U.S. patent application number 15/591468 was filed with the patent office on 2017-11-02 for engineered dendritic cells and uses for the treatment of cancer.
This patent application is currently assigned to Intrexon Corporation. The applicant listed for this patent is Intrexon Corporation, University of Pittsburgh-Of The Commonwealth System Of Higher Education. Invention is credited to Mark BRAUGHLER, Prasanna Kumar, Hideho Okada, Walter J. Storkus.
Application Number | 20170312315 15/591468 |
Document ID | / |
Family ID | 40549463 |
Filed Date | 2017-11-02 |
United States Patent
Application |
20170312315 |
Kind Code |
A1 |
BRAUGHLER; Mark ; et
al. |
November 2, 2017 |
Engineered Dendritic Cells and Uses for the Treatment of Cancer
Abstract
This invention provides the field of therapeutics. Most
specifically present invention provides methods of generating in
vitro engineered dendritic cells conditionally expressing
interleukin-12 (IL-12) under the control of a gene expression
modulation system in the presence of activating ligand and uses for
therapeutic purposes in animals including human.
Inventors: |
BRAUGHLER; Mark;
(Pittsburgh, PA) ; Kumar; Prasanna; (Collegeville,
PA) ; Storkus; Walter J.; (Glenshaw, PA) ;
Okada; Hideho; (Pittsburgh, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Intrexon Corporation
University of Pittsburgh-Of The Commonwealth System Of Higher
Education |
Blacksburg
Pittsburgh |
VA
PA |
US
US |
|
|
Assignee: |
Intrexon Corporation
Blacksburg
VA
University of Pittsburgh-Of The Commonwealth System Of Higher
Education
Pittsburgh
PA
|
Family ID: |
40549463 |
Appl. No.: |
15/591468 |
Filed: |
May 10, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13737316 |
Jan 9, 2013 |
9675642 |
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15591468 |
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12247738 |
Oct 8, 2008 |
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13737316 |
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61019089 |
Jan 4, 2008 |
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60991807 |
Dec 3, 2007 |
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60990689 |
Nov 28, 2007 |
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60990167 |
Nov 26, 2007 |
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60979485 |
Oct 12, 2007 |
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60979480 |
Oct 12, 2007 |
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60978509 |
Oct 9, 2007 |
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60978394 |
Oct 8, 2007 |
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60978224 |
Oct 8, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/4245 20130101;
C12N 2510/00 20130101; A61K 31/00 20130101; A61K 31/166 20130101;
A61K 31/4245 20130101; A61K 31/00 20130101; A61K 45/06 20130101;
A61K 35/15 20130101; C12N 2840/203 20130101; A61K 31/166 20130101;
C12N 2501/23 20130101; C12N 2830/002 20130101; C12N 2710/10343
20130101; G01N 33/6866 20130101; A61P 43/00 20180101; A61K 2300/00
20130101; A61K 2300/00 20130101; A61K 2300/00 20130101; A61K
2300/00 20130101; C07K 2319/715 20130101; C07K 14/5434 20130101;
A61K 38/00 20130101; A61P 35/00 20180101; G01N 2800/52 20130101;
C12N 5/0639 20130101; C12N 2501/24 20130101; A61K 35/15 20130101;
G01N 2333/57 20130101 |
International
Class: |
A61K 35/15 20060101
A61K035/15; A61K 31/4245 20060101 A61K031/4245; G01N 33/68 20060101
G01N033/68; C07K 14/54 20060101 C07K014/54; A61K 31/00 20060101
A61K031/00; C12N 5/0784 20100101 C12N005/0784; A61K 31/166 20060101
A61K031/166; A61K 45/06 20060101 A61K045/06 |
Claims
1-38. (canceled)
39. A method for determining the efficacy of an in vitro engineered
dendritic cell based therapeutic regime in a patient comprising:
(a) measuring the level of expression or the level of activity or
both of interferon-gamma (IFN-.gamma.) in a first biological sample
obtained from said patient in need thereof before administration of
in vitro engineered dendritic cells, thereby generating a control
level; (b) administering to a patient in need thereof, the in vitro
engineered dendritic cell of claim 18 or 19; (c) administering to
said patient in need thereof an effective amount of an activating
ligand; (d) measuring the level of expression or the level of
activity or both of IFN-.gamma. in a second biological sample
obtained from said patient in need thereof following administration
of in vitro engineered DC and activating ligand, thereby generating
a test level; and (e) comparing the control level to the test level
of IFN-.gamma., wherein an increase in the test level of
expression, activity or both of IFN-.gamma. relative to the control
level indicates that the therapeutic regime is effective in said
patient in need thereof.
40. The method of claim 39, wherein said patient in need thereof is
a human patient.
41. The method of claim 39, wherein said patient is a cancer
patient.
42. The method of claim 41, wherein said cancer patient is a
melanoma patient.
43. The method of claim 39, wherein the level of IFN-.gamma. is
measured by ELISA.
44. The method of any one of claims 39-43, wherein said ligand is a
diacylhydrazine.
45. The method of claim 44, wherein said ligand is selected from
the group consisting of RG-115819, RG-115932, and RG-115830.
46. The method of claim 39, wherein said ligand is an amidoketone
or oxadiazoline.
47. The method of any one of claims 39-46, wherein said ligand is
administered intratumorally, orally, intraperitoneally, or
subcutaneously.
48. The method of any one of claims 39-47, wherein said ligand is
administered less than one hour before or after said in vitro
engineered dendritic cell.
49. The method of any one of claims 39-47, wherein said ligand is
administered within less than 24 hours after said in vitro
engineered dendritic cell.
50. The method of any one of claims 39-47, wherein said ligand is
administered within less than 48 hours after said in vitro
engineered dendritic cell.
51-73. (canceled)
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims the benefit of the filing
date of U.S. Provisional Application No. 61/019,089, filed Jan. 4,
2008; U.S. Provisional Application No. 60/991,807, filed Dec. 3,
2007; U.S. Provisional Application No. 60/990,689, filed Nov. 28,
2007; U.S. Provisional Application No. 60/990,167, filed Nov. 26,
2007, U.S. Provisional Application No. 60/979,485, filed Oct. 12,
2007; U.S. Provisional Application No. 60/979,480, filed Oct. 12,
2007; U.S. Provisional Application No. 60/978,509, filed Oct. 9,
2007; U.S. Provisional Application No. 60/978,394, filed Oct. 8,
2007; and U.S. Provisional Application No. 60/978,224, filed Oct.
8, 2007, all of which are incorporated herein by reference in their
entireties.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY VIA
EFS-WEB
[0002] The content of the electronically submitted sequence listing
(Name: Seq_List.ascii.txt; Size: 62,000 bytes; and Date of
Creation: Oct. 8, 2008) filed herewith with the application is
incorporated herein by reference in its entirety.
BACKGROUND OF THE INVENTION
Field of the Invention
[0003] This invention relates to the field of gene therapy for the
treatment of cancer. In one embodiment, the invention relates to
the engineering of dendritic cells to conditionally express
interleukin-12 (IL-12) and use of the cells for therapeutics. In
another embodiment, the invention relates to the engineering of
dendritic cells to conditionally express interleukin-12 (IL-12)
and/or interferon-alpha (IFN-alpha) and use of the cells for
therapeutics.
Background
[0004] Various patents, patent applications, and publications are
cited herein, the disclosures of which are incorporated by
reference in their entireties. However, the citation of any
reference herein should not be construed as an admission that such
reference is available as "Prior Art" to the present
application.
[0005] Interleukin-12 (IL-12) is a member of the type I cytokine
family involved in contributing to a number of biological processes
including, but not limited to, protective immune response and
suppression of tumorigenesis (Abdi et al., 2006; Adorini, 1999;
Adorini, 2001; Adorini et al., 2002; Adorini et al., 1996; Akhtar
et al., 2004; Akiyama et al., 2000; Al-Mohanna et al., 2002;
Aliberti et al., 1996; Allavena et al., 1994; Alli and Khar, 2004;
Aizona et al., 1996; Amemiya et al., 2006; Araujo et al., 2001;
Arulanandam et al., 1999; Athie et al., 2000; Athie-Morales et al.,
2004; Bertagnolli et al., 1992; Bhardwaj et al., 1996; Biedermann
et al., 2006; Brunda and Gately, 1994; Buchanan et al., 1995;
Romani et al., 1997; Rothe et al., 1996; Satoskar et al., 2000;
Schopf et al., 1999; Thomas et al., 2000; Tsung et al., 1997; Wolf
et al., 1994; Yuminamochi et al., 2007). A growing body of evidence
suggests that IL-12 may be a promising target to control human
diseases (e.g., cancer).
[0006] Despite the fact that IL-12 remains promising as a cancer
therapeutic agent based on its potent supportive activity on Type-1
anti-tumor NK cells, CD4.sup.+ T cells and CD8.sup.+ T cells
(Trinchieri, 2003), the reported toxicity of recombinant human
IL-12 (rhIL-12) in patients (Atkins et al., 1997), together with
limited sources of GMP-grade rhIL-12 for clinical application, have
prevented successful IL-12-based therapeutic approaches. Thus it
seems reasonable that gene therapy approaches may represent safer,
more tenable treatment options. Indeed, phase I clinical trials
implementing intra- or peri-tumoral delivery of recombinant viral-
(Sangro et al., 2004; Triozzi et al., 2005) or plasmid-based IL-12
cDNA (Heinzerling et al., 2005), or IL-12 gene modified autologous
fibroblasts (Kang et al., 2001) have been found safe and
well-tolerated.
[0007] However, objective clinical responses in patients with
melanoma or a diverse range of carcinomas receiving these gene
therapies have been rare, variable, transient and largely focused
at the site of treatment (Heinzerling et al., 2005; Kang et al.,
2001; Sangro et al., 2004; Triozzi et al., 2005). In cases where
disease resolution was partial or complete, increased frequencies
of tumor-infiltrating lymphocytes (Heinzerling et al., 2005; Sangro
et al., 2004) and elevated levels of circulating tumor-specific
CD8.sup.+ T cells (Heinzerling et al., 2005) have been noted,
consistent with the improved cross-priming of antigen-specific T
cells in these patients.
[0008] In addition, there raised several residual concerns, e.g.,
unanticipated toxicities associated with DC-based IL-12 gene
therapy and potential IL-12-dependent limitations in therapeutic
DC.IL12 migration after intratumoral administration. Furthermore,
there are further concerns regarding a timing of IL-12 production
in transduced DC most important for therapeutic efficacy (Murphy et
al, 2005)
[0009] Since the cross-priming of specific T cells is best
accomplished by dendritic cells (DC) that serve as a natural but
regulated source of IL-12 (Berard et al., 2000), recent reports of
the superior pre-clinical efficacy of DC-based IL-12 gene therapy
have been of great interest (Satoh et al., 2002; Tatsumi et al.,
2003; Yamanaka et al., 2002). For example, it was shown that
intratumoral (i.t.) injection of DC engineered to produce IL-12p70
(via recombinant adenovirus infection) results in the dramatically
improved cross-priming of a broadly-reactive, tumor-specific
CD8.sup.+ T cell repertoire in concert with tumor rejection in
murine models (Tatsumi et al., 2003). Given the previous use of a
recombinant adenovirus encoding mIL-12 under a CMV-based promoter
(rAd.cIL12, (Tatsumi et al., 2003)), engineered DC production of
IL-12 was constitutive, hence the immunologic impact of this
cytokine early within the tumor lesion and later within
tumor-draining lymph nodes could not be resolved with regards to
therapeutic outcome. Thus, a need exists for DC engineered for
conditional expression of IL-12. The invention provides a promising
therapeutic outcome for the use of such cells.
SUMMARY OF THE INVENTION
[0010] The invention provides a recombinant vector encoding a
protein having the function of IL-12 under the control of a
conditional promoter. In one embodiment, the vector is an
adenovirus vector encoding IL-12p70 driven off a promoter that can
be conditionally activated by provision of a soluble small molecule
ligand such as a diacylhyrdazine, e.g., RG-115819, RG-115830 or
RG-115932. This vector allows for the control of expression of
IL-12 from DC (rAD.RheoIL12).
[0011] In one embodiment, the invention provides a vector for
conditionally expressing a protein having the function of IL-12
comprising a polynucleotide encoding a gene switch, said gene
switch comprising at least one transcription factor sequence,
wherein said at least one transcription factor sequence encodes a
ligand-dependent transcription factor, operably linked to a
promoter, and (2) a polynucleotide encoding a protein having the
function of IL-12 linked to a promoter which is activated by said
ligand-dependent transcription factor. In another embodiment, the
invention relates to a vector for conditionally expressing proteins
having the function of IL-12 and/or IFN-alpha comprising a
polynucleotide encoding a gene switch, said gene switch comprising
at least one transcription factor sequence, wherein said at least
one transcription factor sequence encodes a ligand-dependent
transcription factor, operably linked to a promoter, and (2) a
polynucleotide encoding a protein having the function of IL-12,
and/or a polynucleotide encoding a protein having the function of
IFN-alpha linked to a promoter which is activated by said
ligand-dependent transcription factor.
[0012] For example, the invention provides a vector for
conditionally expressing a protein having the function of IL-12
comprising a polynucleotide encoding a gene switch, wherein the
polynucleotide comprises (1) at least one transcription factor
sequence operably linked to a promoter, wherein said at least one
transcription factor sequence encodes a ligand-dependent
transcription factor, and (2) a polynucleotide encoding a protein
having the function of IL-12 linked to a promoter which is
activated by said ligand-dependent transcription factor. The
invention also provides a vector for conditionally expressing
proteins having the function of IL-12 and/or IFN-alpha comprising a
polynucleotide encoding a gene switch, wherein the polynucleotide
comprises (1) at least one transcription factor sequence operably
linked to a promoter, wherein said at least one transcription
factor sequence encodes a ligand-dependent transcription factor,
and (2) a polynucleotide encoding a protein having the function of
IL-12, and/or a polynucleotide encoding a protein having the
function of IFN-alpha linked to a promoter which is activated by
said ligand-dependent transcription factor.
[0013] The invention further provides a method of producing a
population of DC conditionally expressing a protein having the
function of IL-12 by modifying the DC with a recombinant vector
conditionally expressing a protein having the function of IL-12,
e.g., rAd.RheoIL12. In another embodiment, the invention provides a
method of producing a population of DC conditionally expressing
proteins having the function of IL-12 and/or IFN-alpha by modifying
the DC with a recombinant vector conditionally expressing proteins
having the function of IL-12 and/or IFN-alpha.
[0014] In one embodiment, the invention provides a method of
producing a population of dendritic cells conditionally expressing
a protein having the function of IL-12, comprising modifying at
least a portion of dendritic cells by introducing into said
dendritic cells a vector comprising a polynucleotide encoding a
gene switch, said gene switch comprising at least one transcription
factor sequence, wherein said at least one transcription factor
sequence encodes a ligand-dependent transcription factor, operably
linked to a promoter, and (2) a polynucleotide encoding a protein
having the function of IL-12 linked to a promoter which is
activated by said ligand-dependent transcription factor. In another
embodiment, the invention provides a method of producing a
population of dendritic cells conditionally expressing proteins
having the function of IL-12 and/or IFN-alpha, comprising modifying
at least a portion of dendritic cells by introducing into said
dendritic cells a vector comprising a polynucleotide encoding a
gene switch, said gene switch comprising at least one transcription
factor sequence, wherein said at least one transcription factor
sequence encodes a ligand-dependent transcription factor, operably
linked to a promoter, and (2) a polynucleotide encoding a protein
having the function of IL-12, and/or a polynucleotide encoding a
protein having the function of IFN-alpha linked to a promoter which
is activated by said ligand-dependent transcription factor.
[0015] For example, the invention provides a method of producing a
population of dendritic cells conditionally expressing a protein
having the function of IL-12, comprising modifying at least a
portion of dendritic cells by introducing into said dendritic cells
a vector comprising a polynucleotide encoding a gene switch,
wherein the polynucleotide comprises (1) at least one transcription
factor sequence operably linked to a promoter, wherein said at
least one transcription factor sequence encodes a ligand-dependent
transcription factor, and (2) a polynucleotide encoding a protein
having the function of IL-12 linked to a promoter which is
activated by said ligand-dependent transcription factor. The
invention provides a method of producing a population of dendritic
cells conditionally expressing proteins having the function of
IL-12 and/or IFN-alpha, comprising modifying at least a portion of
dendritic cells by introducing into said dendritic cells a vector
comprising a polynucleotide encoding a gene switch, wherein the
polynucleotide comprises (1) at least one transcription factor
sequence operably linked to a promoter, wherein said at least one
transcription factor sequence encodes a ligand-dependent
transcription factor, and (2) a polynucleotide encoding a protein
having the function of IL-12, and/or a polynucleotide encoding a
protein having the function of IFN-alpha linked to a promoter which
is activated by said ligand-dependent transcription factor.
[0016] The invention also provides a population of DC modified to
conditionally express a protein having the function of IL-12 with a
recombinant vector conditionally expressing a protein having the
function of IL-12, e.g., the rAd.RheoIL12 vector. It has been found
that DC infected with rAd.RheoIL12 produced elevated levels of
IL-12 only after provision of an activating ligand. Another
embodiment provides a population of DC modified to conditionally
express a protein having the function of IL-12 and/or a protein
having the function of IFN-alpha with a recombinant vector
conditionally expressing a protein having the function of IL-12
and/or the function of IFN-alpha. Useful ligands include, but are
not limited to RG-115830, RG-115932, RG-115819, RSL1, and other
diacylhydrazines.
[0017] In one embodiment, the invention provides an in vitro
engineered dendritic cell comprising a vector comprising a
polynucleotide encoding a gene switch, said gene switch comprising
at least one transcription factor sequence, wherein said at least
one transcription factor sequence encodes a ligand-dependent
transcription factor, operably linked to a promoter, and (2) a
polynucleotide encoding a protein having the function of IL-12
linked to a promoter which is activated by said ligand-dependent
transcription factor. In another embodiment, the invention provides
an in vitro engineered dendritic cell comprising a vector
comprising a polynucleotide encoding a gene switch, said gene
switch comprising at least one transcription factor sequence,
wherein said at least one transcription factor sequence encodes a
ligand-dependent transcription factor, operably linked to a
promoter, and (2) a polynucleotide encoding a protein having the
function of IL-12, and/or a polynucleotide encoding a protein
having the function of IFN-alpha linked to a promoter which is
activated by said ligand-dependent transcription factor.
