14082-14087; Xiao et al., 1996, J. Virol.
70:8098-8108; Koeberl et al., 1997, Proc. Natl. Acad. Sci. USA
94:1426-1431; Snyder et al., 1997, Nat. Genet. 16:270-276; and
Kaplitt et al., 1994, Nat. Genet. 8:148-154. In several cases,
expression of a reporter transgene delivered with an rAAV vector
has been documented for greater than one year. Animal studies with
the AAV vector system have in general shown little or no
pathogenicity or immunogenicity, in contrast to other viral vector
systems.
[0221] In a pilot study, 5 normal rats were injected in the hind
paw joints with 10.sup.11 DNase resistant particles (DRP) of an
rAAV containing the LacZ gene, rAAV-LacZ. Detection of
incorporation of the rAAV vector into the genome would be monitored
by the production of the LacZ encoded polypeptide,
.beta.-galactosidase. The rats were observed for 30 days for
indications of inflammation such as joint redness, swelling and
pain. No indication of inflammation was seen in these animals in
contrast to rats injected with M. tuberculosis in incomplete
Freund's adjuvant which developed overt inflammation as indicated
by joint swelling, redness, tenderness.
[0222] The animals were sacrificed at day 30, the joints examined
and joint tissue scraped for assessment of gene expression by
luminescence readout of .beta.-galactosidase activity. No gross
inflammation was seen, the joints appeared identical to uninjected
joints, in contrast with adjuvant injected rats which exhibited
marked cellularity. Luminescence measurement showed
52.times.10.sup.4 RLU in the rAAV injected joint while the
background level was 3.5.times.10.sup.4 RLU. Despite the high
background of endogenous .beta.-galactosidase found in joint
tissue, the results of this experiment indicate that rAAV vectors
are capable of successfully transducing cells found in the
joint.
[0223] In summary, preliminary experiments in normal rats suggest
that rAAV vectors mediates the transduction of cells found in
proximity to the joint space following intra-articular injection of
vector.
Example 5
[0224] rAAV Vector Delivery to Joints in a Rodent Model of
Arthritis
[0225] A study was conducted using rAAV vector gene transfer in the
streptococcus cell wall model of arthritis. The rat model used in
these studies is an art-accepted and FDA-accepted model for
studying arthritis and is used for evaluating anti-cytokine
therapies.
[0226] In this study, rats treated with intraperitoneal injection
of Group A streptococcus cell wall to induce arthritis were also
co-administered an intra-articular injection of 8.6.times.10.sup.9
DRP of rAAV-LacZ vector. Animals were sacrificed on day 5 following
vector administration. Rats that received the streptococcal cell
wall preparation developed arthritis irrespective of rAAV-LacZ
vector administration.
[0227] Histochemical staining for .beta.-galactosidase activity
resulted in the presence of .beta.-galactosidase activity (blue
reaction product) in rAAV-LacZ treated (FIGS. 13 and 14) but not
control treated (FIG. 15) joints. Very dark blue-black cells were
seen in synovium of rAAV-LacZ treated animals and lighter blue
cells were localized to the bone stroma underlying the joint space.
At this time point, neither the cartilage nor cancellous bone
appeared to be transduced by the vector.
[0228] In summary, preliminary experiments in a rat model of
arthritis suggest that rAAV vectors mediates the transduction of
cells found in proximity to the joint space following
intra-articular injection of vector.
Example 6
[0229] rAAV-ratTNFR:Fc Vector Gene Therapy in Rodent Model of
Arthritis
[0230] Vectors. Recombinant AAV-ratTNFR:Fc (see above examples) and
AAV-EGFP vectors were produced from their corresponding stable HeLa
C12 producer cell lines, C12/AAV-ratTNFR:Fc and C12/AAV-EGFP,
respectively. AAV-EGFP encodes the red-shifted enhanced green
fluorescent protein (EGFP) form the bioluminescent jellyfish
Aequorea Victoria (Heim et al., 1995, Nature 373:663-664; Cormack
et al., 1996, Gene 173:33-38). This gene cassette includes a CMV
inmmediate-early (IE) enhancer/promoter and a bovine growth hormone
(BGH) polyadenylation (poly A) signal. It was included in the
experiments as vector control (unrelated gene). Cells were grown in
cell factories and vectors were produced from lysates prepared 3
days after infection with helper Ad5 (moi 10). Cell lysates were
microfluidized through an 18 gauge orfice at 10,000 PSI. The vector
was then banded by CsCl gradient centrifugation, dialyzed and
further purified through a PI column. Finally, the purified vector
bulk was dialyzed against Ringer's buffer saline solution (RBSS)
plus 4% Glycerol, sterile filtered, aliquated and stored at
-80.degree. C. DNase I-Resistant Particle (DRP) titers were
determined by slot blot analyses and were 7.6.times.10.sup.11
DRP/mL and 2.8.times.10.sup.12 DRP/mL for AAV-ratTNFR:Fc and
AAV-EGFP vectors, respectively. Clark et al., 1995, Human Gene
Therapy 6:1329-1341. Infectious titers were determined by
infectious center assays and were 1.times.10.sup.10 i.u/mL and
5.2.times.10.sup.9 i.u./mL for AAV-ratTNFR:Fc and AAV-EGFP vectors,
respectively. Yakobson et al., 1987, J. Virol. 61:972-981;
Zolotukhin et al., 1999, Gene therapy 6:973-985.
[0231] SCW-induced arthritis model. In this experimental model of
arthritis, the disease was initiated by a single intraperitoneal
(i.p.) injection of group A SCW peptidoglycan-polysaccharide
(PG-APS) (30 .mu.g/gr body weight) (Lee Laboratories Inc., Grayson,
Ga.) into 4-week old (100 gr) genetically susceptible female Lewis
rats (Charles River Breeding Laboratories, Wilmington, Mass.)
(Cromartie, et al., 1977, J. Exp. Med. 146:1585-1602). Typically,
this model exhibits a peripheral and symmetrical, biphasic
polyarthritis with cycles of exacerbated recurrence and remission
and is clinically and histologically similar to RA (Cromartie, et
al., 1977). An acute inflammation of the rear ankles developed
within 24-48 hours, which persisted for 4-5 days, and then
partially resolved. This acute, neutrophil-predominant,
inflammatory response was then followed by a spontaneously
reactivating chronic inflammation at approximately day 15, which
developed into a chronic, progressive, erosive synovitis. In
addition to polyarthritis, this PG-APS model induced chronic
granulomatous inflammation of the liver and spleen. The severity of
arthritis (articular index, AI) was determined by scoring each
ankle based on the degree of swelling, erythema, and distortion on
a scale of 0-4 and summing the scores for all limbs.
[0232] Intra-muscular and intra-articular injections. Rats were
anaesthetized with Isoflurane (5% with O.sub.2 for induction and 3%
for maintenance). Twenty microliters of either AAV-ratTNFR or
AAV-EGFP vectors (2.times.10.sup.10 DRP) or an equivalent volume of
RBSS plus 4% Glycerol (vehicle) were injected into the rear ankle
joint using a 30-gauge needle adapted to a Hamilton syringe.
Intra-muscular injections of either vehicle or recombinant AAV
vectors (1.2.times.10.sup.11 DRP in 150 .mu.L) were carried out
using a 25-gauge needle.
[0233] TNFR:Fc bioassay. Blood samples (300 .mu.L) were collected
from tail-vein before (pre-bleed), and 5 (acute phase), 11
(remission) and 33 (chronic phase) days after SCW-injection. Serum
samples (50 .mu.L) were assayed for bioactive rat TNFR:Fc fusion
protein in a standard TNF-.alpha. bioassay adapted for inhibition
studies (Khabar et al., 1995, Immunol. Lett. 46:107-110). In this
assay, inhibition of TNF-.alpha. (750 pg/mL)-mediated killing of
sensitive WEHI-13VAR cells by soluble rat TNFR:Fc is determined by
increased absorbance at OD490 nm.
[0234] Summary. We evaluated AAV-ratTNFR:Fc vector gene therapy in
an experimental rat model of arthritis. The streptococcal cell wall
(SCW)-induced arthritis model in Lewis rats was employed to
evaluate the effect of AAV-ratTNFR:Fc vector administration on the
severity of arthritis on both the ipsilateral and the contralateral
joints.
[0235] Intra-peritoneal injection of SCW followed by a single
intra-articular administration of 2.times.10.sup.10 DNase
I-resistant particles (DRP) of AAV-ratTNFR:Fc vector to both rear
ankle joints resulted in significant reduction of hind paw swelling
as measured by arthritis index (AI) scores. Moreover,
intra-peritoneal injection of SCW followed by administration of
AAV-ratTNFR:Fc vector to a single joint also resulted in
significant reduction of paw swelling in the contralateral joint. A
single intramuscular administration of 1.2.times.10.sup.11 DRP of
AAV-ratTNFR:Fc vector resulted in a similar effect. As expected,
intra-peritoneal injection of SCW followed by intra-articular or
intra-muscular administration-of an AAV vector encoding an
unrelated gene expression cassette (AAV-EGFP) did not exacerbate
joint inflammation but also did not result in any therapeutic
effect. Bioactive rat TNFR:Fc protein was readily detectable at day
33 in serum samples of rats injected intramuscularly with
AAV-ratTNFR:Fc vector. In contrast, serum bioactive rat TNFR:Fc
protein levels in intra-articularly-injected rats were not
significantly different from control rats (RBSS or AAV-EGFP-treated
rats), suggesting that local administration of AAV-ratTNFR:Fc
vector does not lead to significant systemic exposure of this
TNF-.alpha. antagonist.
[0236] Results. The experiments described below were carried out
using the group A SCW-induced arthritis model in rats. A total of
65 four-week old female Lewis rats were divided into 3 groups and
treated as follows:
[0237] Group 1
[0238] N=8, Day 0: SCW (i.p.) and AAV-ratTNFR:Fc (intra-articular,
both rear ankles; 2.times.10.sup.10 DRP/joint)
[0239] N=8, Day 0: SCW (i.p.) and AAV-ratTNFR:Fc (intra-articular,
one rear ankle joint; 2.times.10.sup.10 DRP/joint)
[0240] N=4, Day 0: SCW (i.p.) and AAV-ratTNFR:Fc (intramuscular;
1.2.times.10.sup.11 DRP/muscle)
[0241] N=6, Day 0: SCW (i.p.) and AAV-EGFP (intra-articular, both
rear ankles; 2.times.10.sup.10 DRP/joint)
[0242] N=4, Day 0: SCW (i.p.) and AAV-EGFP (intra-muscular;
1.2.times.10.sup.11 DRP/muscle)
[0243] Group 2
[0244] N=4, Day 0: SCW (i.p.,) and RBSS (intra-articular, both rear
ankles) N=5, Day 0: SCW (i.p.)
[0245] Group 3
[0246] N=4, Day 0: AAV-ratTNFR:Fc (intra-articular, both rear
ankles; 2.times.10.sup.10 DRP/joint)
[0247] N=4, Day 0: AAV-ratTNFR:Fc (intra-muscular;
1.2.times.10.sup.11 DRP/muscle)
[0248] N=4, Day 0: AAV-EGFP (intra-articular, both rear ankles;
2.times.10.sup.10 DRP/joint)
[0249] N=4, Day 0: AAV-EGFP (intra-muscular; 1.2.times.10.sup.10
DRP/muscle)
[0250] N=3, Day 0: RBSS (intra-articular, both rear ankles)
[0251] Rats were inspected daily for disease onset and progression,
and the severity of arthritis (AI) was recorded every 2 to 3 days.
FIG. 19 shows that intra-peritoneal injection of arthritogenic dose
(30 .mu.g/gr body weight) of SCW on Day 0 either alone or in
combination with intra-articular administration of RBSS into both
rear ankle joints resulted in a typical acute inflammatory response
that peaked on day 4 (mean AI=6) and then decreased to its minimum
by day 11 (mean AI=2). Remission was followed by recurrence of
joint swelling that plateaued by day 22 (mean AI=7) and remained
chronic until the animals were sacrificed (day 35). As expected,
intra-peritoneal injection of SCW followed by a single
intra-articular administration of 2.times.10.sup.10 DRP of
AAV-ratTNFR:Fc vector to both rear ankle joints did not have a
significant affect on joint swelling during the acute phase. In
contrast, the effect of the latter treatments resulted in
significant reduction of hind paw swelling during the chronic phase
as measured by Al scores (mean AI=2). Interestingly, administration
of AAV-ratTNFR:Fc vector to one joint produced significant and
similar therapeutic effects on both the ipsilateral as well as the
contralateral joint (see also FIG. 20). FIG. 20 shows that animals
were injected intra-peritoneally with SCW (30 .mu.g/gr body weight)
on day 0 followed by a single administration of AAV-ratTNFR:Fc
(total of 2.times.10.sup.10 DRP) into the left rear ankle joint.
The AI scores for each rear ankle paw was separately recorded. A
single intra-muscular administration of 1.2.times.10.sup.11 DRP of
AAV-ratTNFR:Fc vector following intra-peritoneal injection of SCW
resulted in a similar effect. Intra-peritoneal injection of SCW
followed by intra-articular or intra-muscular administration of an
AAV vector encoding the green fluorescent gene (AAV-EGFP) did not
exacerbate joint inflammation but also did not result in any
therapeutic effect. Finally, administration of either
AAV-ratTNFR:Fc or AAV-EGFP to naive rat joints did not induce
visible joint swelling. From this experiment we concluded that
administration of AAV-ratTNFR:Fc but not AAV-EGFP vector either to
the joint or to the muscle results in production of therapeutic
levels of soluble bioactive rat TNFR:Fc protein that binds and
significantly inhibit the inflammatory activity of TNF-.alpha..
Although administration of AAV-EGFP vector did not result in any
therapeutic effect, it did not exacerbate the inflammatory process
in the affected joints, and did not induce inflammation in the
joints of naive animals, indicating that recombinant AAV vector
delivery locally to the joint is safe. One possible explanation for
the noted contralateral effect is that expression of the rat
TNFR:Fc gene in transduced joint tissue leads to secretion of this
protein to the circulation which then gains access to uninjected
inflamed joints. To test this hypothesis, serum samples from both
naive and SCW-treated animals were assayed for bioactive rat
TNFR:Fc protein in a TNF-.alpha. inhibition bioassay (Khabar et
al., 1995) after administration of AAV-ratTNFR:Fc to the joint or
to the muscle. FIGS. 21 and 22 show that bioactive rat TNFR:Fc
protein was readily detectable by day 33 after intra-muscular
administration of AAV-ratTNFR:Fc vector. In contrast, the
circulating levels of bioactive rat TNFR:Fc protein from
intra-articularly injected animals were low and non-significantly
different from those of control animals (AAV-EGFP or RBSS-treated
rats). We concluded that the contralateral effect is unlikely due
to secretion and systemic distribution of the rat TNFR:Fc
protein.
[0252] Discussion. We described here an in vivo study using an
art-accepted model of arthritis aimed at evaluating recombinant
AAV-mediated TNFR:Fc gene delivery for the treatment of
inflammatory joint disease. We employed the SCW-induced arthritis
model in rats to evaluate the therapeutic effect of local
(intra-articular) and systemic (intra-muscular) administration of
AAV-ratTNFR:Fc vector on the severity of arthritis.
[0253] Our results show that intra-articular administration of
AAV-ratTNFR:Fc vector significantly reduced the severity of
SCW-induced arthritis in the absence of detectable bioactive rat
TNFR:Fc protein in the circulation. Intra-muscular administration
of AAV-ratTNFR:Fc vector was also effective in reducing arthritis
symptoms and as expected bioactive rat TNFR:Fc protein was readily
detectable in the serum.
[0254] Administration of AAV-ratTNFR:Fc or AAV-EGFP to the joints
of naive rats did not induce a detectable inflammatory response in
the injected paws and intra-articular administration of AAV-EGFP
vector to SCW-treated rats did not exacerbate the inflammatory
joint disease, indicating that local intra-articular administration
of recombinant AAV vectors is safe.
[0255] Interestingly, a single administration of this vector to one
joint resulted in a similar therapeutic effect on both the
ipsilateral and the uninjected contalateral joint. The phenomenon
of a therapeutic contralateral effect was first reported by
Ghivizzani et al. (1998, Proc. Natl. Acad. Sci. USA 95:4613-4618)
who noted that adenoviral delivery of soluble interleukin 1
receptor (IL-1sR) to one knee joint of rabbits with bilateral
antigen-induced arthritis suppressed disease in both the
ipsilateral as well as the contralateral uninjected knee. A similar
phenomenon has been noted in this model using the viral interleukin
10 (vIL-10) gene (Lechman, 1999, MS Thesis, University of
Pittsburgh). Moreover, adenoviral delivery of vIL-10 to the paws of
mice with collagen-induced arthritis (CIA) (Whalen et al., 1999, J
Immunol. 162:3625-32) and delivery of IkB to the ankle joints of
rats with SCW-induced arthritis (Miagkov et al., 1998, Proc. Natl.