[0018] For example, the invention provides an in vitro engineered
dendritic cell comprising a vector comprising a polynucleotide
encoding a gene switch, wherein the polynucleotide comprises (1) at
least one transcription factor sequence operably linked to a
promoter, wherein said at least one transcription factor sequence
encodes a ligand-dependent transcription factor, and (2) a
polynucleotide encoding a protein having the function of IL-12
linked to a promoter which is activated by said ligand-dependent
transcription factor. The invention provides an in vitro engineered
dendritic cell comprising a vector comprising a polynucleotide
encoding a gene switch, wherein the polynucleotide comprises (1) at
least one transcription factor sequence operably linked to a
promoter, wherein said at least one transcription factor sequence
encodes a ligand-dependent transcription factor, and (2) a
polynucleotide encoding a protein having the function of IL-12,
and/or a polynucleotide encoding a protein having the function of
IFN-alpha linked to a promoter which is activated by said
ligand-dependent transcription factor.
[0019] The current invention also provides a pharmaceutical
composition comprising a population of DC modified to conditionally
express a protein having the function of IL-12 with a recombinant
vector conditionally expressing a protein having the function of
IL-12, e.g., the rAd.RheoIL12 vector. In another embodiment, the
invention provides a pharmaceutical composition comprising a
population of DC modified to conditionally express a protein having
the function of IL-12 and/or a protein having the function of
IFN-alpha with a recombinant vector conditionally expressing a
protein having the function of IL-12 and/or a protein having the
function of IFN-alpha.
[0020] The invention also provides a treatment of cancer, such as
melanoma tumors or glioma tumors. IL-12 gene therapy has
demonstrated anti-tumor efficacy in animal model studies when
applied as a recombinant cDNA vector (Faure et al., 1998; Sangro et
al., 2005), but even more so, when applied in the context of
gene-modified DC (Satoh et al., 2002; Svane et al., 1999; Tatsumi
et al., 2003; Yamanaka et al., 2002). To date, however, human phase
I trials of IL-12 gene therapy implementing plasmids or viral
vectors have failed to achieve durable, objective clinical
responses in the cancer setting (Heinzerling et al., 2005; Kang et
al., 2001; Sangro et al., 2004; Triozzi et al., 2005). DC-based
IL-12 gene therapy (with or without IFN-alpha) described herein
provides a promising therapeutic modality.
[0021] In one embodiment, the invention provides a method for
treating a tumor in a mammal, comprising (a) administering
intratumorally to tumor microenvironments a population of an in
vitro engineered dendritic cells, wherein said dendritic cells
comprise a vector comprising a polynucleotide encoding a gene
switch, wherein the polynucleotide comprises (1) at least one
transcription factor sequence operably linked to a promoter,
wherein said at least one transcription factor sequence encodes a
ligand-dependent transcription factor, and (2) a polynucleotide
encoding a protein having the function of IL-12 linked to a
promoter which is activated by said ligand-dependent transcription
factor and (b) administering to said mammal an effective amount of
a ligand, which activates the ligand-dependent transcription
factor; thereby inducing expression of a protein having the
function of IL-12 and treating said tumor.
[0022] For example, the invention provides a method for treating a
tumor in a mammal, comprising the steps of: [0023] (a) engineering
dendritic cells in vitro to conditionally express a protein having
the function of IL-12; [0024] (b) administering intratumorally to
tumor microenvironments said in vitro engineered dendritic cells;
and [0025] (c) administering to said mammal a therapeutically
effective amount of an activating ligand; thereby inducing
expression of a protein having the function of IL-12 and treating
said tumor.
[0026] In further embodiments, the invention provides a method for
treating a tumor in a mammal, comprising (a) administering
intratumorally to tumor microenvironments an in vitro engineered
dendritic cells, wherein said dendritic cells comprise a vector
comprising a polynucleotide encoding a gene switch, wherein the
polynucleotide comprises (1) at least one transcription factor
sequence operably linked to a promoter, wherein said at least one
transcription factor sequence encodes a ligand-dependent
transcription factor, and (2) a polynucleotide encoding a protein
having the function of IL-12 and/or a protein having the function
of IFN-alpha, linked to a promoter activated by said
ligand-dependent transcription factor and (b) administering to said
mammal a therapeutically effective amount of an activating ligand;
thereby inducing expression of a protein having the function of
IL-12 and/or a protein having the function of IFN-alpha and
treating said tumor.
[0027] For example, the invention provides a method for treating a
tumor in a mammal, comprising the steps of: [0028] (a) engineering
dendritic cells in vitro to conditionally express a protein having
the function of IL-12 and/or a protein having the function of
IFN-alpha; [0029] (b) administering intratumorally to tumor
microenvironments said in vitro engineered dendritic cells; and
[0030] (c) administering to said mammal a therapeutically effective
amount of an activating ligand; thereby inducing expression of a
protein having the function of IL-12 and/or a protein having the
function of IFN-alpha and treating said tumor.
[0031] The invention also provides a method for determining the
efficacy of engineered DC-based therapy by measuring the level of
expression or activity of IFN-.gamma. in a patient before the start
of therapy, thereby generating a control level, followed by the
administration of DC engineered to conditionally express a protein
having the function of IL-12 and an effective amount of an
activating ligand and then measuring the level of expression of
IFN-.gamma. to generate a test level, and comparing the control
level to the test level to determine if the therapeutic regime is
effective.
[0032] In one embodiment, the invention provides a method for
determining the efficacy of an in vitro engineered dendritic cell
based therapeutic regime in a patient comprising: [0033] (a)
measuring the level of expression or the level of activity or both
of interferon-gamma (IFN-65 ) in a first biological sample obtained
from said patient in need thereof before administration of in vitro
engineered dendritic cells, thereby generating a control level;
[0034] (b) administering to a patient in need thereof, in vitro
engineered dendritic cells engineered to conditionally express a
protein having the function of IL-12; [0035] (c) administering to
said patient in need thereof an effective amount of an activating
ligand; [0036] (d) measuring the level of expression or the level
of activity or both of IFN-.gamma. in a second biological sample
obtained from said patient in need thereof following administration
of in vitro engineered DC and activating ligand, thereby generating
a test level; and [0037] (e) comparing the control level to the
test level of IFN-.gamma., wherein an increase in the test level of
expression, activity or both of IFN-.gamma. relative to the control
level indicates that the therapeutic regime is effective in said
patient in need thereof.
[0038] In another embodiment, the invention provides a method of
inducing conditional expression of a protein having the function of
interleukin-12 (IL-12) in a dendritic cell comprising: (1)
administering to a mammal in need thereof an effective amount of a
population of the in vitro engineered dendritic cells of the
invention; and (2) administering to said mammal in need thereof an
effective amount of a ligand, which activates the ligand-dependent
transcription factor.
[0039] In advance of clinical implementation, previous studies
performed in a CMS4 sarcoma model in BALB/c mice were extended and
it was observed that intratumoral delivery of syngenic bone
marrow-derived DC pre-infected with Ad.cIL12 (constitutive
expression), resulted in effective tumor rejection (Tatsumi et al.,
2003). Rejection was associated with systemic CD8.sup.+ T
cell-mediated immunity against CMS4 tumors, (Tatsumi et al.,
2003).
DETAILED DESCRIPTION OF DRAWINGS
[0040] FIG. 1 shows the structure of the vector rAd.RheoIL12 in
which the E1 and E3 regions have been deleted and the
RheoSwitch.RTM. Therapeutic System (RTS)-IL-12 components replace
the E1 region. The box labeled "IL12" represents the IL-12p40 and
IL-12p35 coding sequences separated by IRES.
[0041] FIGS. 2A-2C show that engineered DCs conditionally express
IL-12 protein in the presence of RG-115830.
[0042] FIG. 3A: shows engineered DC administered into melanoma
tumor microenvironments cause tumor regression when RG-115830 is
intraperitoneally injected into C57B1/6 mice bearing established 7
day B16 subcutaneous tumors within 24 hours of DC injection 3B-3C:
tumor regression occurred when RG-115830 was administered from days
1 to 5, but not when administered only on days 1 to 2 or 1 to 3
post-DC injection
[0043] FIG. 4 shows that engineered DC exhibit prolonged survival
in tumor and tumor-draining lymph node after intraperitoneal
injection of the activating ligand within 24 hours after DC
injection, much less survival and no survival of DC by ligand at 48
hours and 72 hours respectively.
[0044] FIG. 5 A shows that engineered DC promote strong peripheral
activation of anti-B16 CD8.sup.+ T cells if activating ligand is
provided within 24 hours of engineered DC injection. FIG. 5B shows
that all mice previously cured of their melanoma exhibited specific
protection against B16 tumor cells but not against MC38 colon
carcinoma cells when tumor-free animals were rechallenged with
relevant B16 melanoma cells or MC38 colon carcinoma cells on day 45
(post-initial B16 challenge).
[0045] FIG. 6 shows the therapeutic benefit induced by
administration of ligand by intraperitoneal or oral routes.
[0046] FIG. 7 shows the Kaplan-Meier plots of the survival of mice
as a result of intratumoral injection of mouse glioma (GL261) with
dendritic cells transduced with polynucleotides encoding IL-12
and/or IFN-alpha under the control of RTS. Abbreviations in this
figure are as follows: Ad-IFNa is Adenoviral vector constitutively
expressing IFN-alpha; Ad-RTS-IFNa is Adenoviral vector encoding
IFN-alpha under RTS control; Ad-RTS-IFNs no ligand is Adenoviral
vector containing RTS and IFN-alpha where no activator ligand was
present; Ad-IFNa/IL-12 corresponds to DC transduced with Adenoviral
vectors encoding IFN-alpha and IL-12; and Ad-RTS-IFNa/IL-12
corresponds to DC transduced with two Adenoviral vectors encoding
IFN-alpha and IL-12 under RTS control.
[0047] FIG. 8 shows a map for the adenoviral vector
Ad-RTS-ML-12.
[0048] FIG. 9 shows IL-12 production by human dendritic cells
transduced with adenoviral vector Ad-RTS-mIL-12 at different MOI
and duration of viral adsorption. Adenoviral transduction of human
DCs at different MOI and for different duration of viral adsorption
showed efficient transduction of these cells by 3 hour viral
adsorption at MOI of 500. The activator drug ("AD" or "activating
ligand") induced IL-12 expression in these transduced human
dendritic cells.
[0049] FIG. 10 shows a comparison of the effects of different
IL-12-containing adenoviral vectors. The SP1-RheoIL-12 variant was
the most effective of the Rheoswitch-containing variants.
Sp1-RheoIL-12 differs from oldRheoIL-12 in that it replaced an
AdEasy-1 vector backbone with the RAPAd vector backbone
(ViraQuest). Similarly, TTR-RheoIL-12 differs from oldRheoIL-12 in
that it contains a TTR minimal promoter downstream of the Gal4
binding sites, replacing the synthetic minimal promoter and the Sp1
binding sites, and the vector backbone is RAPAd (ViraQuest). As
FIG. 10 illustrates, Sp1-RheoIL-12 was comparable to oldRheoIL-12
and more effective than TTR-RheoIL-12 in reducing B16 melanoma
tumor size.
[0050] FIG. 11 shows lack of B16 melanoma tumor formation after
rechallenge of mice previously treated with dendritic cells
containing recombinant adenoviral Rheoswitch inducible IL-12. This
shows that B16 melanoma tumors were prevented from growing for up
to 25 days when B16 immune mice were re-inoculated 45 days after
the first inoculation with B16 cells. Murine dendritic cells were
generated from bone marrow of B6 mice by 7 day culture in complete
media (RPMI-1640, 10% FBS) containing rmIL-4 plus rmGM-CSF. CD11c
positive dendritic cells were then isolated using specific MACS
beads per manufacturer's protocol (Miltenyi Biotech) and infected
at MOI of 100 using rAd.IL-12 (RheoIL-12 vs. SP1 vs. TTR) for 24
hours prior to injection of 10E6 DC into established day 9 s.c. B16
melanoma tumors (5 mice per group, tumor on right flank). Mice were
treated or not with daily i.p. injections of the activating ligand
RG-115830 (30 mg/kg in 50 microliter DMSO) on days 0-4 post DC
injection. Tumor size was monitored every 3-4 days and is reported
in mm.sup.2 as product of orthogonal diameters. To evaluate the
specificity of therapy-associated protection, all tumor-free
animals were rechallenged with 10E5 B16 melanoma cells on the left
flank versus MC38 colon carcinoma cells on the right flank on day
45 post initial B16 tumor challenge. MC38 tumors formed but B16
tumors did not form.
[0051] FIG. 12 shows a comparison among numbers of dendritic cells
injected into the B16 tumor (10E5, 10E6, 10E7) and length of time
of ligand administration (6 days or 13 days) and the resulting
tumor regression in B16 melanoma tumor mouse model Ligand
administered daily for 13 days in combination with 10E7 dendritic
cells was most effective in causing tumor regression over a 25 day
period.
[0052] FIG. 13 shows that the therapy described herein was not
associated with untoward loss in animal weight due to wasting.
Wasting and weight loss is often associated with high levels of
interferon-gamma and TNF-alpha which are known to be upregulated in
response to IL-12.
[0053] FIG. 14 shows lack of B16 melanoma tumor formation after
rechallenge of mice previously treated with dendritic cells
containing recombinant adenoviral Rheoswitch.RTM. inducible IL-12
and activator ligand RG-115932. B16 melanomas were established s.c.
for 7 days in the right flanks of 5 syngeneic B6 mice. On day 7,
DC.SP1-IL-12 (bone marrow derived DC infected at an MOI of 100
using the SP1 optimal switch) were injected intratumorally (i.t.)
at doses of 10.sup.5, 10.sup.6 or 10.sup.7. RG-115932 was provided
by i.p. injection beginning on the day of DC injection (and daily
thereafter for either 6 days or 13 days). Each cohort contained 5
animals, with tumor growth monitored every 3-4 days and reported as
mean size (mm squared based on the product of orthogonal
measurements). Individual animal weights were also assessed at the
time of tumor measurements (FIG. 13). All animals rendered free of
disease by any therapy were rechallenged on day 50 (post-initial
B16 tumor inoculation) with 10.sup.5 B16 melanoma cells on the
opposite flank (left flank) of the original tumor and with 10.sup.5
MC38 colon carcinoma cells on the right flank. Tumor growth was
monitored every 3-4 days and compared against growth observed in
naive (untreated) animals (see FIG. 12). FIG. 14 therefore shows
that B16 melanoma tumors were prevented from growing for up to 24
days when B16 immune mice were re-inoculated with B16 cells. FIG.
14 also illustrates that B16 naive mice were not protected from
tumor formation, as were MC38 immune mice and MC38 nave mice. MC38
is a colon carcinoma known in the art. This demonstrates the
specificity of immunization caused by the original B16 tumor
injection with dendritic cells containing recombinant adenoviral
Rheoswitch.RTM. inducible IL-12.
[0054] FIG. 15 shows Activator Drug (RG-115932) dose-dependent
IL-12 expression in mouse dendritic cells transduced with
Ad-RTS-mIL-12.
[0055] FIG. 16 shows On/Off response of mIL-12 expression to the
presence or absence of RG-115932 in HT1080 cells transduced with
Ad-RTS-mIL-12.
[0056] FIG. 17 shows that CD8+ T cell response to immunization by
intratumoral injection of adenoviral transduced DC in the presence
or absence of the Activating Drug (AD) corresponds with antitumor
response.
[0057] FIG. 18 shows human IL-12 induction in human DCs from three
volunteers transduced with the adenoviral vector cencoding the
human IL-12 under RTS control.
DETAILED DESCRIPTION OF SEQUENCES
[0058] SEQ ID NO: 1 is a full length nucleotide sequence of wild
type mouse IL-12 p35 gene.
[0059] SEQ ID NO: 2 is a full length nucleotide sequence of wild
type mouse IL-12 p40 gene.
[0060] SEQ ID NO: 3 is a full length nucleotide sequence of wild
type human IL-12 p35 gene.
[0061] SEQ ID NO: 4 is a full length nucleotide sequence of wild
type human IL-12 p40 gene.
[0062] SEQ ID NO: 5 is a full length polypeptide sequence of wild
type mouse IL-12 p35 protein.
[0063] SEQ ID NO: 6 is a full length amino acid sequence of wild
type mouse LL-12 p40 protein.
[0064] SEQ ID NO: 7 is a full length amino acid sequence of wild
type human IL-12 p35 protein.
[0065] SEQ ID NO: 8 is a full length amino acid sequence of wild
type human IL-12 p40 protein.
[0066] SEQ ID NO: 9 is a DNA sequence for an ecdysone response
element found in Drosophila.
[0067] SEQ ID NO: 10 is a DNA sequence for an ecdysone response
element found in Drosophila melanogaster.
[0068] SEQ ID NO: 11 is a DNA sequence for an ecdysone response
element found in Drosophila melanogaster.
[0069] SEQ ID NO: 12 is I-SceI restriction site in a homing
endonuclease (HE) enzyme.
[0070] SEQ ID NO: 13 is a DNA sequence of adenovirus vector
comprising human IL-12 coding sequence: Ad-RTS-hIL-12
(SP1-RheoIL-12).
[0071] The amino acid sequence of interferon alpha (IFN-alpha) is
available from public databases as accession number AAA52724, the
sequence of which is incorporated by reference herein. See also
Capon et al., Mol. Cell. Biol. 5, 768-779 (1985).