Acad. Sci. USA 95:13859-13864) also suppressed disease in
non-injected joints on the same animal. One possible explanation
for this contralateral effect is that expression of the rat TNFR:Fc
gene in transduced joint tissue leads to secretion of this protein
to the circulation which then gains access to uninjected inflamed
joints. Our results indicate that the contralateral effect is
unlikely due to secretion and systemic distribution of the rat
TNFR:Fc protein. These results are also consistent with those of
Ghivizzani et al. (1998) who ruled out the likelihood that the gene
product or even the adenoviral vector travels to the other joints
via systemic circulation or the lymphatics. Thus, our results are
most likely consistent with a model that suggests that direct
introduction of genes into an arthritic joint leads to the
transduction of cells with the ability to traffic to other joints
(Ghivizzani et al., 1998).
[0256] The circulating levels of bioactive rat TNFR:Fc protein in
naive animals (injected intra-muscularly with AAV-ratTNFR:Fc
vector) were significantly higher than in the corresponding
SCW-treated animals. The possible explanation for this difference
is that in SCW-treated animals, the levels of TNF-.alpha. are
considerably higher than in naive animals as a result of the
ongoing systemic inflammatory process. In these diseased animals,
the TNF-.alpha. molecules are most likely being bound and
neutralized by soluble TNFR:Fc protein molecules and cannot be
detected in the bioassay.
Example 7
[0257] rAAV Vector for Co-Delivery of TNF Antagonist and IL-1
Antagonist
[0258] A polynucleotide encoding a TNFR:Fc polypeptide (as
described herein) is cloned into an rAAV vector plasmid as
described in Example 2 to generate an rAAVTNFR:Fc plasmid. A
polynucleotide encoding an IL-1R, GenBank entry U74649, is cloned
into the rAAVTNFR:Fc plasmid. Both the TNFR:Fc encoding sequence
and the IL-1R encoding sequence are operably linked to
transcriptional regulatory sequences and both are enclosed between
the AAV ITRs. This rAAV vector plasmid is denoted
pAAVTNFR:FcIL-1R.
[0259] rAAV producer cell lines are generated by transfection of
cells with the pAAVTNFR:FcIL-1R plasmid as described in Example 2.
rAAV vector particles are prepared as described herein. Expression
of TNFR:Fc activity and of IL-1R activity after transduction of
cells with rAAVTNFR:FcIL-1R viral particles is assessed using
methods described herein. An IL-1 bioassay is described in Kuiper
et al., 1998.
[0260] The effect of administration of rAAVTNFR:FcIL-1R viral
particles is assessed in the context of a animal arthritis model.
rAAV viral particles are administered by different routes including
intra-articular, intramuscular and intravenous injections.
Assessment of treatment includes determination of inflammation and
cartilage destruction in the joints.
Sequence CWU 1
1
20 1 518 PRT Homo sapien 1 Ala Arg Gln Ala Ala Trp Arg Glu Gly Ala
Gly Leu Arg Gly Arg Glu 1 5 10 15 Gly Ala Arg Ala Gly Gly Asn Arg
Thr Pro Pro Ala Ser Met Ala Pro 20 25 30 Val Ala Val Trp Ala Ala
Leu Ala Val Gly Leu Glu Leu Trp Ala Ala 35 40 45 Ala His Ala Leu
Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu 50 55 60 Pro Gly
Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln 65 70 75 80
Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val Phe Cys 85
90 95 Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu Asp Ser Thr
Tyr 100 105 110 Thr Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys
Gly Ser Arg 115 120 125 Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys
Thr Arg Glu Gln Asn 130 135 140 Arg Ile Cys Thr Cys Arg Pro Gly Trp
Tyr Cys Ala Leu Ser Lys Gln 145 150 155 160 Glu Gly Cys Arg Leu Cys
Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 165 170 175 Gly Val Ala Arg
Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro 180 185 190 Cys Ala
Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys 195 200 205
Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn Ala Ser 210
215 220 Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg Ser Met Ala
Pro 225 230 235 240 Gly Ala Val His Leu Pro Gln Pro Val Ser Thr Arg
Ser Gln His Thr 245 250 255 Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro
Ser Thr Ser Phe Leu Leu 260 265 270 Pro Met Gly Pro Ser Pro Pro Ala
Glu Gly Ser Thr Gly Asp Glu Pro 275 280 285 Lys Ser Cys Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu 290 295 300 Leu Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 305 310 315 320 Thr
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 325 330
335 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
340 345 350 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn 355 360 365 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp 370 375 380 Leu Asn Gly Lys Asp Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro 385 390 395 400 Ala Pro Met Gln Lys Thr Ile
Ser Lys Ala Lys Gly Gln Pro Arg Glu 405 410 415 Pro Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 420 425 430 Gln Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Arg His Ile 435 440 445 Ala
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 450 455
460 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
465 470 475 480 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
Phe Ser Cys 485 490 495 Ser Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu 500 505 510 Ser Leu Ser Pro Gly Lys 515 2 1557
DNA Homo sapien CDS (1)...(1554) 2 gcg agg cag gca gcc tgg aga gaa
ggc gct ggg ctg cga ggg cgc gag 48 Ala Arg Gln Ala Ala Trp Arg Glu
Gly Ala Gly Leu Arg Gly Arg Glu 1 5 10 15 ggc gcg agg gca ggg ggc
aac cgg acc ccg ccc gca tcc atg gcg ccc 96 Gly Ala Arg Ala Gly Gly
Asn Arg Thr Pro Pro Ala Ser Met Ala Pro 20 25 30 gtc gcc gtc tgg
gcc gcg ctg gcc gtc gga ctg gag ctc tgg gct gcg 144 Val Ala Val Trp
Ala Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Ala 35 40 45 gcg cac
gcc ttg ccc gcc cag gtg gca ttt aca ccc tac gcc ccg gag 192 Ala His
Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu 50 55 60
ccc ggg agc aca tgc cgg ctc aga gaa tac tat gac cag aca gct cag 240
Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr Ala Gln 65
70 75 80 atg tgc tgc agc aaa tgc tcg ccg ggc caa cat gca aaa gtc
ttc tgt 288 Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala Lys Val
Phe Cys 85 90 95 acc aag acc tcg gac acc gtg tgt gac tcc tgt gag
gac agc aca tac 336 Thr Lys Thr Ser Asp Thr Val Cys Asp Ser Cys Glu
Asp Ser Thr Tyr 100 105 110 acc cag ctc tgg aac tgg gtt ccc gag tgc
ttg agc tgt ggc tcc cgc 384 Thr Gln Leu Trp Asn Trp Val Pro Glu Cys
Leu Ser Cys Gly Ser Arg 115 120 125 tgt agc tct gac cag gtg gaa act
caa gcc tgc act cgg gaa cag aac 432 Cys Ser Ser Asp Gln Val Glu Thr
Gln Ala Cys Thr Arg Glu Gln Asn 130 135 140 cgc atc tgc acc tgc agg
ccc ggc tgg tac tgc gcg ctg agc aag cag 480 Arg Ile Cys Thr Cys Arg
Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 145 150 155 160 gag ggg tgc
cgg ctg tgc gcg ccg ctg cgc aag tgc cgc ccg ggc ttc 528 Glu Gly Cys
Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 165 170 175 ggc
gtg gcc aga cca gga act gaa aca tca gac gtg gtg tgc aag ccc 576 Gly
Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro 180 185
190 tgt gcc ccg ggg acg ttc tcc aac acg act tca tcc acg gat att tgc
624 Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp Ile Cys
195 200 205 agg ccc cac cag atc tgt aac gtg gtg gcc atc cct ggg aat
gca agc 672 Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro Gly Asn
Ala Ser 210 215 220 atg gat gca gtc tgc acg tcc acg tcc ccc acc cgg
agt atg gcc cca 720 Met Asp Ala Val Cys Thr Ser Thr Ser Pro Thr Arg
Ser Met Ala Pro 225 230 235 240 ggg gca gta cac tta ccc cag cca gtg
tcc aca cga tcc caa cac acg 768 Gly Ala Val His Leu Pro Gln Pro Val
Ser Thr Arg Ser Gln His Thr 245 250 255 cag cca act cca gaa ccc agc
act gct cca agc acc tcc ttc ctg ctc 816 Gln Pro Thr Pro Glu Pro Ser
Thr Ala Pro Ser Thr Ser Phe Leu Leu 260 265 270 cca atg ggc ccc agc
ccc cca gct gaa ggg agc act ggc gac gag ccc 864 Pro Met Gly Pro Ser
Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro 275 280 285 aaa tct tgt
gac aaa act cac aca tgc cca ccg tgc cca gca cct gaa 912 Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 290 295 300 ctc
ctg ggg gga ccg tca gtc ttc ctc ttc ccc cca aaa ccc aag gac 960 Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 305 310
315 320 acc ctc atg atc tcc cgg acc cct gag gtc aca tgc gtg gtg gtg
gac 1008 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp 325 330 335 gtg agc cac gaa gac cct gag gtc aag ttc aac tgg
tac gtg gac ggc 1056 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 340 345 350 gtg gag gtg cat aat gcc aag aca aag
ccg cgg gag gag cag tac aac 1104 Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn 355 360 365 agc acg tac cgg gtg gtc
agc gtc ctc acc gtc ctg cac cag gac tgg 1152 Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp 370 375 380 ctg aat ggc
aag gac tac aag tgc aag gtc tcc aac aaa gcc ctc cca 1200 Leu Asn
Gly Lys Asp Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 385 390 395
400 gcc ccc atg cag aaa acc atc tcc aaa gcc aaa ggg cag ccc cga gaa
1248 Ala Pro Met Gln Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
Glu 405 410 415 cca cag gtg tac acc ctg ccc cca tcc cgg gat gag ctg
acc aag aac 1296 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
Leu Thr Lys Asn 420 425 430 cag gtc agc ctg acc tgc ctg gtc aaa ggc
ttc tat ccc agg cac atc 1344 Gln Val Ser Leu Thr Cys Leu Val Lys
Gly Phe Tyr Pro Arg His Ile 435 440 445 gcc gtg gag tgg gag agc aat
ggg cag ccg gag aac aac tac aag acc 1392 Ala Val Glu Trp Glu Ser
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 450 455 460 acg cct ccc gtg
ctg gac tcc gac ggc tcc ttc ttc ctc tac agc aag 1440 Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 465 470 475 480
ctc acc gtg gac aag agc agg tgg cag cag ggg aac gtc ttc tca tgc
1488 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
Cys 485 490 495 tcc gtg atg cat gag gct ctg cac aac cac tac acg cag
aag agc ctc 1536 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
Gln Lys Ser Leu 500 505 510 tcc ctg tct ccg ggt aaa tga 1557 Ser
Leu Ser Pro Gly Lys 515 3 518 PRT Homo sapien 3 Ala Arg Gln Ala Ala
Trp Arg Glu Gly Ala Gly Leu Arg Gly Arg Glu 1 5 10 15 Gly Ala Arg
Ala Gly Gly Asn Arg Thr Pro Pro Ala Ser Met Ala Pro 20 25 30 Val
Ala Val Trp Ala Ala Leu Ala Val Gly Leu Glu Leu Trp Ala Ala 35 40
45 Ala His Ala Leu Pro Ala Gln Val Ala Phe Thr Pro Tyr Ala Pro Glu
50 55 60 Pro Gly Ser Thr Cys Arg Leu Arg Glu Tyr Tyr Asp Gln Thr
Ala Gln 65 70 75 80 Met Cys Cys Ser Lys Cys Ser Pro Gly Gln His Ala
Lys Val Phe Cys 85 90 95 Thr Lys Thr Ser Asp Thr Val Cys Asp Ser
Cys Glu Asp Ser Thr Tyr 100 105 110 Thr Gln Leu Trp Asn Trp Val Pro
Glu Cys Leu Ser Cys Gly Ser Arg 115 120 125 Cys Ser Ser Asp Gln Val
Glu Thr Gln Ala Cys Thr Arg Glu Gln Asn 130 135 140 Arg Ile Cys Thr
Cys Arg Pro Gly Trp Tyr Cys Ala Leu Ser Lys Gln 145 150 155 160 Glu
Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys Arg Pro Gly Phe 165 170
175 Gly Val Ala Arg Pro Gly Thr Glu Thr Ser Asp Val Val Cys Lys Pro
180 185 190 Cys Ala Pro Gly Thr Phe Ser Asn Thr Thr Ser Ser Thr Asp
Ile Cys 195 200 205 Arg Pro His Gln Ile Cys Asn Val Val Ala Ile Pro
Gly Asn Ala Ser 210 215 220 Met Asp Ala Val Cys Thr Ser Thr Ser Pro