DETAILED DESCRIPTION OF INVENTION
[0072] Unless otherwise defined, all terms of art, notations and
other scientific terms or terminology used herein are intended to
have the meanings commonly understood by those of skill in the art
to which this invention pertains. In some cases, terms with
commonly understood meanings are defined herein for clarity and/or
for ready reference and understanding, and the inclusion of such
definitions herein should not necessarily be construed to mean a
substantial difference over what is generally understood in the
art. Commonly understood definitions of molecular biology terms
and/or methods and/or protocols can be found in Rieger et al.,
Glossary of Genetics: Classical and Molecular, 5th edition,
Springer-Verlag: New York, 1991; Lewin, Genes V, Oxford University
Press: New York, 1994; Sambrook et al., Molecular Cloning, A
Laboratory Manual (3d ed. 2001) and Ausubel et al., Current
Protocols in Molecular Biology (1994). As appropriate, procedures
involving the use of commercially available kits and/or reagents
are generally carried out in accordance with manufacturer's
guidance and/or protocols and/or parameters unless otherwise
noted.
[0073] The term "isolated" for the purposes of the invention
designates a biological material (cell, nucleic acid or protein)
that has been removed from its original environment (the
environment in which it is naturally present). For example, a
polynucleotide present in the natural state in a plant or an animal
is not isolated, however the same polynucleotide separated from the
adjacent nucleic acids in which it is naturally present, is
considered "isolated."
[0074] The term "purified," as applied to biological materials does
not require the material to be present in a form exhibiting
absolute purity, exclusive of the presence of other compounds. It
is rather a relative definition.
[0075] "Nucleic acid," "nucleic acid molecule," "oligonucleotide,"
and "polynucleotide" are used interchangeably and refer to the
phosphate ester polymeric form of ribonucleosides (adenosine,
guanosine, uridine or cytidine; "RNA molecules") or
deoxyribonucleosides (deoxyadenosine, deoxyguanosine,
deoxythymidine, or deoxycytidine; "DNA molecules"), or any
phosphoester analogs thereof, such as phosphorothioates and
thioesters, in either single stranded form, or a double-stranded
helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are
possible. The term nucleic acid molecule, and in particular DNA or
RNA molecule, refers only to the primary and secondary structure of
the molecule, and does not limit it to any particular tertiary
forms. Thus, this term includes double-stranded DNA found, inter
alfa, in linear or circular DNA molecules (e.g., restriction
fragments), plasmids, supercoiled DNA and chromosomes. In
discussing the structure of particular double-stranded DNA
molecules, sequences may be described herein according to the
normal convention of giving only the sequence in the 5' to 3'
direction along the non-transcribed strand of DNA (i.e., the strand
having a sequence homologous to the mRNA). A "recombinant DNA
molecule" is a DNA molecule that has undergone a molecular
biological manipulation. DNA includes, but is not limited to, cDNA,
genomic DNA, plasmid DNA, synthetic DNA, and semi-synthetic
DNA.
[0076] The term "fragment," as applied to polynucleotide sequences,
refers to a nucleotide sequence of reduced length relative to the
reference nucleic acid and comprising, over the common portion, a
nucleotide sequence identical to the reference nucleic acid. Such a
nucleic acid fragment according to the invention may be, where
appropriate, included in a larger polynucleotide of which it is a
constituent. Such fragments comprise, or alternatively consist of,
oligonucleotides ranging in length from at least 6, 8, 9, 10, 12,
15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54,
57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200,
300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000, or more
consecutive nucleotides of a nucleic acid according to the
invention.
[0077] As used herein, an "isolated nucleic acid fragment" refers
to a polymer of RNA or DNA that is single- or double-stranded,
optionally containing synthetic, non-natural or altered nucleotide
bases. An isolated nucleic acid fragment in the form of a polymer
of DNA may be comprised of one or more segments of cDNA, genomic
DNA or synthetic DNA.
[0078] A "gene" refers to a polynucleotide comprising nucleotides
that encode a functional molecule, including functional molecules
produced by transcription only (e.g., a bioactive RNA species) or
by transcription and translation (e.g., a polypeptide). The term
"gene" encompasses cDNA and genomic DNA nucleic acids. "Gene" also
refers to a nucleic acid fragment that expresses a specific RNA,
protein or polypeptide, including regulatory sequences preceding
(5' non-coding sequences) and following (3' non-coding sequences)
the coding sequence. "Native gene" refers to a gene as found in
nature with its own regulatory sequences. "Chimeric gene" refers to
any gene that is not a native gene, comprising regulatory and/or
coding sequences that are not found together in nature.
Accordingly, a chimeric gene may comprise regulatory sequences and
coding sequences that are derived from different sources, or
regulatory sequences and coding sequences derived from the same
source, but arranged in a manner different than that found in
nature. A chimeric gene may comprise coding sequences derived from
different sources and/or regulatory sequences derived from
different sources. "Endogenous gene" refers to a native gene in its
natural location in the genome of an organism. A "foreign" gene or
"heterologous" gene refers to a gene not normally found in the host
organism, but that is introduced into the host organism by gene
transfer. Foreign genes can comprise native genes inserted into a
non-native organism, or chimeric genes. A "transgene" is a gene
that has been introduced into the genome by a transformation
procedure. For example, the interleukin-12 (IL-12) gene encodes the
IL-12 protein. IL-12 is a heterodimer of a 35-kD subunit (p35) and
a 40-kD subunit (p40) linked through a disulfide linkage to make
fully functional IL-12p70. The IL-12 gene encodes both the p35 and
p40 subunits.
[0079] "Heterologous DNA" refers to DNA not naturally located in
the cell, or in a chromosomal site of the cell. The heterologous
DNA may include a gene foreign to the cell.
[0080] The term "genome" includes chromosomal as well as
mitochondrial, chloroplast and viral DNA or RNA.
[0081] A nucleic acid molecule is "hybridizable" to another nucleic
acid molecule, such as a cDNA, genomic DNA, or RNA, when a single
stranded form of the nucleic acid molecule can anneal to the other
nucleic acid molecule under the appropriate conditions of
temperature and solution ionic strength. Hybridization and washing
conditions are well known and exemplified in Sambrook et al. in
Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring
Harbor Laboratory Press, Cold Spring Harbor (1989), particularly
Chapter 11 and Table 11.1 therein. The conditions of temperature
and ionic strength determine the "stringency" of the
hybridization.
[0082] Stringency conditions can be adjusted to screen for
moderately similar fragments, such as homologous sequences from
distantly related organisms, to highly similar fragments, such as
genes that duplicate functional enzymes from closely related
organisms. For preliminary screening for homologous nucleic acids,
low stringency hybridization conditions, corresponding to a T.sub.m
of 55.degree., can be used, e.g., 5.times.SSC, 0.1% SDS, 0.25%
milk, and no formamide; or 30% formamide, 5.times.SSC, 0.5% SDS.
Moderate stringency hybridization conditions correspond to a higher
T.sub.m, e.g., 40% formamide, with 5.times. or 6.times.SSC. High
stringency hybridization conditions correspond to the highest
T.sub.m, e.g., 50% formamide, 5.times. or 6.times.SSC.
[0083] Hybridization requires that the two nucleic acids contain
complementary sequences, although depending on the stringency of
the hybridization, mismatches between bases are possible. The term
"complementary" is used to describe the relationship between
nucleotide bases that are capable of hybridizing to one another.
For example, with respect to DNA, adenosine is complementary to
thymine and cytosine is complementary to guanine. Accordingly, the
invention also includes isolated nucleic acid fragments that are
complementary to the complete sequences as disclosed or used herein
as well as those substantially similar nucleic acid sequences.
[0084] In one embodiment of the invention, polynucleotides are
detected by employing hybridization conditions comprising a
hybridization step at T.sub.m of 55.degree. C., and utilizing
conditions as set forth above. In other embodiments, the T.sub.m is
60.degree. C., 63.degree. C., or 65.degree. C.
[0085] Post-hybridization washes also determine stringency
conditions. One set of conditions uses a series of washes starting
with 6.times.SSC, 0.5% SDS at room temperature for 15 minutes
(min), then repeated with 2.times.SSC, 0.5% SDS at 45.degree. C.
for 30 min, and then repeated twice with 0.2.times.SSC, 0.5% SDS at
50.degree. C. for 30 min. A preferred set of stringent conditions
uses higher temperatures in which the washes are identical to those
above except for the temperature of the final two 30 min washes in
0.2.times.SSC, 0.5% SDS is increased to 60.degree. C. Another
preferred set of highly stringent conditions uses two final washes
in 0.1.times.SSC, 0.1% SDS at 65.degree. C.
[0086] The appropriate stringency for hybridizing nucleic acids
depends on the length of the nucleic acids and the degree of
complementation, variables well known in the art. The greater the
degree of similarity or homology between two nucleotide sequences,
the greater the value of T.sub.m for hybrids of nucleic acids
having those sequences. The relative stability (corresponding to
higher T.sub.m) of nucleic acid hybridizations decreases in the
following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater
than 100 nucleotides in length, equations for calculating T.sub.m
have been derived (see Sambrook et al., supra, 9.50-0.51). For
hybridization with shorter nucleic acids, i.e., oligonucleotides,
the position of mismatches becomes more important, and the length
of the oligonucleotide determines its specificity (see Sambrook et
al., supra, 11.7-11.8).
[0087] In one embodiment of the invention, polynucleotides are
detected by employing hybridization conditions comprising a
hybridization step in less than 500 mM salt and at least 37.degree.
C., and a washing step in 2.times. SSPE at a temperature of at
least 63.degree. C. In another embodiment, the hybridization
conditions comprise less than 200 mM salt and at least 37.degree.
C. for the hybridization step. In a further embodiment, the
hybridization conditions comprise 2.times. SSPE and 63.degree. C.
for both the hybridization and washing steps.
[0088] In another embodiment, the length for a hybridizable nucleic
acid is at least about 10 nucleotides. Preferably a minimum length
for a hybridizable nucleic acid is at least about 15 nucleotides;
e.g., at least about 20 nucleotides; e.g., at least 30 nucleotides.
Furthermore, the skilled artisan will recognize that the
temperature and wash solution salt concentration may be adjusted as
necessary according to factors such as length of the probe.
[0089] The term "probe" refers to a single-stranded nucleic acid
molecule that can base pair with a complementary single stranded
target nucleic acid to form a double-stranded molecule.
[0090] As used herein, the term "oligonucleotide" refers to a short
nucleic acid that is hybridizable to a genomic DNA molecule, a cDNA
molecule, a plasmid DNA or an mRNA molecule. Oligonucleotides can
be labeled, e.g., with .sup.32P-nucleotides or nucleotides to which
a label, such as biotin, has been covalently conjugated. A labeled
oligonucleotide can be used as a probe to detect the presence of a
nucleic acid. Oligonucleotides (one or both of which may be
labeled) can be used as PCR primers, either for cloning full length
or a fragment of a nucleic acid, for DNA sequencing, or to detect
the presence of a nucleic acid. An oligonucleotide can also be used
to form a triple helix with a DNA molecule. Generally,
oligonucleotides are prepared synthetically, preferably on a
nucleic acid synthesizer. Accordingly, oligonucleotides can be
prepared with non-naturally occurring phosphoester analog bonds,
such as thioester bonds, etc.
[0091] A "primer" refers to an oligonucleotide that hybridizes to a
target nucleic acid sequence to create a double stranded nucleic
acid region that can serve as an initiation point for DNA synthesis
under suitable conditions. Such primers may be used in a polymerase
chain reaction or for DNA sequencing.
[0092] "Polymerase chain reaction" is abbreviated PCR and refers to
an in vitro method for enzymatically amplifying specific nucleic
acid sequences. PCR involves a repetitive series of temperature
cycles with each cycle comprising three stages: denaturation of the
template nucleic acid to separate the strands of the target
molecule, annealing a single stranded PCR oligonucleotide primer to
the template nucleic acid, and extension of the annealed primer(s)
by DNA polymerase. PCR provides a means to detect the presence of
the target molecule and, under quantitative or semi-quantitative
conditions, to determine the relative amount of that target
molecule within the starting pool of nucleic acids.
[0093] "Reverse transcription-polymerase chain reaction" is
abbreviated RT-PCR and refers to an in vitro method for
enzymatically producing a target cDNA molecule or molecules from an
RNA molecule or molecules, followed by enzymatic amplification of a
specific nucleic acid sequence or sequences within the target cDNA
molecule or molecules as described above. RT-PCR also provides a
means to detect the presence of the target molecule and, under
quantitative or semi-quantitative conditions, to determine the
relative amount of that target molecule within the starting pool of
nucleic acids.
[0094] A DNA "coding sequence" refers to a double-stranded DNA
sequence that encodes a polypeptide and can be transcribed and
translated into a polypeptide in a cell in vitro or in vivo when
placed under the control of suitable regulatory sequences.
"Suitable regulatory sequences" refers to nucleotide sequences
located upstream (5' non-coding sequences), within, or downstream
(3' non-coding sequences) of a coding sequence, and which influence
the transcription, RNA processing or stability, or translation of
the associated coding sequence. Regulatory sequences may include
promoters, translation leader sequences, introns, polyadenylation
recognition sequences, RNA processing sites, effector binding sites
and stem-loop structures. The boundaries of the coding sequence are
determined by a start codon at the 5' (amino) terminus and a
translation stop codon at the 3' (carboxyl) terminus. A coding
sequence can include, but is not limited to, prokaryotic sequences,
cDNA from mRNA, genomic DNA sequences, and even synthetic DNA
sequences. If the coding sequence is intended for expression in an
eukaryotic cell, a polyadenylation signal and transcription
termination sequence will usually be located 3' to the coding
sequence.
[0095] "Open reading frame" is abbreviated ORF and refers to a
length of nucleic acid sequence, either DNA, cDNA or RNA, that
comprises a translation start signal or initiation codon, such as
an ATG or AUG, and a termination codon and can be potentially
translated into a polypeptide sequence.
[0096] The term "head-to-head" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a head-to-head
orientation when the 5' end of the coding strand of one
polynucleotide is adjacent to the 5' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds away from the 5' end of the other
polynucleotide. The term "head-to-head" may be abbreviated
(5')-to-(5') and may also be indicated by the symbols
(.rarw..fwdarw.) or (3'.rarw.5'5'.fwdarw.3').
[0097] The term "tail-to-tail" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a tail-to-tail
orientation when the 3' end of the coding strand of one
polynucleotide is adjacent to the 3' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds toward the other polynucleotide. The
term "tail-to-tail" may be abbreviated (3')-to-(3') and may also be
indicated by the symbols (.fwdarw..rarw.) or
(5'.fwdarw.3'3'.revreaction.5').
[0098] The term "head-to-tail" is used herein to describe the
orientation of two polynucleotide sequences in relation to each
other. Two polynucleotides are positioned in a head-to-tail
orientation when the 5' end of the coding strand of one
polynucleotide is adjacent to the 3' end of the coding strand of
the other polynucleotide, whereby the direction of transcription of
each polynucleotide proceeds in the same direction as that of the
other polynucleotide. The term "head-to-tail" may be abbreviated
(5')-to-(3') and may also be indicated by the symbols
(.fwdarw..fwdarw.) or (5'.fwdarw.3'5'.fwdarw.3').
[0099] The term "downstream" refers to a nucleotide sequence that
is located 3' to a reference nucleotide sequence. In particular,
downstream nucleotide sequences generally relate to sequences that
follow the starting point of transcription. For example, the
translation initiation codon of a gene is located downstream of the
start site of transcription.
[0100] The term "upstream" refers to a nucleotide sequence that is
located 5' to a reference nucleotide sequence. In particular,
upstream nucleotide sequences generally relate to sequences that
are located on the 5' side of a coding sequence or starting point
of transcription. For example, most promoters are located upstream
of the start site of transcription.
[0101] The terms "restriction endonuclease" and "restriction
enzyme" are used interchangeably and refer to an enzyme that binds
and cuts within a specific nucleotide sequence within double
stranded DNA.
[0102] "Homologous recombination" refers to the insertion of a
foreign DNA sequence into another DNA molecule, e.g., insertion of
a vector in a chromosome. Preferably, the vector targets a specific
chromosomal site for homologous recombination. For specific
homologous recombination, the vector will contain sufficiently long
regions of homology to sequences of the chromosome to allow
complementary binding and incorporation of the vector into the
chromosome. Longer regions of homology, and greater degrees of
sequence similarity, may increase the efficiency of homologous
recombination.
[0103] Several methods known in the art may be used to propagate a
polynucleotide according to the invention. Once a suitable host
system and growth conditions are established, recombinant
expression vectors can be propagated and prepared in quantity. As
described herein, the expression vectors which can be used include,
but are not limited to, the following vectors or their derivatives:
human or animal viruses such as vaccinia virus or adenovirus;
insect viruses such as baculovirus; yeast vectors; bacteriophage
vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name
but a few.
[0104] A "vector" refers to any vehicle for the cloning of and/or
transfer of a nucleic acid into a host cell. A vector may be a
replicon to which another DNA segment may be attached so as to
bring about the replication of the attached segment. A "replicon"
refers to any genetic element (e.g., plasmid, phage, cosmid,
chromosome, virus) that functions as an autonomous unit of DNA
replication in vivo, i.e., capable of replication under its own
control. The term "vector" includes both viral and nonviral
vehicles for introducing the nucleic acid into a cell in vitro, ex
vivo or in vivo. A large number of vectors known in the art may be
used to manipulate nucleic acids, incorporate response elements and
promoters into genes, etc. Possible vectors include, for example,
plasmids or modified viruses including, for example bacteriophages
such as lambda derivatives, or plasmids such as pBR322 or pUC
plasmid derivatives, or the Bluescript vector. Another example of
vectors that are useful in the invention is the UltraVector.TM.
Production System (Intrexon Corp., Blacksburg, Va.) as described in
WO 2007/038276. For example, the insertion of the DNA fragments
corresponding to response elements and promoters into a suitable
vector can be accomplished by ligating the appropriate DNA
fragments into a chosen vector that has complementary cohesive
termini. Alternatively, the ends of the DNA molecules may be
enzymatically modified or any site may be produced by ligating
nucleotide sequences (linkers) into the DNA termini. Such vectors
may be engineered to contain selectable marker genes that provide
for the selection of cells that have incorporated the marker into
the cellular genome. Such markers allow identification and/or
selection of host cells that incorporate and express the proteins
encoded by the marker.
[0105] Viral vectors, and particularly retroviral vectors, have
been used in a wide variety of gene delivery applications in cells,
as well as living animal subjects. Viral vectors that can be used
include, but are not limited to, retrovirus, adeno-associated
virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr,
adenovirus, geminivirus, and caulimovirus vectors. Non-viral
vectors include plasmids, liposomes, electrically charged lipids
(cytofectins), DNA-protein complexes, and biopolymers. In addition
to a nucleic acid, a vector may also comprise one or more
regulatory regions, and/or selectable markers useful in selecting,
measuring, and monitoring nucleic acid transfer results (transfer
to which tissues, duration of expression, etc.).
[0106] The term "plasmid" refers to an extra-chromosomal element
often carrying a gene that is not part of the central metabolism of
the cell, and usually in the form of circular double-stranded DNA
molecules. Such elements may be autonomously replicating sequences,
genome integrating sequences, phage or nucleotide sequences,
linear, circular, or supercoiled, of a single- or double-stranded
DNA or RNA, derived from any source, in which a number of
nucleotide sequences have been joined or recombined into a unique
construction which is capable of introducing a promoter fragment
and DNA sequence for a selected gene product along with appropriate
3' untranslated sequence into a cell.
[0107] A "cloning vector" refers to a "replicon," which is a unit
length of a nucleic acid, preferably DNA, that replicates
sequentially and which comprises an origin of replication, such as
a plasmid, phage or cosmid, to which another nucleic acid segment
may be attached so as to bring about the replication of the
attached segment. Cloning vectors may be capable of replication in
one cell type and expression in another ("shuttle vector"). Cloning
vectors may comprise one or more sequences that can be used for
selection of cells comprising the vector and/or one or more
multiple cloning sites for insertion of sequences of interest.
10097] The term "expression vector" refers to a vector, plasmid or
vehicle designed to enable the expression of an inserted nucleic
acid sequence following transformation into the host. The cloned
gene, i.e., the inserted nucleic acid sequence, is usually placed
under the control of control elements such as a promoter, a minimal
promoter, an enhancer, or the like. Initiation control regions or
promoters, which are useful to drive expression of a nucleic acid
in the desired host cell are numerous and familiar to those skilled
in the art. Virtually any promoter capable of driving expression of
these genes can be used in an expression vector, including but not
limited to, viral promoters, bacterial promoters, animal promoters,
mammalian promoters, synthetic promoters, constitutive promoters,
tissue specific promoters, pathogenesis or disease related
promoters, developmental specific promoters, inducible promoters,
light regulated promoters; CYC1, HIS3, GAL1, GAL4, GAL10, ADH1,
PGK, PHO5, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI, alkaline
phosphatase promoters (useful for expression in Saccharomyces);
AOX1 promoter (useful for expression in Pichia); .beta.-lactamase,
lac, ara, tet, trp, lP.sub.L, lP.sub.R, T7, tac, and trc promoters
(useful for expression in Escherichia coli); light regulated-, seed
specific-, pollen specific-, ovary specific-, cauliflower mosaic
virus 35S, CMV 35S minimal, cassava vein mosaic virus (CsVMV),
chlorophyll a/b binding protein, ribulose 1,5-bisphosphate
carboxylase, shoot-specific, root specific, chitinase, stress
inducible, rice tungro bacilliform virus, plant super-promoter,
potato leucine aminopeptidase, nitrate reductase, mannopine
synthase, nopaline synthase, ubiquitin, zein protein, and
anthocyanin promoters (useful for expression in plant cells);
animal and mammalian promoters known in the art including, but are
not limited to, the SV40 early (SV40e) promoter region, the
promoter contained in the 3' long terminal repeat (LTR) of Rous
sarcoma virus (RSV), the promoters of the E1A or major late
promoter (MLP) genes of adenoviruses (Ad), the cytomegalovirus
(CMV) early promoter, the herpes simplex virus (HSV) thymidine
kinase (TK) promoter, a baculovirus 1E1 promoter, an elongation
factor 1 alpha (EF1) promoter, a phosphoglycerate kinase (PGK)
promoter, a ubiquitin (Ubc) promoter, an albumin promoter, the
regulatory sequences of the mouse metallothionein-L promoter and
transcriptional control regions, the ubiquitous promoters (HPRT,
vimentin, .alpha.-actin, tubulin and the like), the promoters of
the intermediate filaments (desmin, neurofilaments, keratin, GFAP,
and the like), the promoters of therapeutic genes (of the MDR, CFTR
or factor VIII type, and the like), pathogenesis or disease
related-promoters, and promoters that exhibit tissue specificity
and have been utilized in transgenic animals, such as the elastase
I gene control region which is active in pancreatic acinar cells;
insulin gene control region active in pancreatic beta cells,
immunoglobulin gene control region active in lymphoid cells, mouse
mammary tumor virus control region active in testicular, breast,
lymphoid and mast cells; albumin gene, Apo AI and Apo AII control
regions active in liver, alpha-fetoprotein gene control region
active in liver, alpha 1-antitrypsin gene control region active in
the liver, beta-globin gene control region active in myeloid cells,
myelin basic protein gene control region active in oligodendrocyte
cells in the brain, myosin light chain-2 gene control region active
in skeletal muscle, and gonadotropic releasing hormone gene control
region active in the hypothalamus, pyruvate kinase promoter, villin
promoter, promoter of the fatty acid binding intestinal protein,
promoter of the smooth muscle cell .alpha.-actin, and the like. In
addition, these expression sequences may be modified by addition of
enhancer or regulatory sequences and the like.
[0108] Vectors may be introduced into the desired host cells by
methods known in the art, e.g., transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, lipofection (lysosome fusion), use of a
gene gun, or a DNA vector transporter (see, e.g., Wu et al., J.
Biol. Chem. 267:963 (1992); Wu et al., J. Biol. Chem. 263:14621
(1988); and Hartmut et al., Canadian Patent Application No.
2,012,311).
[0109] A polynucleotide according to the invention can also be
introduced in vivo by lipofection. For the past decade, there has
been increasing use of liposomes for encapsulation and transfection
of nucleic acids in vitro. Synthetic cationic lipids designed to
limit the difficulties and dangers encountered with
liposome-mediated transfection can be used to prepare liposomes for
in vivo transfection of a gene encoding a marker (Feigner et al.,
Proc. Natl. Acad. Sci. USA. 84:7413 (1987); Mackey et al., Proc.
Natl. Acad. Sci. USA 85:8027 (1988); and Ulmer et al., Science
259:1745 (1993)). The use of cationic lipids may promote
encapsulation of negatively charged nucleic acids, and also promote
fusion with negatively charged cell membranes (Feigner et al.,
Science 337:387 (1989)). Particularly useful lipid compounds and
compositions for transfer of nucleic acids are described in
WO95/18863, WO96/17823 and U.S. Pat. No. 5,459,127. The use of
lipofection to introduce exogenous genes into the specific organs
in vivo has certain practical advantages. Molecular targeting of
liposomes to specific cells represents one area of benefit. It is
clear that directing transfection to particular cell types would be
particularly preferred in a tissue with cellular heterogeneity,
such as pancreas, liver, kidney, and the brain. Lipids may be
chemically coupled to other molecules for the purpose of targeting
(Mackey et al. 1988, supra). Targeted peptides, e.g., hormones or
neurotransmitters, and proteins such as antibodies, or non-peptide
molecules could be coupled to liposomes chemically.
[0110] Other molecules are also useful for facilitating
transfection of a nucleic acid in vivo, such as a cationic
oligopeptide (e.g., WO95/21931), peptides derived from DNA binding
proteins (e.g., WO96/25508), or a cationic polymer (e.g.,
WO95/21931).
[0111] It is also possible to introduce a vector in vivo as a naked
DNA plasmid (see U.S. Pat. Nos. 5,693,622, 5,589,466 and
5,580,859). Receptor-mediated DNA delivery approaches can also be
used (Curiel et al., Hum. Gene Ther. 3:147 (1992); and Wu et al.,
J. Biol. Chem. 262:4429 (1987)).
[0112] The term "transfection" refers to the uptake of exogenous or
heterologous RNA or DNA by a cell. A cell has been "transfected" by
exogenous or heterologous RNA or DNA when such RNA or DNA has been
introduced inside the cell. A cell has been "transformed" by
exogenous or heterologous RNA or DNA when the transfected RNA or
DNA effects a phenotypic change. The transforming RNA or DNA can be
integrated (covalently linked) into chromosomal DNA making up the
genome of the cell.
[0113] "Transformation" refers to the transfer of a nucleic acid
fragment into the genome of a host organism, resulting in
genetically stable inheritance. Host organisms containing the
transformed nucleic acid fragments are referred to as "transgenic"
or "recombinant" or "transformed" organisms.
[0114] In addition, the recombinant vector comprising a
polynucleotide according to the invention may include one or more
origins for replication in the cellular hosts in which their
amplification or their expression is sought, markers or selectable
markers.
[0115] The term "selectable marker" refers to an identifying
factor, usually an antibiotic or chemical resistance gene, that is
able to be selected for based upon the marker gene's effect, i.e.,
resistance to an antibiotic, resistance to a herbicide,
colorimetric markers, enzymes, fluorescent markers, and the like,
wherein the effect is used to track the inheritance of a nucleic
acid of interest and/or to identify a cell or organism that has
inherited the nucleic acid of interest. Examples of selectable
marker genes known and used in the art include: genes providing
resistance to ampicillin, streptomycin, gentamycin, kanamycin,
hygromycin, bialaphos herbicide, sulfonamide, and the like; and
genes that are used as phenotypic markers, i.e., anthocyanin
regulatory genes, isopentanyl transferase gene, and the like.
[0116] The term "reporter gene" refers to a nucleic acid encoding
an identifying factor that is able to be identified based upon the
reporter gene's effect, wherein the effect is used to track the
inheritance of a nucleic acid of interest, to identify a cell or
organism that has inherited the nucleic acid of interest, and/or to
measure gene expression induction or transcription. Examples of
reporter genes known and used in the art include: luciferase (Luc),
green fluorescent protein (GFP), chloramphenicol acetyltransferase
(CAT), .beta.-galactosidase (LacZ), .beta.-glucuronidase (Gus), and
the like. Selectable marker genes may also be considered reporter
genes.
[0117] "Promoter" and "promoter sequence" are used interchangeably
and refer to a DNA sequence capable of controlling the expression
of a coding sequence or functional RNA. In general, a coding
sequence is located 3' to a promoter sequence. Promoters may be
derived in their entirety from a native gene, or be composed of
different elements derived from different promoters found in
nature, or even comprise synthetic DNA segments. It is understood
by those skilled in the art that different promoters may direct the
expression of a gene in different tissues or cell types, or at
different stages of development, or in response to different
environmental or physiological conditions. Promoters that cause a
gene to be expressed in most cell types at most times are commonly
referred to as "constitutive promoters." Promoters that cause a
gene to be expressed in a specific cell type are commonly referred
to as "cell-specific promoters" or "tissue-specific promoters."
Promoters that cause a gene to be expressed at a specific stage of
development or cell differentiation are commonly referred to as
"developmentally-specific promoters" or "cell
differentiation-specific promoters." Promoters that are induced and
cause a gene to be expressed following exposure or treatment of the
cell with an agent, biological molecule, chemical, ligand, light,
or the like that induces the promoter are commonly referred to as
"inducible promoters" or "regulatable promoters." It is further
recognized that since in most cases the exact boundaries of
regulatory sequences have not been completely defined, DNA
fragments of different lengths may have identical promoter
activity.
[0118] The promoter sequence is typically bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site (conveniently defined for example, by
mapping with nuclease S1), as well as protein binding domains
(consensus sequences) responsible for the binding of RNA
polymerase.
[0119] A coding sequence is "under the control" of transcriptional
and translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then trans-RNA
spliced (if the coding sequence contains introns) and translated
into the protein encoded by the coding sequence.
[0120] "Transcriptional and translational control sequences" refer
to DNA regulatory sequences, such as promoters, enhancers,
terminators, and the like, that provide for the expression of a
coding sequence in a host cell. In eukaryotic cells,
polyadenylation signals are control sequences.
[0121] The term "response element" refers to one or more cis-acting
DNA elements which confer responsiveness on a promoter mediated
through interaction with the DNA-binding domains of a transcription
factor. This DNA element may be either palindromic (perfect or
imperfect) in its sequence or composed of sequence motifs or half
sites separated by a variable number of nucleotides. The half sites
can be similar or identical and arranged as either direct or
inverted repeats or as a single half site or multimers of adjacent
half sites in tandem. The response element may comprise a minimal
promoter isolated from different organisms depending upon the
nature of the cell or organism into which the response element will
be incorporated. The DNA binding domain of the transcription factor
binds, in the presence or absence of a ligand, to the DNA sequence
of a response element to initiate or suppress transcription of
downstream gene(s) under the regulation of this response element.
Examples of DNA sequences for response elements of the natural
ecdysone receptor include: RRGG/TTCANTGAC/ACYY (SEQ ID NO: 9) (see
Cherbas et. al., Genes Dev. 5:120 (1991)); AGGTCAN.sub.(n)AGGTCA,
where N.sub.(n) can be one or more spacer nucleotides (SEQ ID NO:
10) (see D'Avino et al., Mol. Cell. Endocrinol. 113:1 (1995)); and
GGGTTGAATGAATTT (SEQ ID NO: 11) (see Antoniewski et al., Mol. Cell
Biol. 14:4465 (1994)).
[0122] The term "operably linked" refers to the association of
nucleic acid sequences on a single nucleic acid fragment so that
the function of one is affected by the other. For example, a
promoter is operably linked with a coding sequence when it is
capable of affecting the expression of that coding sequence (i.e.,
that the coding sequence is under the transcriptional control of
the promoter). Coding sequences can be operably linked to
regulatory sequences in sense or antisense orientation.
[0123] The term "expression" as used herein refers to the
transcription and stable accumulation of sense (mRNA) or antisense
RNA derived from a nucleic acid or polynucleotide. Expression may
also refer to translation of mRNA into a protein or
polypeptide.
[0124] The terms "cassette," "expression cassette" and "gene
expression cassette" refer to a segment of DNA that can be inserted
into a nucleic acid or polynucleotide at specific restriction sites
or by homologous recombination. The segment of DNA comprises a
polynucleotide that encodes a polypeptide of interest, and the
cassette and restriction sites are designed to ensure insertion of
the cassette in the proper reading frame for transcription and
translation. "Transformation cassette" refers to a specific vector
comprising a polynucleotide that encodes a polypeptide of interest
and having elements in addition to the polynucleotide that
facilitate transformation of a particular host cell. Cassettes,
expression cassettes, gene expression cassettes and transformation
cassettes of the invention may also comprise elements that allow
for enhanced expression of a polynucleotide encoding a polypeptide
of interest in a host cell. These elements may include, but are not
limited to: a promoter, a minimal promoter, an enhancer, a response
element, a terminator sequence, a polyadenylation sequence, and the
like.
[0125] For purposes of this invention, the term "gene switch"
refers to the combination of a response element associated with a
promoter, and a ligand-dependent transcription factor-based system
which, in the presence of one or more ligands, modulates the
expression of a gene into which the response element and promoter
are incorporated. The term "a polynucleotide encoding a gene
switch" refers to the combination of a response element associated
with a promoter, and a polynucleotide encoding a ligand-dependent
transcription factor-based system which, in the presence of one or
more ligands, modulates the expression of a gene into which the
response element and promoter are incorporated.
[0126] The term "ecdysone receptor-based," with respect to a gene
switch, refers to a gene switch comprising at least a functional
part of a naturally occurring or synthetic ecdysone receptor ligand
binding domain and which regulates gene expression in response to a
ligand that binds to the ecdysone receptor ligand binding domain.
Examples of ecdysone-responsive systems are described in U.S. Pat.
Nos. 7,091,038 and 6,258,603. In one embodiment, the system is the
RheoSwitch.RTM. Therapeutic System (RTS), which contains two fusion
proteins, the DEF domains of a mutagenized ecdysone receptor (EcR)
fused with a Gal4 DNA binding domain and the EF domains of a
chimeric RXR fused with a VP16 transcription activation domain,
expressed under a constitutive promoter as illustrated in FIG.
1.
[0127] The terms "modulate" and "modulates" mean to induce, reduce
or inhibit nucleic acid or gene expression, resulting in the
respective induction, reduction or inhibition of protein or
polypeptide production.
[0128] The polynucleotides or vectors according to the invention
may further comprise at least one promoter suitable for driving
expression of a gene in a host cell.
[0129] Enhancers that may be used in embodiments of the invention
include but are not limited to: an SV40 enhancer, a cytomegalovirus
(CMV) enhancer, an elongation factor 1 (EF1) enhancer, yeast
enhancers, viral gene enhancers, and the like.
[0130] Termination control regions, i.e., terminator or
polyadenylation sequences, may also be derived from various genes
native to the preferred hosts. Optionally, a termination site may
be unnecessary, however, it is most preferred if included. In one
embodiment of the invention, the termination control region may be
comprised or be derived from a synthetic sequence, synthetic
polyadenylation signal, an SV40 late polyadenylation signal, an
SV40 polyadenylation signal, a bovine growth hormone (BGH)
polyadenylation signal, viral terminator sequences, or the
like.
[0131] The terms "3' non-coding sequences" or "3' untranslated
region (UTR)" refer to DNA sequences located downstream (3') of a
coding sequence and may comprise polyadenylation [poly(A)]
recognition sequences and other sequences encoding regulatory
signals capable of affecting mRNA processing or gene expression.
The polyadenylation signal is usually characterized by affecting
the addition of polyadenylic acid tracts to the 3' end of the mRNA
precursor.
[0132] "Regulatory region" refers to a nucleic acid sequence that
regulates the expression of a second nucleic acid sequence. A
regulatory region may include sequences which are naturally
responsible for expressing a particular nucleic acid (a homologous
region) or may include sequences of a different origin that are
responsible for expressing different proteins or even synthetic
proteins (a heterologous region). In particular, the sequences can
be sequences of prokaryotic, eukaryotic, or viral genes or derived
sequences that stimulate or repress transcription of a gene in a
specific or non-specific manner and in an inducible or
non-inducible manner. Regulatory regions include origins of
replication, RNA splice sites, promoters, enhancers,
transcriptional termination sequences, and signal sequences which
direct the polypeptide into the secretory pathways of the target
cell.