Thr Arg Ser Met Ala Pro 225 230 235 240 Gly Ala Val His Leu Pro Gln
Pro Val Ser Thr Arg Ser Gln His Thr 245 250 255 Gln Pro Thr Pro Glu
Pro Ser Thr Ala Pro Ser Thr Ser Phe Leu Leu 260 265 270 Pro Met Gly
Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly Asp Glu Pro 275 280 285 Lys
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 290 295
300 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
305 310 315 320 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val Asp 325 330 335 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn
Trp Tyr Val Asp Gly 340 345 350 Val Glu Val His Asn Ala Lys Thr Lys
Pro Arg Glu Glu Gln Tyr Asn 355 360 365 Ser Thr Tyr Arg Val Val Ser
Val Leu Thr Val Leu His Gln Asp Trp 370 375 380 Leu Asn Gly Lys Asp
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 385 390 395 400 Ala Pro
Met Gln Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 405 410 415
Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn 420
425 430 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Arg His
Ile 435 440 445 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
Tyr Lys Thr 450 455 460 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys 465 470 475 480 Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser Cys 485 490 495 Ser Val Met His Glu Ala
Leu His Asn His Tyr Thr Gln Lys Ser Leu 500 505 510 Ser Leu Ser Pro
Gly Lys 515 4 398 PRT Homo sapien 4 Met Leu Arg Leu Tyr Val Leu Val
Met Gly Val Ser Ala Phe Thr Leu 1 5 10 15 Gln Pro Ala Ala His Thr
Gly Ala Ala Arg Ser Cys Arg Phe Arg Gly 20 25 30 Arg His Tyr Lys
Arg Glu Phe Arg Leu Glu Gly Glu Pro Val Ala Leu 35 40 45 Arg Cys
Pro Gln Val Pro Tyr Trp Leu Trp Ala Ser Val Ser Pro Arg 50 55 60
Ile Asn Leu Thr Trp His Lys Asn Asp Ser Ala Arg Thr Val Pro Gly 65
70 75 80 Glu Glu Glu Thr Arg Met Trp Ala Gln Asp Gly Ala Leu Trp
Leu Leu 85 90 95 Pro Ala Leu Gln Glu Asp Ser Gly Thr Tyr Val Cys
Thr Thr Arg Asn 100 105 110 Ala Ser Tyr Cys Asp Lys Met Ser Ile Glu
Phe Arg Val Phe Glu Asn 115 120 125 Thr Asp Ala Phe Leu Pro Phe Ile
Ser Tyr Pro Gln Ile Leu Thr Leu 130 135 140 Ser Thr Ser Gly Val Leu
Val Cys Pro Asp Leu Ser Glu Phe Thr Arg 145 150 155 160 Asp Lys Thr
Asp Val Lys Ile Gln Trp Tyr Arg Asp Ser Leu Leu Leu 165 170 175 Asp
Lys Asp Asn Glu Lys Phe Leu Ser Val Arg Gly Thr Thr His Leu 180 185
190 Leu Val His Asp Val Ala Gln Glu Asp Ala Gly Tyr Tyr Arg Cys Val
195 200 205 Leu Thr Phe Ala His Glu Gly Gln Gln Tyr Asn Ile Thr Arg
Ser Ile 210 215 220 Glu Leu Arg Ile Lys Lys Lys Lys Glu Glu Thr Ile
Pro Val Ile Ile 225 230 235 240 Ser Pro Leu Lys Thr Ile Ser Ala Ser
Leu Gly Ser Arg Leu Thr Ile 245 250 255 Pro Cys Lys Val Phe Leu Gly
Thr Gly Thr Pro Leu Thr Thr Met Leu 260 265 270 Trp Trp Thr Ala Asn
Asp Thr His Ile Glu Ser Ala Tyr Pro Gly Gly 275 280 285 Arg Val Thr
Glu Gly Pro Arg Gln Glu Tyr Ser Glu Asn Asn Glu Asn 290 295 300 Tyr
Ile Glu Val Pro Leu Ile Phe Asp Pro Val Thr Arg Glu Asp Leu 305 310
315 320 His Met Asp Phe Lys Cys Val Val His Asn Thr Leu Ser Phe Gln
Thr 325 330 335 Leu Arg Thr Thr Val Lys Glu Ala Ser Ser Thr Phe Ser
Trp Gly Ile 340 345 350 Val Leu Ala Pro Leu Ser Leu Ala Phe Leu Val
Leu Gly Gly Ile Trp 355 360 365 Met His Arg Arg Cys Lys His Arg Thr
Gly Lys Ala Asp Gly Leu Thr 370 375 380 Val Leu Trp Pro His His Gln
Asp Phe Gln Ser Tyr Pro Lys 385 390 395 5 1264 DNA Homo sapien 5
cgggatcccg tgtcctctgg aagttgtcag gagcaatgtt gcgcttgtac gtgttggtaa
60 tgggagtttc tgccttcacc cttcagcctg cggcacacac aggggctgcc
agaagctgcc 120 ggtttcgtgg gaggcattac aagcgggagt tcaggctgga
aggggagcct gtagccctga 180 ggtgccccca ggtgccctac tggttgtggg
cctctgtcag cccccgcatc aacctgacat 240 ggcataaaaa tgactctgct
aggacggtcc caggagaaga agagacacgg atgtgggccc 300 aggacggtgc
tctgtggctt ctgccagcct tgcaggagga ctctggcacc tacgtctgca 360
ctactagaaa tgcttcttac tgtgacaaaa tgtccattga gttcagagtc tttgagaata
420 cagatgcttt cctgccgttc atctcatacc cgcaaatttt aaccttgtca
acctctgggg 480 tattagtatg ccctgacctg agtgaattca cccgtgacaa
aactgacgtg aagattcaat 540 ggtacaggga ttctcttctt ttggataaag
acaatgagaa atttctaagt gtgaggggga 600 ccactcactt actcgtacac
gatgtggccc aggaagatgc tggctattac cgctgtgtcc 660 tgacatttgc
ccatgaaggc cagcaataca acatcactag gagtattgag ctacgcatca 720
agaaaaaaaa agaagagacc attcctgtga tcatttcccc cctcaagacc atatcagctt
780 ctctggggtc aagactgaca atcccgtgta aggtgtttct gggaaccggc
acacccttaa 840 ccaccatgct gtggtggacg gccaatgaca cccacataga
gagcgcctac ccgggaggcc 900 gcgtgaccga ggggccacgc caggaatatt
cagaaaataa tgagaactac attgaagtgc 960 cattgatttt tgatcctgtc
acaagagagg atttgcacat ggattttaaa tgtgttgtcc 1020 ataataccct
gagttttcag acactacgca ccacagtcaa ggaagcctcc tccacgttct 1080
cctggggcat tgtgctggcc ccactttcac tggccttctt ggttttgggg ggaatatgga
1140 tgcacagacg gtgcaaacac agaactggaa aagcagatgg tctgactgtg
ctatggcctc 1200 atcatcaaga ctttcaatcc tatcccaagt gaaataaatg
gaatgaaata attcaaacac 1260 aaaa 1264 6 774 DNA Rattus rattus CDS
(1)...(774) 6 atg gcg ccc gcc gcc ctc tgg gtc gcg ctg gtc gtc gaa
ctg cag ctg 48 Met Ala Pro Ala Ala Leu Trp Val Ala Leu Val Val Glu
Leu Gln Leu 1 5 10 15 tgg gcc acc ggg cac aca gtg ccc gcc aag gtt
gtc ttg aca ccc tac 96 Trp Ala Thr Gly His Thr Val Pro Ala Lys Val
Val Leu Thr Pro Tyr 20 25 30 aag cca gaa cct ggg aac cag tgc cag
atc tca cag gag tac tat gac 144 Lys Pro Glu Pro Gly Asn Gln Cys Gln
Ile Ser Gln Glu Tyr Tyr Asp 35 40 45 aag aag gct cag atg tgc tgt
gct aag tgt ccc cct ggc cag tat gca 192 Lys Lys Ala Gln Met Cys Cys
Ala Lys Cys Pro Pro Gly Gln Tyr Ala 50 55 60 aaa cac ttc tgc aac
aag act tca gac acc gtg tgt gcg gac tgt gcg 240 Lys His Phe Cys Asn
Lys Thr Ser Asp Thr Val Cys Ala Asp Cys Ala 65 70 75 80 gca ggc atg
ttt acc cag gtc tgg aac cat ctg cat aca tgc ctg agc 288 Ala Gly Met
Phe Thr Gln Val Trp Asn His Leu His Thr Cys Leu Ser 85 90 95 tgc