[0133] A regulatory region from a "heterologous source" refers to a
regulatory region that is not naturally associated with the
expressed nucleic acid. Included among the heterologous regulatory
regions are regulatory regions from a different species, regulatory
regions from a different gene, hybrid regulatory sequences, and
regulatory sequences which do not occur in nature, but which are
designed by one having ordinary skill in the art.
[0134] "RNA transcript" refers to the product resulting from RNA
polymerase-catalyzed transcription of a DNA sequence. When the RNA
transcript is a perfect complementary copy of the DNA sequence, it
is referred to as the primary transcript or it may be a RNA
sequence derived from post-transcriptional processing of the
primary transcript and is referred to as the mature RNA. "Messenger
RNA (mRNA)" refers to the RNA that is without introns and that can
be translated into protein by the cell. "cDNA" refers to a
double-stranded DNA that is complementary to and derived from mRNA.
"Sense" RNA refers to RNA transcript that includes the mRNA and so
can be translated into protein by the cell. "Antisense RNA" refers
to a RNA transcript that is complementary to all or part of a
target primary transcript or mRNA and that blocks the expression of
a target gene. The complementarity of an antisense RNA may be with
any part of the specific gene transcript, i.e., at the 5'
non-coding sequence, 3' non-coding sequence, or the coding
sequence. "Functional RNA" refers to antisense RNA, ribozyme RNA,
or other RNA that is not translated yet has an effect on cellular
processes.
[0135] "Polypeptide," "peptide" and "protein" are used
interchangeably and refer to a polymeric compound comprised of
covalently linked amino acid residues.
[0136] An "isolated polypeptide," "isolated peptide" or "isolated
protein" refer to a polypeptide or protein that is substantially
free of those compounds that are normally associated therewith in
its natural state (e.g., other proteins or polypeptides, nucleic
acids, carbohydrates, lipids). "Isolated" is not meant to exclude
artificial or synthetic mixtures with other compounds, or the
presence of impurities which do not interfere with biological
activity, and which may be present, for example, due to incomplete
purification, addition of stabilizers, or compounding into a
pharmaceutically acceptable preparation.
[0137] A "substitution mutant polypeptide" or a "substitution
mutant" will be understood to mean a mutant polypeptide comprising
a substitution of at least one wild-type or naturally occurring
amino acid with a different amino acid relative to the wild-type or
naturally occurring polypeptide. A substitution mutant polypeptide
may comprise only one wild-type or naturally occurring amino acid
substitution and may be referred to as a "point mutant" or a
"single point mutant" polypeptide. Alternatively, a substitution
mutant polypeptide may comprise a substitution of two or more
wild-type or naturally occurring amino acids with two or more amino
acids relative to the wild-type or naturally occurring polypeptide.
According to the invention, a Group H nuclear receptor ligand
binding domain polypeptide comprising a substitution mutation
comprises a substitution of at least one wild-type or naturally
occurring amino acid with a different amino acid relative to the
wild-type or naturally occurring Group H nuclear receptor ligand
binding domain polypeptide.
[0138] When the substitution mutant polypeptide comprises a
substitution of two or more wild-type or naturally occurring amino
acids, this substitution may comprise either an equivalent number
of wild-type or naturally occurring amino acids deleted for the
substitution, i.e., 2 wild-type or naturally occurring amino acids
replaced with 2 non-wild-type or non-naturally occurring amino
acids, or a non-equivalent number of wild-type amino acids deleted
for the substitution, i.e., 2 wild-type amino acids replaced with 1
non-wild-type amino acid (a substitution+deletion mutation), or 2
wild-type amino acids replaced with 3 non-wild-type amino acids (a
substitution+insertion mutation).
[0139] Substitution mutants may be described using an abbreviated
nomenclature system to indicate the amino acid residue and number
replaced within the reference polypeptide sequence and the new
substituted amino acid residue. For example, a substitution mutant
in which the twentieth (20.sup.th) amino acid residue of a
polypeptide is substituted may be abbreviated as "x20z", wherein
"x" is the amino acid to be replaced, "20" is the amino acid
residue position or number within the polypeptide, and "z" is the
new substituted amino acid. Therefore, a substitution mutant
abbreviated interchangeably as "E20A" or "Glu20Ala" indicates that
the mutant comprises an alanine residue (commonly abbreviated in
the art as "A" or "Ala") in place of the glutamic acid (commonly
abbreviated in the art as "E" or "Glu") at position 20 of the
polypeptide.
[0140] A substitution mutation may be made by any technique for
mutagenesis known in the art, including but not limited to, in
vitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem.
253:6551 (1978); Zoller et al., DNA 3:479 (1984); Oliphant et al.,
Gene 44:177 (1986); Hutchinson et al., Proc. Natl. Acad. Sci. USA
83:710 (1986)), use of TAB.RTM. linkers (Pharmacia), restriction
endonuclease digestion/fragment deletion and substitution,
PCR-mediated/oligonucleotide-directed mutagenesis, and the like.
PCR-based techniques are preferred for site-directed mutagenesis
(see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology:
Principles and Applications for DNA Amplification, H. Erlich, ed.,
Stockton Press, Chapter 6, pp. 61-70).
[0141] The term "fragment," as applied to a polypeptide, refers to
a polypeptide whose amino acid sequence is shorter than that of the
reference polypeptide and which comprises, over the entire portion
with these reference polypeptides, an identical amino acid
sequence. Such fragments may, where appropriate, be included in a
larger polypeptide of which they are a part. Such fragments of a
polypeptide according to the invention may have a length of at
least 2, 3, 4, 5, 6, 8, 10, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,
25, 26, 30, 35, 40, 45, 50, 100, 200, 240, or 300 or more amino
acids.
[0142] A "variant" of a polypeptide or protein refers to any
analogue, fragment, derivative, or mutant which is derived from a
polypeptide or protein and which retains at least one biological
property of the polypeptide or protein. Different variants of the
polypeptide or protein may exist in nature. These variants may be
allelic variations characterized by differences in the nucleotide
sequences of the structural gene coding for the protein, or may
involve differential splicing or post-translational modification.
The skilled artisan can produce variants having single or multiple
amino acid substitutions, deletions, additions, or replacements.
These variants may include, inter alia: (a) variants in which one
or more amino acid residues are substituted with conservative or
non-conservative amino acids, (b) variants in which one or more
amino acids are added to the polypeptide or protein, (c) variants
in which one or more of the amino acids includes a substituent
group, and (d) variants in which the polypeptide or protein is
fused with another polypeptide such as serum albumin. The
techniques for obtaining these variants, including genetic
(suppressions, deletions, mutations, etc.), chemical, and enzymatic
techniques, are known to persons having ordinary skill in the art.
In one embodiment, a variant polypeptide comprises at least about
14 amino acids.
[0143] The term "homology" refers to the percent of identity
between two polynucleotide or two polypeptide moieties. The
correspondence between the sequence from one moiety to another can
be determined by techniques known to the art. For example, homology
can be determined by a direct comparison of the sequence
information between two polypeptide molecules by aligning the
sequence information and using readily available computer programs.
Alternatively, homology can be determined by hybridization of
polynucleotides under conditions that form stable duplexes between
homologous regions, followed by digestion with
single-stranded-specific nuclease(s) and size determination of the
digested fragments.
[0144] As used herein, the term "homologous" in all its grammatical
forms and spelling variations refers to the relationship between
proteins that possess a "common evolutionary origin," including
proteins from superfamilies (e.g., the immunoglobulin superfamily)
and homologous proteins from different species (e.g., myosin light
chain, etc.) (Reeck et al., Cell 50:667 (1987)). Such proteins (and
their encoding genes) have sequence homology, as reflected by their
high degree of sequence similarity. However, in common usage and in
the application, the term "homologous," when modified with an
adverb such as "highly," may refer to sequence similarity and not a
common evolutionary origin.
[0145] Accordingly, the term "sequence similarity" in all its
grammatical forms refers to the degree of identity or
correspondence between nucleic acid or amino acid sequences of
proteins that may or may not share a common evolutionary origin
(see Reeck et al., Cell 50:667 (1987)). In one embodiment, two DNA
sequences are "substantially homologous" or "substantially similar"
when at least about 50% (e.g., at least about 75%, 90%, or 95%) of
the nucleotides match over the defined length of the DNA sequences.
Sequences that are substantially homologous can be identified by
comparing the sequences using standard software available in
sequence data banks, or in a Southern hybridization experiment
under, for example, stringent conditions as defined for that
particular system. Defining appropriate hybridization conditions is
within the skill of the art (see e.g., Sambrook et al., 1989,
supra).
[0146] As used herein, "substantially similar" refers to nucleic
acid fragments wherein changes in one or more nucleotide bases
results in substitution of one or more amino acids, but do not
affect the functional properties of the protein encoded by the DNA
sequence. "Substantially similar" also refers to nucleic acid
fragments wherein changes in one or more nucleotide bases do not
affect the ability of the nucleic acid fragment to mediate
alteration of gene expression by antisense or co-suppression
technology. "Substantially similar" also refers to modifications of
the nucleic acid fragments of the invention such as deletion or
insertion of one or more nucleotide bases that do not substantially
affect the functional properties of the resulting transcript. It is
therefore understood that the invention encompasses more than the
specific exemplary sequences. Each of the proposed modifications is
well within the routine skill in the art, as is determination of
retention of biological activity of the encoded products.
[0147] Moreover, the skilled artisan recognizes that substantially
similar sequences encompassed by this invention are also defined by
their ability to hybridize, under stringent conditions
(0.1.times.SSC, 0.1% SDS, 65.degree. C. and washed with
2.times.SSC, 0.1% SDS followed by 0.1.times.SSC, 0.1% SDS), with
the sequences exemplified herein. Substantially similar nucleic
acid fragments of the invention are those nucleic acid fragments
whose DNA sequences are at least about 70%, 80%, 90% or 95%
identical to the DNA sequence of the nucleic acid fragments
reported herein.
[0148] Two amino acid sequences are "substantially homologous" or
"substantially similar" when greater than about 40% of the amino
acids are identical, or greater than 60% are similar (functionally
identical). Preferably, the similar or homologous sequences are
identified by alignment using, for example, the GCG (Genetics
Computer Group, Program Manual for the GCG Package, Version 7,
Madison, Wis.) pileup program.
[0149] The term "corresponding to" is used herein to refer to
similar or homologous sequences, whether the exact position is
identical or different from the molecule to which the similarity or
homology is measured. A nucleic acid or amino acid sequence
alignment may include spaces. Thus, the term "corresponding to"
refers to the sequence similarity, and not the numbering of the
amino acid residues or nucleotide bases.
[0150] A "substantial portion" of an amino acid or nucleotide
sequence comprises enough of the amino acid sequence of a
polypeptide or the nucleotide sequence of a gene to putatively
identify that polypeptide or gene, either by manual evaluation of
the sequence by one skilled in the art, or by computer-automated
sequence comparison and identification using algorithms such as
BLAST (Basic Local Alignment Search Tool; Altschul et al., J. Mol.
Biol. 215:403 (1993)); available at ncbi.nlm.nih.gov/BLAST/). In
general, a sequence of ten or more contiguous amino acids or thirty
or more nucleotides is necessary in order to putatively identify a
polypeptide or nucleic acid sequence as homologous to a known
protein or gene. Moreover, with respect to nucleotide sequences,
gene specific oligonucleotide probes comprising 20-30 contiguous
nucleotides may be used in sequence-dependent methods of gene
identification (e.g., Southern hybridization) and isolation (e.g.,
in situ hybridization of bacterial colonies or bacteriophage
plaques). In addition, short oligonucleotides of 12-15 bases may be
used as amplification primers in PCR in order to obtain a
particular nucleic acid fragment comprising the primers.
Accordingly, a "substantial portion" of a nucleotide sequence
comprises enough of the sequence to specifically identify and/or
isolate a nucleic acid fragment comprising the sequence.
[0151] The term "percent identity," as known in the art, is a
relationship between two or more polypeptide sequences or two or
more polynucleotide sequences, as determined by comparing the
sequences. In the art, "identity" also means the degree of sequence
relatedness between polypeptide or polynucleotide sequences, as the
case may be, as determined by the match between strings of such
sequences. "Identity" and "similarity" can be readily calculated by
known methods, including but not limited to those described in:
Computational Molecular Biology (Lesk, A. M., ed.) Oxford
University Press, New York (1988); Biocomputing: Informatics and
Genome Projects (Smith, D. W., ed.) Academic Press, New York
(1993); Computer Analysis of Sequence Data, Part I (Griffin, A. M.,
and Griffin, H. G., eds.) Humana Press, New Jersey (1994); Sequence
Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press
(1987); and Sequence Analysis Primer (Gribskov, M. and Devereux,
J., eds.) Stockton Press, New York (1991). Preferred methods to
determine identity are designed to give the best match between the
sequences tested. Methods to determine identity and similarity are
codified in publicly available computer programs. Sequence
alignments and percent identity calculations may be performed using
sequence analysis software such as the Megalign program of the
LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison,
Wis.). Multiple alignment of the sequences may be performed using
the Clustal method of alignment (Higgins et al., CABIOS. 5:151
(1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH
PENALTY=10). Default parameters for pairwise alignments using the
Clustal method may be selected: KTUPLE 1, GAP PENALTY=3, WINDOW=5
and DIAGONALS SAVED=5.
[0152] The term "sequence analysis software" refers to any computer
algorithm or software program that is useful for the analysis of
nucleotide or amino acid sequences. "Sequence analysis software"
may be commercially available or independently developed. Typical
sequence analysis software includes, but is not limited to, the GCG
suite of programs (Wisconsin Package Version 9.0, Genetics Computer
Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et
al., J. Mol. Biol. 215:403 (1990)), and DNASTAR (DNASTAR, Inc. 1228
S. Park St. Madison, Wis. 53715 USA). Within the context of this
application it will be understood that where sequence analysis
software is used for analysis, that the results of the analysis
will be based on the "default values" of the program referenced,
unless otherwise specified. As used herein "default values" will
mean any set of values or parameters which originally load with the
software when first initialized.
[0153] "Chemically synthesized," as related to a sequence of DNA,
means that the component nucleotides were assembled in vitro.
Manual chemical synthesis of DNA may be accomplished using
well-established procedures, or automated chemical synthesis can be
performed using one of a number of commercially available machines.
Accordingly, the genes can be tailored for optimal gene expression
based on optimization of nucleotide sequence to reflect the codon
bias of the host cell. The skilled artisan appreciates the
likelihood of successful gene expression if codon usage is biased
towards those codons favored by the host. Determination of
preferred codons can be based on a survey of genes derived from the
host cell where sequence information is available.
[0154] As used herein, two or more individually operable gene
regulation systems are said to be "orthogonal" when; a) modulation
of each of the given systems by its respective ligand, at a chosen
concentration, results in a measurable change in the magnitude of
expression of the gene of that system, and b) the change is
statistically significantly different than the change in expression
of all other systems simultaneously operable in the cell, tissue,
or organism, regardless of the simultaneity or sequentially of the
actual modulation. Preferably, modulation of each individually
operable gene regulation system effects a change in gene expression
at least 2-fold greater than all other operable systems in the
cell, tissue, or organism, e.g., at least 5-fold, 10-fold,
100-fold, or 500-fold greater. Ideally, modulation of each of the
given systems by its respective ligand at a chosen concentration
results in a measurable change in the magnitude of expression of
the gene of that system and no measurable change in expression of
all other systems operable in the cell, tissue, or organism. In
such cases the multiple inducible gene regulation system is said to
be "fully orthogonal." Useful orthogonal ligands and orthogonal
receptor-based gene expression systems are described in US
2002/0110861 A.
[0155] The term "exogenous gene" means a gene foreign to the
subject, that is, a gene which is introduced into the subject
through a transformation process, an unmutated version of an
endogenous mutated gene or a mutated version of an endogenous
unmutated gene. The method of transformation is not critical to
this invention and may be any method suitable for the subject known
to those in the art. Exogenous genes can be either natural or
synthetic genes which are introduced into the subject in the form
of DNA or RNA which may function through a DNA intermediate such as
by reverse transcriptase. Such genes can be introduced into target
cells, directly introduced into the subject, or indirectly
introduced by the transfer of transformed cells into the
subject.
[0156] The term "therapeutic product" refers to a therapeutic polyp
eptide or therapeutic polynucleotide which imparts a beneficial
function to the host cell in which such product is expressed.
Therapeutic polypeptides may include, without limitation, peptides
as small as three amino acids in length, single- or multiple-chain
proteins, and fusion proteins. Therapeutic polynucleotides may
include, without limitation, antisense oligonucleotides, small
interfering RNAs, ribozymes, and RNA external guide sequences. The
therapeutic product may comprise a naturally occurring sequence, a
synthetic sequence or a combination of natural and synthetic
sequences.
[0157] The term "ecdysone receptor complex" generally refers to a
heterodimeric protein complex having at least two members of the
nuclear receptor family, ecdysone receptor ("EcR") and
ultraspiracle ("USP") proteins (see Yao et al., Nature 366:476
(1993)); Yao et al., Cell 71:63 (1992)). The functional EcR complex
may also include additional protein(s) such as immunophilins.
Additional members of the nuclear receptor family of proteins,
known as transcriptional factors (such as DHR38, betaFTZ-1 or other
insect homologs), may also be ligand dependent or independent
partners for EcR and/or USP. The EcR complex can also be a
heterodimer of EcR protein and the vertebrate homolog of
ultraspiracle protein, retinoic acid-X-receptor ("RXR") protein or
a chimera of USP and RXR. The term EcR complex also encompasses
homodimer complexes of the EcR protein or USP.
[0158] An EcR complex can be activated by an active ecdysteroid or
non-steroidal ligand bound to one of the proteins of the complex,
inclusive of EcR, but not excluding other proteins of the complex.