agt tct tcc tgt agt gat gac cag gtg gag acc cac aac tgc act 336 Cys
Ser Ser Ser Cys Ser Asp Asp Gln Val Glu Thr His Asn Cys Thr 100 105
110 aaa aaa cag aac cga gtg tgt gct tgc aac gct gac agt tac tgt gcc
384 Lys Lys Gln Asn Arg Val Cys Ala Cys Asn Ala Asp Ser Tyr Cys Ala
115 120 125 ttg aaa ttg cat tct ggg aac tgt cga cag tgc atg aag ctg
agc aag 432 Leu Lys Leu His Ser Gly Asn Cys Arg Gln Cys Met Lys Leu
Ser Lys 130 135 140 tgt ggc cct ggc ttc gga gtg gcc cgt tca aga acc
tca aat gga aac 480 Cys Gly Pro Gly Phe Gly Val Ala Arg Ser Arg Thr
Ser Asn Gly Asn 145 150 155 160 gtg ata tgc agt gcc tgt gcc cca ggg
acg ttc tct gac acc aca tca 528 Val Ile Cys Ser Ala Cys Ala Pro Gly
Thr Phe Ser Asp Thr Thr Ser 165 170 175 tcc aca gat gtg tgc agg ccc
cac ggc att tgt agc atc ctg gct att 576 Ser Thr Asp Val Cys Arg Pro
His Gly Ile Cys Ser Ile Leu Ala Ile 180 185 190 cct gga aat gca agc
acg gat gca gtc tgt gca tcc gag tcc cca act 624 Pro Gly Asn Ala Ser
Thr Asp Ala Val Cys Ala Ser Glu Ser Pro Thr 195 200 205 cca agc gct
gtt cca agg aca atc tac gta tct cag cca gag ccc aca 672 Pro Ser Ala
Val Pro Arg Thr Ile Tyr Val Ser Gln Pro Glu Pro Thr 210 215 220 aga
tcc cag ccc atg gat caa gag cca ggg cct agc caa act cca cac 720 Arg
Ser Gln Pro Met Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro His 225 230
235 240 atc cct gtg tcc ttg ggt tca acc ccc atc att gaa cca agc atc
acg 768 Ile Pro Val Ser Leu Gly Ser Thr Pro Ile Ile Glu Pro Ser Ile
Thr 245 250 255 ggt ggg 774 Gly Gly 7 258 PRT Rattus rattus 7 Met
Ala Pro Ala Ala Leu Trp Val Ala Leu Val Val Glu Leu Gln Leu 1 5 10
15 Trp Ala Thr Gly His Thr Val Pro Ala Lys Val Val Leu Thr Pro Tyr
20 25 30 Lys Pro Glu Pro Gly Asn Gln Cys Gln Ile Ser Gln Glu Tyr
Tyr Asp 35 40 45 Lys Lys Ala Gln Met Cys Cys Ala Lys Cys Pro Pro
Gly Gln Tyr Ala 50 55 60 Lys His Phe Cys Asn Lys Thr Ser Asp Thr
Val Cys Ala Asp Cys Ala 65 70 75 80 Ala Gly Met Phe Thr Gln Val Trp
Asn His Leu His Thr Cys Leu Ser 85 90 95 Cys Ser Ser Ser Cys Ser
Asp Asp Gln Val Glu Thr His Asn Cys Thr 100 105 110 Lys Lys Gln Asn
Arg Val Cys Ala Cys Asn Ala Asp Ser Tyr Cys Ala 115 120 125 Leu Lys
Leu His Ser Gly Asn Cys Arg Gln Cys Met Lys Leu Ser Lys 130 135 140
Cys Gly Pro Gly Phe Gly Val Ala Arg Ser Arg Thr Ser Asn Gly Asn 145
150 155 160 Val Ile Cys Ser Ala Cys Ala Pro Gly Thr Phe Ser Asp Thr
Thr Ser 165 170 175 Ser Thr Asp Val Cys Arg Pro His Gly Ile Cys Ser
Ile Leu Ala Ile 180 185 190 Pro Gly Asn Ala Ser Thr Asp Ala Val Cys
Ala Ser Glu Ser Pro Thr 195 200 205 Pro Ser Ala Val Pro Arg Thr Ile
Tyr Val Ser Gln Pro Glu Pro Thr 210 215 220 Arg Ser Gln Pro Met Asp
Gln Glu Pro Gly Pro Ser Gln Thr Pro His 225 230 235 240 Ile Pro Val
Ser Leu Gly Ser Thr Pro Ile Ile Glu Pro Ser Ile Thr 245 250 255 Gly
Gly 8 258 PRT Rattus rattus VARIANT (1)...(258) Xaa = Any Amino
Acid 8 Met Ala Pro Ala Ala Leu Trp Val Ala Leu Val Val Glu Leu Gln
Leu 1 5 10 15 Trp Ala Thr Gly His Thr Val Pro Ala Lys Val Val Leu
Thr Pro Tyr 20 25 30 Lys Pro Xaa Pro Gly Asn Gln Cys Gln Ile Ser
Gln Glu Tyr Tyr Asp 35 40 45 Lys Xaa Ala Gln Met Cys Cys Ala Lys
Cys Pro Pro Gly Gln Tyr Ala 50 55 60 Lys His Phe Cys Asn Lys Thr
Ser Asp Thr Val Cys Ala Asp Cys Ala 65 70 75 80 Ala Xaa Met Phe Thr
Gln Val Trp Asn His Leu His Thr Cys Leu Ser 85 90 95 Cys Ser Ser
Ser Cys Ser Asp Asp Gln Val Glu Xaa His Asn Cys Thr 100 105 110 Lys
Lys Xaa Asn Arg Val Cys Gly Cys Lys Ala Asp Ser Tyr Xaa Ala 115 120
125 Leu Lys Leu His Xaa Gly Asn Cys Arg Gln Cys Met Lys Leu Ser Lys
130 135 140 Xaa Gly Pro Gly Phe Gly Val Ala Arg Ser Arg Thr Ser Asn
Gly Lys 145 150 155 160 Val Ile Cys Ser Ala Cys Ala Pro Gly Thr Phe
Ser Asp Thr Thr Ser 165 170 175 Ser Thr Asp Val Cys Arg Pro His Gly
Ile Cys Ser Ile Leu Ala Ile 180 185 190 Arg Gly Asn Ala Ser Thr Asp
Ala Val Cys Ala Ser Glu Ser Pro Thr 195 200 205 Pro Ser Ala Val Pro
Arg Thr Leu Tyr Val Phe Gln Pro Glu Pro Thr 210 215 220 Arg Ser Gln
Pro Met Asp Gln Glu Pro Gly Xaa Ser Gln Thr Pro His 225 230 235 240
Ile Pro Val Ser Leu Gly Ser Thr Pro Ile Ile Glu Pro Ser Ile Thr 245
250 255 Gly Gly 9 258 PRT Mus musculus 9 Met Ala Pro Ala Ala Leu
Trp Val Ala Leu Val Phe Glu Leu Gln Leu 1 5 10 15 Trp Ala Thr Gly
His Thr Val Pro Ala Gln Val Val Leu Thr Pro Tyr 20 25 30 Lys Pro
Glu Pro Gly Tyr Glu Cys Gln Ile Ser Gln Glu Tyr Tyr Asp 35 40 45
Arg Lys Ala Gln Met Cys Cys Ala Lys Cys Pro Pro Gly Gln Tyr Val 50
55 60 Lys His Phe Cys Asn Lys Thr Ser Asp Thr Val Cys Ala Asp Cys
Glu 65 70 75 80 Ala Ser Met Tyr Thr Gln Val Trp Asn Gln Phe Arg Thr
Cys Leu Ser 85 90 95 Cys Ser Ser Ser Cys Thr Thr Asp Gln Val Glu
Ile Arg Ala Cys Thr 100 105 110 Lys Gln Gln Asn Arg Val Cys Ala Cys
Glu Ala Gly Arg Tyr Cys Ala 115 120 125 Leu Lys Thr His Ser Gly Ser
Cys Arg Gln Cys Met Arg Leu Ser Lys 130 135 140 Cys Gly Pro Gly Phe
Gly Val Ala Ser Ser Arg Ala Pro Asn Gly Asn 145 150 155 160 Val Leu
Cys Lys Ala Cys Ala Pro Gly Thr Phe Ser Asp Thr Thr Ser 165 170 175
Ser Thr Asp Val Cys Arg Pro His Arg Ile Cys Ser Ile Leu Ala Ile 180
185 190 Pro Gly Asn Ala Ser Thr Asp Ala Val Cys Ala Pro Glu Ser Pro
Thr 195 200 205 Leu Ser Ala Ile Pro Arg Thr Leu Tyr Val Ser Gln Pro
Glu Pro Thr 210 215 220 Arg Ser Gln Pro Leu Asp Gln Glu Pro Gly Pro
Ser Gln Thr Pro Ser 225 230 235 240 Ile Leu Thr Ser Leu Gly Ser Thr
Pro Ile Ile Glu Gln Ser Thr Lys 245 250 255 Gly Gly 10 257 PRT Homo
sapien 10 Met Ala Pro Val Ala Val Trp Ala Ala Leu Ala Val Gly Leu
Glu Leu 1 5 10 15 Trp Ala Ala Ala His Ala Leu Pro Ala Gln Val Ala
Phe Thr Pro Tyr 20 25 30 Ala Pro Glu Pro Gly Ser Thr Cys Arg Leu
Arg Glu Tyr Tyr Asp Gln 35 40 45 Thr Ala Gln Met Cys Cys Ser Lys
Cys Ser Pro Gly Gln His Ala Lys 50 55 60 Val Phe Cys Thr Lys Thr
Ser Asp Thr Val Cys Asp Ser Cys Glu Asp 65 70 75 80 Ser Thr Tyr Thr