As used herein, the term "ligand," as applied to EcR-based gene
switches, describes small and soluble molecules having the
capability of activating a gene switch to stimulate expression of a
polypeptide encoded therein. Examples of ligands include, without
limitation, an ecdysteroid, such as ecdysone, 20-hydroxyecdysone,
ponasterone A, muristerone A, and the like, 9-cis-retinoic acid,
synthetic analogs of retinoic acid, N,N'-diacylhydrazines such as
those disclosed in U.S. Pat. Nos. 6,013,836; 5,117,057; 5,530,028;
and 5,378,726 and U.S. Published Application Nos. 2005/0209283 and
2006/0020146; oxadiazolines as described in U.S. Published
Application No. 2004/0171651; dibenzoylalkyl cyanohydrazines such
as those disclosed in European Application No. 461,809;
N-alkyl-N,N'-diaroylhydrazines such as those disclosed in U.S. Pat.
No. 5,225,443; N-acyl-N-alkylcarbonylhydrazines such as those
disclosed in European Application No. 234,994;
N-aroyl-N-alkyl-N'-aroylhydrazines such as those described in U.S.
Pat. No. 4,985,461; arnidoketones such as those described in U.S.
Published Application No. 2004/0049037; and other similar materials
including 3,5-di-tert-butyl-4-hydroxy-N-isobutyl-benzamide,
8-O-acetylharpagide, oxysterols, 22(R) hydroxycholesterol, 24(S)
hydroxycholesterol, 25-epoxycholesterol, T0901317,
5-alpha-6-alpha-epoxycholesterol-3-sulfate (ECHS),
7-ketocholesterol-3-sulfate, famesol, bile acids, 1,1-biphosphonate
esters, juvenile hormone III, and the like. Examples of
diacylhydrazine ligands useful in the invention include RG-115819
(3,5-Dimethyl-benzoic acid
N-(1-ethyl-2,2-dimethyl-propyl)-N'-(2-methyl-3-methoxy-benzoyl)-hydrazide-
), RG-115932 ((R)-3,5-Dimethyl-benzoic acid
N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide),
and RG-115830 (3,5-Dimethyl-benzoic acid
N-(1-tert-butyl-butyl)-N'-(2-ethyl-3-methoxy-benzoyl)-hydrazide).
See U.S. application Ser. No. 12/155,111, filed May 29, 2008, and
PCT/US2008/006757 filed May 29, 2008, for additional
diacylhydrazines that are useful in the practice of the
invention.
[0159] The EcR complex includes proteins which are members of the
nuclear receptor superfamily wherein all members are characterized
by the presence of an amino-terminal transactivation domain ("TA"),
a DNA binding domain ("DBD"), and a ligand binding domain ("LBD")
separated by a hinge region. Some members of the family may also
have another transactivation domain on the carboxy-terminal side of
the LBD. The DBD is characterized by the presence of two cysteine
zinc fingers between which are two amino acid motifs, the P-box and
the D-box, which confer specificity for ecdysone response elements.
These domains may be either native, modified, or chimeras of
different domains of heterologous receptor proteins.
[0160] The DNA sequences making up the exogenous gene, the response
element, and the EcR `complex may be incorporated into
archaebacteria, procaryotic cells such as Escherichia coli,
Bacillus subtilis, or other enterobacteria, or eucaryotic cells
such as plant or animal cells. However, because many of the
proteins expressed by the gene are processed incorrectly in
bacteria, eucaryotic cells are preferred. The cells may be in the
form of single cells or multicellular organisms. The nucleotide
sequences for the exogenous gene, the response element, and the
receptor complex can also be incorporated as RNA molecules,
preferably in the form of functional viral RNAs such as tobacco
mosaic virus. Of the eucaryotic cells, vertebrate cells are
preferred because they naturally lack the molecules which confer
responses to the ligands of this invention for the EcR. As a
result, they are "substantially insensitive" to the ligands of this
invention. Thus, the ligands useful in this invention will have
negligible physiological or other effects on transformed cells, or
the whole organism. Therefore, cells can grow and express the
desired product, substantially unaffected by the presence of the
ligand itself.
[0161] EcR ligands, when used with the EcR complex which in turn is
bound to the response element linked to an exogenous gene (e.g.,
IL-12), provide the means for external temporal regulation of
expression of the exogenous gene. The order in which the various
components bind to each other, that is, ligand to receptor complex
and receptor complex to response element, is not critical.
Typically, modulation of expression of the exogenous gene is in
response to the binding of the EcR complex to a specific control,
or regulatory, DNA element. The EcR protein, like other members of
the nuclear receptor family, possesses at least three domains, a
transactivation domain, a DNA binding domain, and a ligand binding
domain. This receptor, like a subset of the nuclear receptor
family, also possesses less well-defined regions responsible for
heterodimerization properties. Binding of the ligand to the ligand
binding domain of EcR protein, after heterodimerization with USP or
RXR protein, enables the DNA binding domains of the heterodimeric
proteins to bind to the response element in an activated form, thus
resulting in expression or suppression of the exogenous gene. This
mechanism does not exclude the potential for ligand binding to
either EcR or USP, and the resulting formation of active homodimer
complexes (e.g. EcR+EcR or USP+USP). In one embodiment, one or more
of the receptor domains can be varied producing a chimeric gene
switch. Typically, one or more of the three domains may be chosen
from a source different than the source of the other domains so
that the chimeric receptor is optimized in the chosen host cell or
organism for transactivating activity, complementary binding of the
ligand, and recognition of a specific response element. In
addition, the response element itself can be modified or
substituted with response elements for other DNA binding protein
domains such as the GAL-4 protein from yeast (see Sadowski et al.,
Nature 335:563 (1988) or LexA protein from E. coli (see Brent et
al., Cell 43:729 (1985)) to accommodate chimeric EcR complexes.
Another advantage of chimeric systems is that they allow choice of
a promoter used to drive the exogenous gene according to a desired
end result. Such double control can be particularly important in
areas of gene therapy, especially when cytotoxic proteins are
produced, because both the timing of expression as well as the
cells wherein expression occurs can be controlled. When exogenous
genes, operatively linked to a suitable promoter, are introduced
into the cells of the subject, expression of the exogenous genes is
controlled by the presence of the ligand of this invention.
Promoters may be constitutively or inducibly regulated or may be
tissue-specific (that is, expressed only in a particular type of
cell) or specific to certain developmental stages of the
organism.
[0162] Numerous genomic and cDNA nucleic acid sequences coding for
a variety of polypeptides, such as transcription factors and
reporter proteins, are well known in the art. Those skilled in the
art have access to nucleic acid sequence information for virtually
all known genes and can either obtain the nucleic acid molecule
directly from a public depository, the institution that published
the sequence, or employ routine methods to prepare the molecule.
See for example the description of the sequence accession numbers,
infra.
[0163] The gene switch may be any gene switch system that regulates
gene expression by addition or removal of a specific ligand. In one
embodiment, the gene switch is one in which the level of gene
expression is dependent on the level of ligand that is present.
Examples of ligand-dependent transcription factors that may be used
in the gene switches of the invention include, without limitation,
members of the nuclear receptor superfamily activated by their
respective ligands (e.g., glucocorticoid, estrogen, progestin,
retinoid, ecdysone, and analogs and mimetics thereof) and rTTA
activated by tetracycline. In one aspect of the invention, the gene
switch is an EcR-based gene switch. Examples of such systems
include, without limitation, the systems described in U.S. Pat.
Nos. 6,258,603, 7,045,315, U.S. Published Patent Application Nos.
2006/0014711, 2007/0161086, and International Published Application
No. WO 01/70816. Examples of chimeric ecdysone receptor systems are
described in U.S. Pat. No. 7,091,038, U.S. Published Patent
Application Nos. 2002/0110861, 2004/0033600, 2004/0096942,
2005/0266457, and 2006/0100416, and International Published
Application Nos. WO 01/70816, WO 02/066612, WO 02/066613, WO
02/066614, WO 02/066615, WO 02/29075, and WO 2005/108617. An
example of a non-steroidal ecdysone agonist-regulated system is the
RheoSwitch.RTM. Mammalian Inducible Expression System (New England
Biolabs, Ipswich, Mass.).
[0164] In one embodiment, a polynucleotide encoding the gene switch
comprises a single transcription factor sequence encoding a
ligand-dependent transcription factor under the control of a
promoter. The transcription factor sequence may encode a
ligand-dependent transcription factor that is a naturally occurring
or an artificial transcription factor. An artificial transcription
factor is one in which the natural sequence of the transcription
factor has been altered, e.g., by mutation of the sequence or by
the combining of domains from different transcription factors. In
one embodiment, the transcription factor comprises a Group H
nuclear receptor ligand binding domain (LBD). In one embodiment,
the Group H nuclear receptor LBD is from an EcR, a ubiquitous
receptor, an orphan receptor 1, a NER-1, a steroid hormone nuclear
receptor 1, a retinoid X receptor interacting protein-15, a liver X
receptor .beta., a steroid hormone receptor like protein, a liver X
receptor, a liver X receptor .alpha., a farnesoid X receptor, a
receptor interacting protein 14, or a farnesol receptor. In another
embodiment, the Group H nuclear receptor LBD is from an ecdysone
receptor.
[0165] The EcR and the other Group H nuclear receptors are members
of the nuclear receptor superfamily wherein all members are
generally characterized by the presence of an amino-terminal
transactivation domain (TD), a DNA binding domain (DBD), and a LBD
separated from the DBD by a hinge region. As used herein, the term
"DNA binding domain" comprises a minimal polypeptide sequence of a
DNA binding protein, up to the entire length of a DNA binding
protein, so long as the DNA binding domain functions to associate
with a particular response element. Members of the nuclear receptor
superfamily are also characterized by the presence of four or five
domains: A/B, C, D, E, and in some members F (see U.S. Pat. No.
4,981,784 and Evans, Science 240:889 (1988)). The "A/B" domain
corresponds to the transactivation domain, "C" corresponds to the
DNA binding domain, "D" corresponds to the hinge region, and "E"
corresponds to the ligand binding domain. Some members of the
family may also have another transactivation domain on the
carboxy-terminal side of the LBD corresponding to "F".
[0166] The DBD is characterized by the presence of two cysteine
zinc fingers between which are two amino acid motifs, the P-box and
the D-box, which confer specificity for response elements. These
domains may be either native, modified, or chimeras of different
domains of heterologous receptor proteins. The EcR, like a subset
of the nuclear receptor family, also possesses less well-defined
regions responsible for heterodimerization properties. Because the
domains of nuclear receptors are modular in nature, the LBD, DBD,
and TD may be interchanged.
[0167] In another embodiment, the transcription factor comprises a
TD, a DBD that recognizes a response element associated with the
exogenous gene whose expression is to be modulated; and a Group H
nuclear receptor LBD. In certain embodiments, the Group H nuclear
receptor LBD comprises a substitution mutation.
[0168] In other embodiments, a polynucleotide encoding the gene
switch comprises a first transcription factor sequence under the
control of a first promoter and a second transcription factor
sequence under the control of a second promoter, wherein the
proteins encoded by said first transcription factor sequence and
said second transcription factor sequence interact to form a
protein complex which functions as a ligand-dependent transcription
factor, i.e., a "dual switch"- or "two-hybrid"-based gene switch.
The first and second promoters may be the same or different.
[0169] A polynucleotide encoding a gene switch may also comprise a
first transcription factor sequence and a second transcription
factor sequence under the control of one promoter, wherein the
proteins encoded by the first transcription factor sequence and the
second transcription factor sequence interact to form a protein
complex which functions as a ligand-dependent transcription factor,
i.e., a "single gene switch." The first transcription factor
sequence and the second transcription factor sequence may be
connected by an internal ribosomal entry site, e.g., EMCV IRES.
[0170] In one embodiment, the first transcription factor sequence
encodes a polypeptide comprising a TD, a DBD that recognizes a
response element associated with the exogenous gene whose
expression is to be modulated; and a Group H nuclear receptor LBD,
and the second transcription factor sequence encodes a
transcription factor comprising a nuclear receptor LBD selected
from a vertebrate RXR LBD, an invertebrate RXR LBD, an
ultraspiracle protein LBD, and a chimeric LBD comprising two
polypeptide fragments, wherein the first polypeptide fragment is
from a vertebrate RXR LBD, an invertebrate RXR LBD, or an
ultraspiracle protein LBD, and the second polypeptide fragment is
from a different vertebrate RXR LBD, invertebrate RXR LBD, or
ultraspiracle protein LBD.
[0171] In another embodiment, the gene switch comprises a first
transcription factor sequence encoding a first polypeptide
comprising a nuclear receptor LBD and a DBD that recognizes a
response element associated with the exogenous gene whose
expression is to be modulated, and a second transcription factor
sequence encoding a second polypeptide comprising a TD and a
nuclear receptor LBD, wherein one of the nuclear receptor LBDs is a
Group H nuclear receptor LBD. In a preferred embodiment, the first
polypeptide is substantially free of a TD and the second
polypeptide is substantially free of a DBD. For purposes of the
invention, "substantially free" means that the protein in question
does not contain a sufficient sequence of the domain in question to
provide activation or binding activity.
[0172] In another aspect of the invention, the first transcription
factor sequence encodes a protein comprising a heterodimer partner
and a TD and the second transcription factor sequence encodes a
protein comprising a DBD and a LBD.
[0173] When only one nuclear receptor LBD is a Group H LBD, the
other nuclear receptor LBD may be from any other nuclear receptor
that forms a dimer with the Group H LBD. For example, when the
Group H nuclear receptor LBD is an EcR LBD, the other nuclear
receptor LBD "partner" may be from an EcR, a vertebrate RXR, an
invertebrate RXR, an ultraspiracle protein (USP), or a chimeric
nuclear receptor comprising at least two different nuclear receptor
LBD polypeptide fragments selected from a vertebrate RXR, an
invertebrate RXR, and a USP (see WO 01/70816 A2, International
Patent Application No. PCT/US02/05235 and US 2004/0096942 A1. The
"partner" nuclear receptor ligand binding domain may further
comprise a truncation mutation, a deletion mutation, a substitution
mutation, or another modification.
[0174] In one embodiment, the vertebrate RXR LBD is from a human
Homo sapiens, mouse Mus musculus, rat Rattus norvegicus, chicken
Gallus gallus, pig Sus scrofa domestica, frog Xenopus laevis,
zebrafish Danio rerio, tunicate Polyandrocarpa misakiensis, or
jellyfish Tripedalia cysophora RXR.
[0175] In one embodiment, the invertebrate RXR ligand binding
domain is from a locust Locusta migratoria ultraspiracle
polypeptide ("LmUSP"), an ixodid tick Amblyomma americanum RXR
homolog 1 ("AmaRXR1"), an ixodid tick Amblyomma americanum RXR
homolog 2 ("AmaRXR2"), a fiddler crab Celuca pugilator RXR homolog
("CpRXR"), a beetle Tenebrio molitor RXR homolog ("TmRXR"), a
honeybee Apis mellifera RXR homolog ("AmRXR"), an aphid Myzus
persicae RXR homolog ("MpRXR"), or a non-Dipteran/non-Lepidopteran
RXR homolog.
[0176] In one embodiment, the chimeric RXR LBD comprises at least
two polypeptide fragments selected from a vertebrate species RXR
polypeptide fragment, an invertebrate species RXR polypeptide
fragment, and a non-Dipteran/non-Lepidopteran invertebrate species
RXR homolog polypeptide fragment. A chimeric RXR ligand binding
domain for use in the invention may comprise at least two different
species RXR polypeptide fragments, or when the species is the same,
the two or more polypeptide fragments may be from two or more
different isoforms of the species RXR polypeptide fragment.
[0177] In one embodiment, the chimeric RXR ligand binding domain
comprises at least one vertebrate species RXR polypeptide fragment
and one invertebrate species RXR polypeptide fragment.
[0178] In another embodiment, the chimeric RXR ligand binding
domain comprises at least one vertebrate species RXR polypeptide
fragment and one non-Dipteran/non-Lepidopteran invertebrate species
RXR homolog polypeptide fragment.
[0179] The ligand, when combined with the LBD of the nuclear
receptor(s), which in turn are bound to the response element linked
to the exogenous gene, provides external temporal regulation of
expression of the exogenous gene. The binding mechanism or the
order in which the various components of this invention bind to
each other, that is, for example, ligand to LBD, DBD to response
element, TD to promoter, etc., is not critical.
[0180] In a specific example, binding of the ligand to the LBD of a
Group H nuclear receptor and its nuclear receptor LBD partner
enables expression of the exogenous gene. This mechanism does not
exclude the potential for ligand binding to the Group H nuclear
receptor (GHNR) or its partner, and the resulting formation of
active homodimer complexes (e.g. GHNR+GHNR or partner+partner).
Preferably, one or more of the receptor domains is varied producing
a hybrid gene switch. Typically, one or more of the three domains,
DBD, LBD, and TD, may be chosen from a source different than the
source of the other domains so that the hybrid genes and the
resulting hybrid proteins are optimized in the chosen host cell or
organism for transactivating activity, complementary binding of the
ligand, and recognition of a specific response element. In
addition, the response element itself can be modified or
substituted with response elements for other DNA binding protein
domains such as the GAL-4 protein from yeast (see Sadowski et al.,
Nature 335:563 (1988)) or LexA protein from Escherichia coli (see
Brent et al., Cell 43:729 (1985)), or synthetic response elements
specific for targeted interactions with proteins designed,
modified, and selected for such specific interactions (see, for
example, Kim et al., Proc. Natl. Acad. Sci. USA, 94:3616 (1997)) to
accommodate hybrid receptors.
[0181] The functional EcR complex may also include additional
protein(s) such as immunophilins. Additional members of the nuclear
receptor family of proteins, known as transcriptional factors (such
as DHR38 or betaFTZ-1), may also be ligand dependent or independent
partners for EcR, USP, and/or RXR. Additionally, other cofactors
may be required such as proteins generally known as coactivators
(also termed adapters or mediators). These proteins do not bind
sequence-specifically to DNA and are not involved in basal
transcription. They may exert their effect on transcription
activation through various mechanisms, including stimulation of
DNA-binding of activators, by affecting chromatin structure, or by
mediating activator-initiation complex interactions. Examples of
such coactivators include RIP140, TIF1, RAP46/Bag-1, ARA70,
SRC-1/NCoA-1, TIF2/GRIP/NCoA-2, ACTR/A1B1/RAC3/pCIP as well as the
promiscuous coactivator C response element B binding protein,
CBP/p300 (for review see Glass et al., Curr. Opin. Cell Biol. 9:222
(1997)). Also, protein cofactors generally known as corepressors
(also known as repressors, silencers, or silencing mediators) may
be required to effectively inhibit transcriptional activation in
the absence of ligand. These corepressors may interact with the
unliganded EcR to silence the activity at the response element.