Gln Leu Trp Asn Trp Val Pro Glu Cys Leu Ser Cys 85 90 95 Gly Ser
Arg Cys Ser Ser Asp Gln Val Glu Thr Gln Ala Cys Thr Arg 100 105 110
Glu Gln Asn Arg Ile Cys Thr Cys Arg Pro Gly Trp Tyr Cys Ala Leu 115
120 125 Ser Lys Gln Glu Gly Cys Arg Leu Cys Ala Pro Leu Arg Lys Cys
Arg 130 135 140 Pro Gly Phe Gly Val Ala Arg Pro Gly Thr Glu Thr Ser
Asp Val Val 145 150 155 160 Cys Lys Pro Cys Ala Pro Gly Thr Phe Ser
Asn Thr Thr Ser Ser Thr 165 170 175 Asp Ile Cys Arg Pro His Gln Ile
Cys Asn Val Val Ala Ile Pro Gly 180 185 190 Asn Ala Ser Met Asp Ala
Val Cys Thr Ser Thr Ser Pro Thr Arg Ser 195 200 205 Met Ala Pro Gly
Ala Val His Leu Pro Gln Pro Val Ser Thr Arg Ser 210 215 220 Gln His
Thr Gln Pro Thr Pro Glu Pro Ser Thr Ala Pro Ser Thr Ser 225 230 235
240 Phe Leu Leu Pro Met Gly Pro Ser Pro Pro Ala Glu Gly Ser Thr Gly
245 250 255 Asp 11 690 DNA Rattus rattus CDS (1)...(687) 11 gta ccc
aga aac tgt gga ggt gat tgc aag cct tgt ata tgt aca ggc 48 Val Pro
Arg Asn Cys Gly Gly Asp Cys Lys Pro Cys Ile Cys Thr Gly 1 5 10 15
tca gaa gta tca tct gtc ttc atc ttc ccc cca aag ccc aaa gat gtg 96
Ser Glu Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val 20
25 30 ctc acc atc act ctg act cct aag gtc acg tgt gtt gtg gta gac
att 144 Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp
Ile 35 40 45 agc cag gac gat ccc gag gtc cat ttc agc tgg ttt gta
gat gac gtg 192 Ser Gln Asp Asp Pro Glu Val His Phe Ser Trp Phe Val
Asp Asp Val 50 55 60 gaa gtc cac aca gct cag act cga cca cca gag
gag cag ttc aac agc 240 Glu Val His Thr Ala Gln Thr Arg Pro Pro Glu
Glu Gln Phe Asn Ser 65 70 75 80 act ttc cgc tca gtc agt gaa ctc ccc
atc ctg cac cag gac tgg ctc 288 Thr Phe Arg Ser Val Ser Glu Leu Pro
Ile Leu His Gln Asp Trp Leu 85 90 95 aat ggc agg acg ttc aga tgc
aag gtc acc agt gca gct ttc cca tcc 336 Asn Gly Arg Thr Phe Arg Cys
Lys Val Thr Ser Ala Ala Phe Pro Ser 100 105 110 ccc atc gag aaa acc
atc tcc aaa ccc gaa ggc aga aca caa gtt ccg 384 Pro Ile Glu Lys Thr
Ile Ser Lys Pro Glu Gly Arg Thr Gln Val Pro 115 120 125 cat gta tac
acc atg tca cct acc aag gaa gag atg acc cag aat gaa 432 His Val Tyr
Thr Met Ser Pro Thr Lys Glu Glu Met Thr Gln Asn Glu 130 135 140 gtc
agt atc acc tgc atg gta aaa ggc ttc tat ccc cca gac att tat 480 Val
Ser Ile Thr Cys Met Val Lys Gly Phe Tyr Pro Pro Asp Ile Tyr 145 150
155 160 gtg gag tgg cag atg aac ggg cag cca cag gaa aac tac aag aac
act 528 Val Glu Trp Gln Met Asn Gly Gln Pro Gln Glu Asn Tyr Lys Asn
Thr 165 170 175 cca cct acg atg gac aca gat ggg agt tac ttc ctc tac
agc aag ctc 576 Pro Pro Thr Met Asp Thr Asp Gly Ser Tyr Phe Leu Tyr
Ser Lys Leu 180 185 190 aat gtg aag aag gaa aaa tgg cag cag gga aac
acg ttc acg tgt tct 624 Asn Val Lys Lys Glu Lys Trp Gln Gln Gly Asn
Thr Phe Thr Cys Ser 195 200 205 gtg ctg cat gaa ggc ctg cac aac cac
cat act gag aag agt ctc tcc 672 Val Leu His Glu Gly Leu His Asn His
His Thr Glu Lys Ser Leu Ser 210 215 220 cac tct ccg ggt aaa tga 690
His Ser Pro Gly Lys 225 12 229 PRT Rattus rattus 12 Val Pro Arg Asn
Cys Gly Gly Asp Cys Lys Pro Cys Ile Cys Thr Gly 1 5 10 15 Ser Glu
Val Ser Ser Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val 20 25 30
Leu Thr Ile Thr Leu Thr Pro Lys Val Thr Cys Val Val Val Asp Ile 35
40 45 Ser Gln Asp Asp Pro Glu Val His Phe Ser Trp Phe Val Asp Asp
Val 50 55 60 Glu Val His Thr Ala Gln Thr Arg Pro Pro Glu Glu Gln
Phe Asn Ser 65 70 75 80 Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Leu
His Gln Asp Trp Leu 85 90 95 Asn Gly Arg Thr Phe Arg Cys Lys Val
Thr Ser Ala Ala Phe Pro Ser 100 105 110 Pro Ile Glu Lys Thr Ile Ser
Lys Pro Glu Gly Arg Thr Gln Val Pro 115 120 125 His Val Tyr Thr Met
Ser Pro Thr Lys Glu Glu Met Thr Gln Asn Glu 130 135 140 Val Ser Ile
Thr Cys Met Val Lys Gly Phe Tyr Pro Pro Asp Ile Tyr 145 150 155 160
Val Glu Trp Gln Met Asn Gly Gln Pro Gln Glu Asn Tyr Lys Asn Thr 165
170 175 Pro Pro Thr Met Asp Thr Asp Gly Ser Tyr Phe Leu Tyr Ser Lys
Leu 180 185 190 Asn Val Lys Lys Glu Lys Trp Gln Gln Gly Asn Thr Phe
Thr Cys Ser 195 200 205 Val Leu His Glu Gly Leu His Asn His His Thr
Glu Lys Ser Leu Ser 210 215 220 His Ser Pro Gly Lys 225 13 1464 DNA
Rattus rattus CDS (1)...(1461) 13 atg gcg ccc gcc gcc ctc tgg gtc
gcg ctg gtc gtc gaa ctg cag ctg 48 Met Ala Pro Ala Ala Leu Trp Val
Ala Leu Val Val Glu Leu Gln Leu 1 5 10 15 tgg gcc acc ggg cac aca
gtg ccc gcc aag gtt gtc ttg aca ccc tac 96 Trp Ala Thr Gly His Thr
Val Pro Ala Lys Val Val Leu Thr Pro Tyr 20 25 30 aag cca gaa cct
ggg aac cag tgc cag atc tca cag gag tac tat gac 144 Lys Pro Glu Pro
Gly Asn Gln Cys Gln Ile Ser Gln Glu Tyr Tyr Asp 35 40 45 aag aag
gct cag atg tgc tgt gct aag tgt ccc cct ggc cag tat gca 192 Lys Lys
Ala Gln Met Cys Cys Ala Lys Cys Pro Pro Gly Gln Tyr Ala 50 55 60
aaa cac ttc tgc aac aag act tca gac acc gtg tgt gcg gac tgt gcg 240
Lys His Phe Cys Asn Lys Thr Ser Asp Thr Val Cys Ala Asp Cys Ala 65
70 75 80 gca ggc atg ttt acc cag gtc tgg aac cat ctg cat aca tgc
ctg agc 288 Ala Gly Met Phe Thr Gln Val Trp Asn His Leu His Thr Cys
Leu Ser 85 90 95 tgc agt tct tcc tgt agt gat gac cag gtg gag acc
cac aac tgc act 336 Cys Ser Ser Ser Cys Ser Asp Asp Gln Val Glu Thr
His Asn Cys Thr 100 105 110 aaa aaa cag aac cga gtg tgt gct tgc aac
gct gac agt tac tgt gcc 384 Lys Lys Gln Asn Arg Val Cys Ala Cys Asn
Ala Asp Ser Tyr Cys Ala 115 120 125 ttg aaa ttg cat tct ggg aac tgt
cga cag tgc atg aag ctg agc aag 432 Leu Lys Leu His Ser Gly Asn Cys
Arg Gln Cys Met Lys Leu Ser Lys 130 135 140 tgt ggc cct ggc ttc gga
gtg gcc cgt tca aga acc tca aat gga aac 480 Cys Gly Pro Gly Phe Gly
Val Ala Arg Ser Arg Thr Ser Asn Gly Asn 145 150 155 160 gtg ata tgc
agt gcc tgt gcc cca ggg acg ttc tct gac acc aca tca 528 Val Ile Cys