Current evidence suggests that the binding of ligand changes the
conformation of the receptor, which results in release of the
corepressor and recruitment of the above described coactivators,
thereby abolishing their silencing activity. Examples of
corepressors include N-CoR and SMRT (for review, see Horwitz et
al., Mol Endocrinol. 10:1167 (1996)). These cofactors may either be
endogenous within the cell or organism, or may be added exogenously
as transgenes to be expressed in either a regulated or unregulated
fashion.
[0182] The exogenous gene is operably linked to a promoter
comprising at least one response element that is recognized by the
DBD of the ligand-dependent transcription factor encoded by the
gene switch. In one embodiment, the promoter comprises 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, or more copies of the response element.
Promoters comprising the desired response elements may be naturally
occurring promoters or artificial promoters created using
techniques that are well known in the art, e.g., one or more
response elements operably linked to a minimal promoter.
[0183] To introduce the polynucleotides into the cells, a vector
can be used. The vector may be, for example, a plasmid vector or a
single-or double-stranded RNA or DNA viral vector. Such vectors may
be introduced into cells by well-known techniques for introducing
DNA and RNA into cells. Viral vectors may be replication competent
or replication defective. In the latter case, viral propagation
generally will occur only in complementing host cells. As used
herein, the term "host cell" or "host" is used to mean a cell of
the invention that is harboring one or more polynucleotides of the
invention.
[0184] Thus, at a minimum, the vectors must include the
polynucleotides of the invention. Other components of the vector
may include, but are not limited to, selectable markers, chromatin
modification domains, additional promoters driving expression of
other polypeptides that may also be present on the vector (e.g., a
lethal polypeptide), genomic integration sites, recombination
sites, and molecular insertion pivots. The vectors may comprise any
number of these additional elements, either within or not within
the polynucleotides, such that the vector can be tailored to the
specific goals of the therapeutic methods desired.
[0185] In one embodiment of the invention, the vectors that are
introduced into the cells further comprise a "selectable marker
gene" which, when expressed, indicates that the gene switch
construct of the invention has been integrated into the genome of
the host cell. In this manner, the selector gene can be a positive
marker for the genome integration. While not critical to the
methods of the invention, the presence of a selectable marker gene
allows the practitioner to select for a population of live cells
where the vector construct has been integrated into the genome of
the cells. Thus, certain embodiments of the invention comprise
selecting cells where the vector has successfully been integrated.
As used herein, the term "select" or variations thereof, when used
in conjunction with cells, is intended to mean standard, well-known
methods for choosing cells with a specific genetic make-up or
phenotype. Typical methods include, but are not limited to,
culturing cells in the presence of antibiotics, such as G418,
neomycin and ampicillin. Other examples of selectable marker genes
include, but are not limited to, genes that confer resistance to
dihydrofolate reductase, hygromycin, or mycophenolic acid. Other
methods of selection include, but are not limited to, a selectable
marker gene that allows for the use of thymidine kinase,
hypoxanthine-guanine phosphoribosyltransferase or adenine
phosphoribosyltransferase as selection agents. Cells comprising a
vector construct comprising an antibiotic resistance gene or genes
would then be capable of tolerating the antibiotic in culture.
Likewise, cells not comprising a vector construct comprising an
antibiotic resistance gene or genes would not be capable of
tolerating the antibiotic in culture.
[0186] As used herein, a "chromatin modification domain" (CMD)
refers to nucleotide sequences that interact with a variety of
proteins associated with maintaining and/or altering chromatin
structure, such as, but not limited to, DNA insulators. See
Ciavatta et al., Proc. Nat'l Acad Sci. U.S.A., 103:9958 (2006).
Examples of CMDs include, but are not limited to, the chicken
.beta.-globulin insulator and the chicken hypersensitive site 4
(cHS4). The use of different CMD sequences between one or more gene
programs (i.e., a promoter, coding sequence, and 3' regulatory
region), for example, can facilitate the use of the differential
CMD DNA sequences as "mini homology arms" in combination with
various microorganism or in vitro recombineering technologies to
"swap" gene programs between existing multigenic and monogenic
shuttle vectors. Other examples of chromatin modification domains
are known in the art or can be readily identified.
[0187] Particular vectors for use with the invention are expression
vectors that code for proteins or polynucleotides. Generally, such
vectors comprise cis-acting control regions effective for
expression in a host operatively linked to the polynucleotide to be
expressed. Appropriate trans-acting factors are supplied by the
host, supplied by a complementing vector or supplied by the vector
itself upon introduction into the host.
[0188] A great variety of expression vectors can be used to express
proteins or polynucleotides. Such vectors include chromosomal,
episomal and virus-derived vectors, e.g., vectors derived from
bacterial plasmids, from bacteriophage, from yeast episomes, from
yeast chromosomal elements, from viruses such as adeno-associated
viruses, lentiviruses, baculoviruses, papova viruses, such as SV40,
vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies
viruses and retroviruses, and vectors derived from combinations
thereof, such as those derived from plasmid and bacteriophage
genetic elements, such as cosmids and phagemids. All may be used
for expression in accordance with this aspect of the invention.
Generally, any vector suitable to maintain, propagate or express
polynucleotides or proteins in a host may be used for expression in
this regard.
[0189] The polynucleotide sequence in the expression vector is
operatively linked to appropriate expression control sequence(s)
including, for instance, a promoter to direct mRNA transcription.
Representatives of additional promoters include, but are not
limited to, constitutive promoters and tissue specific or inducible
promoters. Examples of constitutive eukaryotic promoters include,
but are not limited to, the promoter of the mouse metallothionein I
gene (Hamer et al., J. Mol. Appl. Gen. 1:273 (1982)); the TK
promoter of Herpes virus (McKnight, Cell 31:355 (1982)); the SV40
early promoter (Benoist et al., Nature 290:304 (1981)); and the
vaccinia virus promoter. Additional examples of the promoters that
could be used to drive expression of a protein or polynucleotide
include, but are not limited to, tissue-specific promoters and
other endogenous promoters for specific proteins, such as the
albumin promoter (hepatocytes), a proinsulin promoter (pancreatic
beta cells) and the like. In general, expression constructs will
contain sites for transcription, initiation and termination and, in
the transcribed region, a ribosome binding site for translation.
The coding portion of the mature transcripts expressed by the
constructs may include a translation initiating AUG at the
beginning and a termination codon (UAA, UGA or UAG) appropriately
positioned at the end of the polypeptide to be translated.
[0190] In addition, the constructs may contain control regions that
regulate, as well as engender expression. Generally, such regions
will operate by controlling transcription, such as repressor
binding sites and enhancers, among others.
[0191] Examples of eukaryotic vectors include, but are not limited
to, pW-LNEO, pSV2CAT, pOG44, pXT1 and pSG available from
Stratagene; pSVK3, pBPV, pMSG and pSVL available from Amersham
Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2,
pDsRed2-Mito, and pCMV-EGFP available from Clontech. Many other
vectors are well-known and commercially available.
[0192] Particularly useful vectors, which comprise molecular
insertion pivots for rapid insertion and removal of elements of
gene programs, are described in United States Published Patent
Application No. 2004/0185556, U.S. patent application Ser. No.
11/233,246 and International Published Application Nos. WO
2005/040336 and WO 2005/116231. An example of such vectors is the
UltraVector.TM. Production System (Intrexon Corp., Blacksburg,
Va.), as described in WO 2007/038276. As used herein, a "gene
program" is a combination of genetic elements comprising a promoter
(P), an expression sequence (E) and a 3' regulatory sequence (3),
such that "PE3" is a gene program. The elements within the gene
program can be easily swapped between molecular pivots that flank
each of the elements of the gene program. A molecular pivot, as
used herein, is defined as a polynucleotide comprising at least two
non-variable rare or uncommon restriction sites arranged in a
linear fashion. In one embodiment, the molecular pivot comprises at
least three non-variable rare or uncommon restriction sites
arranged in a linear fashion. Typically any one molecular pivot
would not include a rare or uncommon restriction site of any other
molecular pivot within the same gene program. Cognate sequences of
greater than 6 nucleotides upon which a given restriction enzyme
acts are referred to as "rare" restriction sites. There are,
however, restriction sites of 6 bp that occur more infrequently
than would be statistically predicted, and these sites and the
endonucleases that cleave them are referred to as "uncommon"
restriction sites. Examples of either rare or uncommon restriction
enzymes include, but are not limited to, AsiS I, Pac I, Sbf I, Fse
I, Asc I, Mlu I, SnaB I, Not I, Sal I, Swa I, Rsr II, BSiW I, Sfo
I, Sgr AI, AflIII, Pvu I, Ngo MW, Ase I, Flp I, Pme I, Sda I, Sgf
I, Srf I, and Sse8781 I.
[0193] The vector may also comprise restriction sites for a second
class of restriction enzymes called homing endonuclease (HE)
enzymes. HE enzymes have large, asymmetric restriction sites (12-40
base pairs), and their restriction sites are infrequent in nature.
For example, the HE known as I-SceI has an 18 bp restriction site
(5TAGGGATAACAGGGTAAT3' (SEQ ID NO:12)), predicted to occur only
once in every 7.times.10.sup.10 base pairs of random sequence. This
rate of occurrence is equivalent to only one site in a genome that
is 20 times the size of a mammalian genome. The rare nature of HE
sites greatly increases the likelihood that a genetic engineer can
cut a gene program without disrupting the integrity of the gene
program if HE sites were included in appropriate locations in a
cloning vector plasmid.
[0194] Selection of appropriate vectors and promoters for
expression in a host cell is a well-known procedure, and the
requisite techniques for vector construction and introduction into
the host, as well as its expression in the host are routine skills
in the art.
[0195] The introduction of the polynucleotides into the cells can
be a transient transfection, stable transfection, or can be a
locus-specific insertion of the vector. Transient and stable
transfection of the vectors into the host cell can be effected by
calcium phosphate transfection, DEAE-dextran mediated transfection,
cationic lipid-mediated transfection, electroporation,
transduction, infection, or other methods. Such methods are
described in many standard laboratory manuals, such as Davis et
al., Basic Methods in Molecular Biology (1986); Keown et al., 1990,
Methods Enzymol. 185: 527-37; Sambrook et al., 2001, Molecular
Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor
Laboratory Press, N.Y. These stable transfection methods result in
random insertion of the vector into the genome of the cell.
Further, the copy number and orientation of the vectors are also,
generally speaking, random.
[0196] In one embodiment of the invention, the vector is inserted
into a bio-neutral site in the genome. A bio-neutral site is a site
in the genome where insertion of the polynucleotides interferes
very little, if any, with the normal function of the cell.
Bio-neutral sites may be analyzed using available bioinformatics.
Many bio-neutral sites are known in the art, e.g., the
ROSA-equivalent locus. Other bio-neutral sites may be identified
using routine techniques well known in the art. Characterization of
the genomic insertion site(s) is performed using methods known in
the art. To control the location, copy number and/or orientation of
the polynucleotides when introducing the vector into the cells,
methods of locus-specific insertion may be used. Methods of
locus-specific insertion are well-known in the art and include, but
are not limited to, homologous recombination and
recombinase-mediated genome insertion. Of course, if locus-specific
insertion methods are to be used in the methods of the invention,
the vectors may comprise elements that aid in this locus-specific
insertion, such as, but not limited to, homologous recombination.
For example, the vectors may comprise one, two, three, four or more
genomic integration sites (GISs). As used herein, a "genomic
integration site" is defined as a portion of the vector sequence
which nucleotide sequence is identical or nearly identical to
portions of the genome within the cells that allows for insertion
of the vector in the genome. hi particular, the vector may comprise
two genomic insertion sites that flank at least the
polynucleotides. Of course, the GISs may flank additional elements,
or even all elements present on the vector.
[0197] In another embodiment, locus-specific insertion may be
carried out by recombinase-site specific gene insertion. Briefly,
bacterial recombinase enzymes, such as, but not limited to, PhiC31
integrase can act on "pseudo" recombination sites within the human
genome. These pseudo recombination sites can be targets for
locus-specific insertion using the recombinases. Recombinase-site
specific gene insertion is described in Thyagarajan et al., Mol.
Cell Biol. 21:3926 (2001). Other examples of recombinases and their
respective sites that may be used for recombinase-site specific
gene insertion include, but are not limited to, serine recombinases
such as R4 and TP901-1 and recombinases described in WO
2006/083253.
[0198] In a further embodiment, the vector may comprise a
chemo-resistance gene, e.g., the multidrug resistance gene mdrl,
dihydrofolate reductase, or O.sup.6-alkylguanine-DNA
alkyltransferase. The chemo-resistance gene may be under the
control of a constitutive (e.g., CMV) or inducible (e.g.,
RheoSwitch.RTM.) promoter. In this embodiment, if it is desired to
treat a disease in a subject while maintaining the modified cells
within the subject, a clinician may apply a chemotherapeutic agent
to destroy diseased cells while the modified cells would be
protected from the agent due to expression of a suitable
chemo-resistance gene and may continue to be used for treatment,
amelioration, or prevention of a disease or disorder. By placing
the chemo-resistance gene under an inducible promoter, the
unnecessary expression of the chemo-resistance gene can be avoided,
yet it will still be available in case continued treatment is
needed. If the modified cells themselves become diseased, they
could still be destroyed by inducing expression of a lethal
polypeptide as described below.
[0199] The methods of the invention are carried out by introducing
the polynucleotides encoding the gene switch and the exogenous gene
into cells of a subject. Any method known for introducing a
polynucleotide into a cell known in the art, such as those
described above, can be used.
[0200] When the polynucleotides are to be introduced into cells ex
vivo, the cells may be obtained from a subject by any technique
known in the art, including, but not limited to, biopsies,
scrapings, and surgical tissue removal. The isolated cells may be
cultured for a sufficient amount of time to allow the
polynucleotides to be introduced into the cells, e.g., 2, 4, 6, 8,
10, 12, 18, 24, 36, 48, hours or more. Methods for culturing
primary cells for short periods of time are well known in the art.
For example, cells may be cultured in plates (e.g., in microwell
plates) either attached or in suspension.
[0201] For ex vivo therapeutic methods, cells are isolated from a
subject and cultured under conditions suitable for introducing the
polynucleotides into the cells. Once the polynucleotides have been
introduced into the cells, the cells are incubated for a sufficient
period of time to allow the ligand-dependent transcription factor
to be expressed, e.g., 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 18,
or 24 hours or more. At some point after the introduction of the
polynucleotides into the cells (either before or after significant
levels of the ligand-dependent transcription factor is expressed),
the cells are introduced back into the subject. Reintroduction may
be carried out by any method known in the art, e.g., intravenous
infusion or direct injection into a tissue or cavity. In one
embodiment, the presence of the polynucleotides in the cells is
determined prior to introducing the cells back into the subject. In
another embodiment, cells containing the polynucleotides are
selected (e.g., based on the presence of a selectable marker in the
polynucleotides) and only those cells containing the
polynucleotides are reintroduced into the subject. After the cells
are reintroduced to the subject, ligand is administered to the
subject to induce expression of the therapeutic polypeptide or
therapeutic polynucleotide. In an alternative embodiment, the
ligand may be added to the cells even before the cells are
reintroduced to the subject such that the therapeutic polypeptide
or therapeutic polynucleotide is expressed prior to reintroduction
of the cells. The ligand may be administered by any suitable
method, either systemically (e.g., orally, intravenously) or
locally (e.g., intraperitoneally, intrathecally,
intraventricularly, direct injection into the tissue or organ where
the cells were reintroduced, for example, intratumorally). The
optimal timing of ligand administration can be determined for each
type of cell and disease or disorder using only routine
techniques.
[0202] The in vivo therapeutic methods of the invention involve
direct in vivo introduction of the polynucleotides into the cells
of the subject. The polynucleotides may be introduced into the
subject systemically or locally (e.g., at the site of the disease
or disorder). Once the polynucleotides have been introduced to the
subject, the ligand may be administered to induce expression of the
therapeutic polypeptide or therapeutic polynucleotide. The ligand
may be administered by any suitable method, either systemically
(e.g., orally, intravenously) or locally (e.g., intraperitoneally,
intrathecally, intraventricularly, or direct injection into the
tissue or organ where the disease or disorder is occurring, for
example, intratumorally). The optimal timing of ligand
administration can be determined for each type of cell and disease
or disorder using only routine techniques.
[0203] For in vivo use, the ligands described herein may be taken
up in pharmaceutically acceptable carriers, such as, for example,
solutions, suspensions, tablets, capsules, ointments, elixirs, and
injectable compositions. Pharmaceutical compositions may contain
from 0.01% to 99% by weight of the ligand. Compositions may be
either in single or multiple dose forms. The amount of ligand in
any particular pharmaceutical composition will depend upon the
effective dose, that is, the dose required to elicit the desired
gene expression or suppression.
[0204] Suitable routes of administering the pharmaceutical
preparations include oral, rectal, topical (including dermal,
buccal and sublingual), vaginal, parenteral (including
subcutaneous, intramuscular, intravenous, intradermal, intrathecal,
intratumoral, and epidural) and by naso-gastric tube. It will be
understood by those skilled in the art that the preferred route of
administration will depend upon the condition being treated and may
vary with factors such as the condition of the recipient.
[0205] As used herein, the term "rAD.RheoIL12" refers to an
adenoviral polynucleotide vector harboring the IL-12 gene under the
control of a gene switch of the RheoSwitch.RTM. Therapeutic System
(RTS), which is capable of producing IL-12 protein in the presence
of activating ligand.
[0206] As used herein, the term "IL-12p70" refers to IL-12 protein,
which naturally has two subunits commonly referred to as p40 and
p35. The term IL-12p70 encompasses fusion proteins comprising the
two subunits of IL-12 (p40 and p35), wherein the fusion protein may
include linker amino acids between subunits.