Ser Ala Cys Ala Pro Gly Thr Phe Ser Asp Thr Thr Ser 165 170 175 tcc
aca gat gtg tgc agg ccc cac ggc att tgt agc atc ctg gct att 576 Ser
Thr Asp Val Cys Arg Pro
His Gly Ile Cys Ser Ile Leu Ala Ile 180 185 190 cct gga aat gca agc
acg gat gca gtc tgt gca tcc gag tcc cca act 624 Pro Gly Asn Ala Ser
Thr Asp Ala Val Cys Ala Ser Glu Ser Pro Thr 195 200 205 cca agc gct
gtt cca agg aca atc tac gta tct cag cca gag ccc aca 672 Pro Ser Ala
Val Pro Arg Thr Ile Tyr Val Ser Gln Pro Glu Pro Thr 210 215 220 aga
tcc cag ccc atg gat caa gag cca ggg cct agc caa act cca cac 720 Arg
Ser Gln Pro Met Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro His 225 230
235 240 atc cct gtg tcc ttg ggt tca acc ccc atc att gaa cca agc atc
acg 768 Ile Pro Val Ser Leu Gly Ser Thr Pro Ile Ile Glu Pro Ser Ile
Thr 245 250 255 ggt ggg gta ccc aga aac tgt gga ggt gat tgc aag cct
tgt ata tgt 816 Gly Gly Val Pro Arg Asn Cys Gly Gly Asp Cys Lys Pro
Cys Ile Cys 260 265 270 aca ggc tca gaa gta tca tct gtc ttc atc ttc
ccc cca aag ccc aaa 864 Thr Gly Ser Glu Val Ser Ser Val Phe Ile Phe
Pro Pro Lys Pro Lys 275 280 285 gat gtg ctc acc atc act ctg act cct
aag gtc acg tgt gtt gtg gta 912 Asp Val Leu Thr Ile Thr Leu Thr Pro
Lys Val Thr Cys Val Val Val 290 295 300 gac att agc cag gac gat ccc
gag gtc cat ttc agc tgg ttt gta gat 960 Asp Ile Ser Gln Asp Asp Pro
Glu Val His Phe Ser Trp Phe Val Asp 305 310 315 320 gac gtg gaa gtc
cac aca gct cag act cga cca cca gag gag cag ttc 1008 Asp Val Glu
Val His Thr Ala Gln Thr Arg Pro Pro Glu Glu Gln Phe 325 330 335 aac
agc act ttc cgc tca gtc agt gaa ctc ccc atc ctg cac cag gac 1056
Asn Ser Thr Phe Arg Ser Val Ser Glu Leu Pro Ile Leu His Gln Asp 340
345 350 tgg ctc aat ggc agg acg ttc aga tgc aag gtc acc agt gca gct
ttc 1104 Trp Leu Asn Gly Arg Thr Phe Arg Cys Lys Val Thr Ser Ala
Ala Phe 355 360 365 cca tcc ccc atc gag aaa acc atc tcc aaa ccc gaa
ggc aga aca caa 1152 Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro
Glu Gly Arg Thr Gln 370 375 380 gtt ccg cat gta tac acc atg tca cct
acc aag gaa gag atg acc cag 1200 Val Pro His Val Tyr Thr Met Ser
Pro Thr Lys Glu Glu Met Thr Gln 385 390 395 400 aat gaa gtc agt atc
acc tgc atg gta aaa ggc ttc tat ccc cca gac 1248 Asn Glu Val Ser
Ile Thr Cys Met Val Lys Gly Phe Tyr Pro Pro Asp 405 410 415 att tat
gtg gag tgg cag atg aac ggg cag cca cag gaa aac tac aag 1296 Ile
Tyr Val Glu Trp Gln Met Asn Gly Gln Pro Gln Glu Asn Tyr Lys 420 425
430 aac act cca cct acg atg gac aca gat ggg agt tac ttc ctc tac agc
1344 Asn Thr Pro Pro Thr Met Asp Thr Asp Gly Ser Tyr Phe Leu Tyr
Ser 435 440 445 aag ctc aat gtg aag aag gaa aaa tgg cag cag gga aac
acg ttc acg 1392 Lys Leu Asn Val Lys Lys Glu Lys Trp Gln Gln Gly
Asn Thr Phe Thr 450 455 460 tgt tct gtg ctg cat gaa ggc ctg cac aac
cac cat act gag aag agt 1440 Cys Ser Val Leu His Glu Gly Leu His
Asn His His Thr Glu Lys Ser 465 470 475 480 ctc tcc cac tct ccg ggt
aaa tga 1464 Leu Ser His Ser Pro Gly Lys 485 14 487 PRT Rattus
rattus 14 Met Ala Pro Ala Ala Leu Trp Val Ala Leu Val Val Glu Leu
Gln Leu 1 5 10 15 Trp Ala Thr Gly His Thr Val Pro Ala Lys Val Val
Leu Thr Pro Tyr 20 25 30 Lys Pro Glu Pro Gly Asn Gln Cys Gln Ile
Ser Gln Glu Tyr Tyr Asp 35 40 45 Lys Lys Ala Gln Met Cys Cys Ala
Lys Cys Pro Pro Gly Gln Tyr Ala 50 55 60 Lys His Phe Cys Asn Lys
Thr Ser Asp Thr Val Cys Ala Asp Cys Ala 65 70 75 80 Ala Gly Met Phe
Thr Gln Val Trp Asn His Leu His Thr Cys Leu Ser 85 90 95 Cys Ser
Ser Ser Cys Ser Asp Asp Gln Val Glu Thr His Asn Cys Thr 100 105 110
Lys Lys Gln Asn Arg Val Cys Ala Cys Asn Ala Asp Ser Tyr Cys Ala 115
120 125 Leu Lys Leu His Ser Gly Asn Cys Arg Gln Cys Met Lys Leu Ser
Lys 130 135 140 Cys Gly Pro Gly Phe Gly Val Ala Arg Ser Arg Thr Ser
Asn Gly Asn 145 150 155 160 Val Ile Cys Ser Ala Cys Ala Pro Gly Thr
Phe Ser Asp Thr Thr Ser 165 170 175 Ser Thr Asp Val Cys Arg Pro His
Gly Ile Cys Ser Ile Leu Ala Ile 180 185 190 Pro Gly Asn Ala Ser Thr
Asp Ala Val Cys Ala Ser Glu Ser Pro Thr 195 200 205 Pro Ser Ala Val
Pro Arg Thr Ile Tyr Val Ser Gln Pro Glu Pro Thr 210 215 220 Arg Ser
Gln Pro Met Asp Gln Glu Pro Gly Pro Ser Gln Thr Pro His 225 230 235
240 Ile Pro Val Ser Leu Gly Ser Thr Pro Ile Ile Glu Pro Ser Ile Thr
245 250 255 Gly Gly Val Pro Arg Asn Cys Gly Gly Asp Cys Lys Pro Cys
Ile Cys 260 265 270 Thr Gly Ser Glu Val Ser Ser Val Phe Ile Phe Pro
Pro Lys Pro Lys 275 280 285 Asp Val Leu Thr Ile Thr Leu Thr Pro Lys
Val Thr Cys Val Val Val 290 295 300 Asp Ile Ser Gln Asp Asp Pro Glu
Val His Phe Ser Trp Phe Val Asp 305 310 315 320 Asp Val Glu Val His
Thr Ala Gln Thr Arg Pro Pro Glu Glu Gln Phe 325 330 335 Asn Ser Thr
Phe Arg Ser Val Ser Glu Leu Pro Ile Leu His Gln Asp 340 345 350 Trp
Leu Asn Gly Arg Thr Phe Arg Cys Lys Val Thr Ser Ala Ala Phe 355 360
365 Pro Ser Pro Ile Glu Lys Thr Ile Ser Lys Pro Glu Gly Arg Thr Gln
370 375 380 Val Pro His Val Tyr Thr Met Ser Pro Thr Lys Glu Glu Met
Thr Gln 385 390 395 400 Asn Glu Val Ser Ile Thr Cys Met Val Lys Gly
Phe Tyr Pro Pro Asp 405 410 415 Ile Tyr Val Glu Trp Gln Met Asn Gly
Gln Pro Gln Glu Asn Tyr Lys 420 425 430 Asn Thr Pro Pro Thr Met Asp
Thr Asp Gly Ser Tyr Phe Leu Tyr Ser 435 440 445 Lys Leu Asn Val Lys
Lys Glu Lys Trp Gln Gln Gly Asn Thr Phe Thr 450 455 460 Cys Ser Val
Leu His Glu Gly Leu His Asn His His Thr Glu Lys Ser 465 470 475 480
Leu Ser His Ser Pro Gly Lys 485 15 26 DNA Rattus rattus 15
ctaacgacgt taacgatgca ggtgac 26 16 27 DNA Rattus rattus 16
cggaattcgt gcccagaaac tgtggag 27 17 27 DNA Rattus rattus 17
gctctagatc atttacccgg agagtgg 27 18 33 DNA Rattus rattus 18
cataagggcc cgcaagagcg ggagctaccg ccg 33 19 30 DNA Rattus rattus 19
ggtaccccac ccgtgatgct tggttcaatg 30 20 29 DNA Rattus rattus 20
gggtacccag aaactgtgga ggtgattgc 29
* * * * *
References