[0207] As used herein, the term "a protein having the function of
IL-12" refers to a protein that has at least 20% (e.g., at least
30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, or 100%) of any
bioactivity of human IL-12. The bioactivities of IL-12 are well
known in the art and include, without limitation, differentiation
of naive T cells into Th1 cells, stimulation of the growth and
function of T cells, production of interferon-gamma (IFN-.gamma.)
and tumor necrosis factor-alpha (TNF-.alpha.) from T and natural
killer (NK) cells, reduction of IL-4 mediated suppression of
IFN-.gamma., enhancement of the cytotoxic activity of NK cells and
CD8.sup.+ cytotoxic T lymphocytes, stimulation of the expression of
IL-12R-.beta.1 and IL-12R-.beta.2, and anti-angiogenic activity.
The term "a protein having the function of IL-12" encompasses
mutants of a wild type IL-12 sequence, wherein the wild type
sequence has been altering by one or more of addition, deletion, or
substitution of amino acids, as well as non-IL-12 proteins that
mimic one or more of the bioactivities of IL-12.
[0208] As used herein, the term "rAd.cIL12" refers to an adenoviral
polynucleotide control vector containing the IL-12 gene under the
control of a constitutive promoter.
[0209] As used herein, the terms "activating" or "activate" refer
to any measurable increase in cellular activity of a gene switch,
resulting in expression of a gene of interest (e.g., IL-12
protein).
[0210] As used herein, the terms "treating" or "treatment" of a
disease refer to executing a protocol, which may include
administering one or more drugs or in vitro engineered cells to a
mammal (human or non-human), in an effort to alleviate signs or
symptoms of the disease. Thus, "treating" or "treatment" should not
necessarily be construed to require complete alleviation of signs
or symptoms, does not require a cure, and specifically includes
protocols that have only marginal effect on the subject.
[0211] As used herein, "immune cells" include dendritic cells,
macrophages, neutrophils, mast cells, eosinophils, basophils,
natural killer cells and lymphocytes (e.g., B and T cells).
[0212] As used herein, the terms "dendritic cells" and "DC" are
interchangeably used.
[0213] As used herein, the term "therapy support cells" (TSC) are
cells that can be modified (e.g., transfected) with the vector of
the invention to deliver the one or more proteins having the
function of an immunomodulator and, optionally, a protein having
the function of IL-12, to tumor microenvironments. Such TSC
include, but are not limited to, stem cells, fibroblasts,
endothelial cells and keratinocytes.
[0214] As used herein, the terms "in vitro engineered dendritic
cells" or "in vitro engineered population of dendritic cells" or
"in vitro engineered DC" or "a population of engineered dendritic
cells" or "DC expressing IL-12" or "DC.RheoIL12" refer to dendritic
cells conditionally expressing IL-12 under the control of a gene
switch, which can be activated by activating ligand.
[0215] As used herein, the terms "in vitro engineered TSC" or "in
vitro engineered population of TSC" or "a population of engineered
TSC" or "TSC expressing an immunomodulator" or "TSC expressing
IL-12" refer to therapy support cells, e.g., stem cells,
fibroblasts, endothelial cells and keratinocytes, conditionally
expressing an immunomodulator and/or IL-12 as the case may be under
the control of a gene switch, which can be activated by activating
ligand.
[0216] As used herein, the terms "MOI" or "Multiplicity of
Infection" refer to the average number of adenovirus particles that
infect a single cell in a specific experiment (e.g., recombinant
adenovirus or control adenovirus)
[0217] As used herein, the term "tumor" refers to all benign or
malignant cell growth and proliferation either in vivo or in vitro,
whether precancerous or cancerous cells and/or tissues.
[0218] Examples of cancers that can be treated according to the
invention include breast cancer, prostate cancer, lymphoma, skin
cancer, pancreatic cancer, colon cancer, melanoma, malignant
melanoma, ovarian cancer, brain cancer, primary brain carcinoma,
head-neck cancer, glioma, glioblastoma, liver cancer, bladder
cancer, non-small cell lung cancer, head or neck carcinoma, breast
carcinoma, ovarian carcinoma, lung carcinoma, small-cell lung
carcinoma, Wilms' tumor, cervical carcinoma, testicular carcinoma,
bladder carcinoma, pancreatic carcinoma, stomach carcinoma, colon
carcinoma, prostatic carcinoma, genitourinary carcinoma, thyroid
carcinoma, esophageal carcinoma, myeloma, multiple myeloma, adrenal
carcinoma, renal cell carcinoma, endometrial carcinoma, adrenal
cortex carcinoma, malignant pancreatic insulinoma, malignant
carcinoid carcinoma, choriocarcinoma, mycosis fungoides, malignant
hypercalcemia, cervical hyperplasia, leukemia, acute lymphocytic
leukemia, chronic lymphocytic leukemia, acute myelogenous leukemia,
chronic myelogenous leukemia, chronic granulocytic leukemia, acute
granulocytic leukemia, hairy cell leukemia, neuroblastoma,
rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera, essential
thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,
soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia,
and retinoblastoma, and the like.
[0219] The invention provides engineering of DC to conditionally
express a protein having the function of IL-12 and therapeutic uses
and/or applications for the treatment of cancer or tumors or both.
In vitro engineered DC that conditionally express a protein having
the function of IL-12 are a safe improvement over constitutive
production of IL-12 protein. Additionally, the ability to control
the timing and level of IL-12 expression provides improved control
of the efficacy of the treatment. Therefore, in vitro engineered DC
may be formulated into pharmaceutical compositions as therapeutics
for the treatment of a cancer or a tumor in a human or a non-human
organism. Alternatively, in vitro engineered populations of DC or
subsets thereof may be used as vehicles to conditionally deliver
IL-12 protein production to a specific area (normal tissue, cancer,
or tumor) in the body of a human or non-human organism. The
engineered dendritic cells may also conditionally express
IFN-alpha. The dendritic cells utilitzed may be autologous or
non-autologous dendritic cells. The dendritic cells may be isolated
from bone marrow or from peripheral blood circulation. In human
patients, dendritic cell populations may be isolated via a
leukophoresis procedure, where a white blood cell fraction is
isolated and removed and other blood components are re-infused to
the patient.
[0220] The invention also provides engineering of immune cells
other than DC such as macrophages, neutrophils, mast cells,
eosinophils, basophils, natural killer cells, and lymphocytes
(e.g., B and T cells) to conditionally express a protein having the
function of IL-12 and therapeutic uses and/or applications for the
treatment of cancer or tumors or both. In vitro engineered immune
cells other than DC, e.g., macrophages, neutrophils, mast cells,
eosinophils, basophils, natural killer cells, and lymphocytes
(e.g., B and T cells) that conditionally express a protein having
the function of IL-12 are a safe improvement over constitutive
production of IL-12 protein. Additionally, the ability to control
the timing and level of IL-12 expression provides improved control
of the efficacy of the treatment. Therefore, in vitro engineered
immune cells other than DC, e.g., macrophages, neutrophils, mast
cells, eosinophils, basophils, natural killer cells, and
lymphocytes (e.g., B and T cells) may be formulated into
pharmaceutical compositions as therapeutics for the treatment of a
cancer or a tumor in a human or a non-human organism.
Alternatively, in vitro engineered populations of immune cells
other than DC, e.g., macrophages, neutrophils, mast cells,
eosinophils, basophils, natural killer cells, and lymphocytes
(e.g., B and T cells) or subsets thereof may be used as vehicles to
conditionally deliver IL-12 protein production to a specific area
(normal tissue, cancer, or tumor) in the body of a human or
non-human organism. The engineered immune cells other than DC,
e.g., macrophages, neutrophils, mast cells, eosinophils, basophils,
natural killer cells, and lymphocytes (e.g., B and T cells) may
also conditionally express IFN-alpha. The immune cells utilitzed
may be autologous or non-autologous immune cells. The immune cells
may be isolated from bone marrow or from peripheral blood
circulation. In human patients, immune cell populations may be
isolated via a leukophoresis procedure, where a white blood cell
fraction is isolated and removed and other blood components are
re-infused to the patient.
[0221] In another embodiment, the dendritic cells may be prepared
by transfecting human hematopoietic stem cells with a vector of the
invention expressing a protein having the function of IL-12, and
differentiating the transfected stem cell to give a dendritic cell.
See U.S. Pat. No. 6,734,014.
[0222] In one embodiment, a nucleic acid adenoviral vector
(rAd.RheoIL12) containing a gene switch, wherein the coding
sequences for VP16-RXR and Gal4-EcR are separated by the EMCV
internal ribosome entry site (IRES) sequence are inserted into the
adenoviral shuttle vector under the control of the human ubiquitin
C promoter. The coding sequences for the p40 and p35 subunits of
IL12 separated by an IRES sequence, and placed under the control of
a synthetic inducible promoter, are inserted upstream of the
ubiquitin C promoter.
[0223] In another embodiment, the invention provides a shuttle
vector carrying transcription units (VP16-RXR and Gal4-EcR) for the
two fusion proteins and inducible IL-12 subunits recombined with
the adenoviral backbone (AdEasyl) in E. coli BJ5183 cells. After
verifying the recombinant clone, the plasmid carrying the
rAd.RheoIL12 genome is grown in and purified from XL 10-Gold cells,
digested off the plasmid backbone and packaged by transfection into
HEK 293 cells.
[0224] In a particular embodiment, the resulting primary viral
stock is amplified by re-infection of HEK 293 cells and is purified
by CsCl density-gradient centrifugation.
[0225] In one embodiment the IL-12 gene is a wild-type IL-12 gene
sequence. In another embodiment, the IL-12 gene is a modified gene
sequence, e.g., a chimeric sequence or a sequence that has been
modified to use preferred codons.
[0226] In one embodiment, the IL-12 gene is the human wild type
IL-12 sequence. In another embodiment, the sequence is at least 85%
identical to wild type human IL-12 sequence, e.g., at least 90%,
95%, or 99% identical to wild type human IL-12. In a further
embodiment, the IL-12 gene sequence encodes the human IL-12
polypeptide. In another embodiment, the gene encodes a polypeptide
that is at least 85% identical to wild type human IL-12 e.g., at
least 90%, 95%, or 99% identical to wild type human IL-12.
[0227] In one embodiment, the IL-12 gene is the wild type mouse
IL-12 sequence. In another embodiment, the sequence is at least 85%
identical to wild type mouse IL-12, e.g., at least 90%, 95%, or 99%
identical to wild type mouse IL-12. In a further embodiment, the
IL-12 gene sequence encodes the mouse IL-12 polypeptide. In another
embodiment, the gene encodes a polypeptide that is at least 85%
identical to wild type mouse IL-12, e.g., at least 90%, 95%, or 99%
identical to wild type mouse IL-12.
[0228] The invention provides a method for producing a population
of in vitro engineered DC conditionally expressing a protein having
the function of IL-12, the method comprising the steps of: (a)
modifying at least a portion of DC, e.g., bone-marrow derived DC,
by introducing into said DC a vector harboring a gene switch
comprising a nucleic acid sequence encoding a protein having the
function of IL-12, thereby producing said population of in vitro
engineered DC that are capable of treating or preventing a
disease.
[0229] In another embodiment, the invention provides a method for
producing a population of in vitro engineered immune cells other
than DC, e.g., macrophages, neutrophils, mast cells, eosinophils,
basophils, natural killer cells and lymphocytes (e.g., B and T
cells) or TSC conditionally expressing a protein having the
function of IL-12, the method comprising the steps of: (a)
modifying at least a portion of the immune cells other than DC or
TSC by introducing into said immune cells other than DC or TSC a
vector harboring a gene switch comprising a nucleic acid sequence
encoding a protein having the function of IL-12, thereby producing
said population of in vitro engineered immune cells other than DC
or TSC that are capable of treating or preventing a disease.
[0230] In other embodiments, the invention provides isolation and
enrichment of DC, immune cells other than DC or TSC. DC may be
isolated from bone marrow from humans, mice, or other mammals. The
dendritic cells may be isolated from the blood of humans, mice or
other mammals. In human patients, dendritic cell populations may be
isolated via a leukophoresis procedure as is known in the art,
where a white blood cell fraction is isolated and removed and other
blood components are re-infused to the patient. In one embodiment,
DC are derived from murine bone marrow as previously described
(Tatsumi et al., 2003). Briefly, wild-type or EGFP Tg mouse bone
marrow (BM) is cultured in conditioned medium (CM) supplemented
with 1000 units/ml recombinant murine granulocyte/macrophage
colony-stimulating factor and recombinant mIL-4 (Peprotech, Rocky
Hill, N.J.) at 37.degree. C. in a humidified, 5% CO.sub.2 incubator
for 7 days. CD11c.sup.+ DC are then isolated, e.g., using specific
MACSTM beads, per the manufacturer's instructions (Miltenyi Biotec,
Auburn, Calif.). CD11c.sup.+ DC produced in this manner were
>95% pure based on morphology and co-expression of the CD11b,
CD40, CD80, and class I and class II MHC antigens.
[0231] One embodiment of the invention provides engineered DC
conditionally expressing a protein having the function of IL-12
suitable for therapeutic applications for the treatment of cancer,
or tumors or both as gene therapy in human or non-human organism.
In another embodiment of the invention provides engineered DC
conditionally expressing a protein having the function of IL-12
and/or a protein having the function of IFN-alpha suitable for
therapeutic applications for the treatment of cancer, or tumors or
both as gene therapy in human or non-human organism.
[0232] In an embodiment, the invention provides engineered DC
containing a gene switch.
[0233] In another embodiment, the invention encompasses method of
treating tumors in a mammal comprising administering an effective
amount of a diacylhydrazine ligand.
[0234] In another embodiment, the invention encompasses method of
treating tumors in a mammal comprising administering an effective
amount of RG-115830 or RG-115932.
[0235] In another embodiment, the invention encompasses kits
comprising dendritic cells engineered to contain a gene switch and
comprising a ligand that activates the gene switch.
[0236] In another embodiment, the invention encompasses kits
comprising dendritic cells engineered to contain a gene switch and
comprising a composition containing RG-115830 or RG-115932.
[0237] In another embodiment, the invention provides engineered DC,
immune cells other than DC, or TSC containing at least a portion of
an ecdysone receptor. In another embodiment, the invention provides
engineered DC, immune cells other than DC, or TSC containing an
ecdysone recptor-based gene switch. In another embodiment, the
invention provides engineered DC, immune cells other than DC, or
TSC containing RheoSwitch. In another embodiment, the invention
provides a kit comprising engineered DC, immune cells other than
DC, or TSC containing a gene switch and a ligand that modulates the
gene switch. In another embodiment, the kits further comprise a
diacylhydrazine ligand. In another embodiment, the kit further
comprises RG-115830 or RG-115932.
[0238] In one embodiment, the invention provides an engineered
population of DC. Day 7 cultured DC were untreated, were infected
with recombinant adenovirus encoding murine IL-12p70 driven off a
constitutive (rAd.cIL12) or inducible (rAd.RheoIL12) promoter, or
were infected with mock, control adenovirus vector (rAdy5), over a
range of multiplicity of infection (MOIs). After 48 h, infected DC
were harvested and analyzed for phenotype and for production of
IL-12p70 using a specific ELISA kit (BD-PharMingen, San Diego,
Calif.), with a lower level of detection of 62.5 pg/ml.
[0239] In another embodiment, the invention provides in vitro
engineered population of
[0240] DC, immune cells other than DC, or TSC comprising a vector,
e.g., a DNA vector, having a gene switch capable of conditionally
expressing a protein having the function of IL-12, and further
comprising activating ligand. In another embodiment, the invention
provides in vitro engineered population of DC, immune cells other
than DC, or TSC comprising a vector having a gene switch capable of
conditionally expressing a protein having the function of IL-12
and/or a protein having the function of IFN-alpha, and further
comprising activating ligand.
[0241] In a further embodiment, the invention provides a method of
treating cancer, e.g., melanoma or glioma, by administering
engineered DC, immune cells other than DC, or TSC to a patient and
then administering an activating ligand, such as RG-115919,
RG-115830 or RG-115932, to said patient. The patient may be a human
or an animal with cancer. The treatment methods and products,
engineered cells, kits, and ligands have application in human
therapy and in veterinary animal therapy. Therefore, the products
and methods are contemplated to be used for human and veterinary
animal purposes.
[0242] The invention provides that conditional expression of IL-12
protein in DC (referred to as DC.RheoIL12), immune cells other than
DC, or TSC can overcome the immunologic impact of IL-12 early
within the tumor lesion and later within tumor-draining lymph nodes
that could not be resolved with regards to therapeutic outcome with
conventional gene therapy schemes. It has further been discovered
that the timing of expression of IL-12 after administration of
engineered DC, immune cells other than DC, or TSC is critical to
the successful treatment of cancer.
[0243] In one aspect, the invention provides a pharmaceutical
composition suitable for administration to a human or a non-human
comprising a population of in vitro engineered DC, immune cells
other than DC, or TSC conditionally expressing a protein having the
function of IL-12, or conditionally expressing IL-12 and/or
IFN-alpha, wherein the formulation is suitable for administration
by intratumoral administration. The invention further provides a
pharmaceutical composition comprising an activating ligand, such as
RG-115830 or RG-115932, wherein the composition is suitable for
administration by intraperitoneal, oral, or subcutaneous
administration.
[0244] In the particular embodiment described herein, the invention
provides a method for treating a tumor, comprising:
[0245] a. administering intratumorally in a mammal the in vitro
engineered DC described above; and
[0246] b. administering to said mammal a therapeutically effective
amount of an activating ligand.
[0247] For example, the invention provides a method for treating a
tumor, comprising the steps in order of:
[0248] a. providing in vitro engineered DC;
[0249] b. administering intratumorally in a mammal said in vitro
engineered DC; and
[0250] c. administering to said mammal a therapeutically effective
amount of an activating ligand.
[0251] In another embodiment, the invention provides a method for
treating a tumor, comprising:
[0252] a. administering intratumorally in a mammal the in vitro
engineered immune cells other than dendritic cells, e.g.,
macrophages, neutrophils, mast cells, eosinophils, basophils,
natural killer cells and lymphocytes (e.g., B and T cells) or
therapy support cells, which are described above; and
[0253] b. administering to said mammal a therapeutically effective
amount of an activating ligand.
[0254] In one embodiment, the in vitro engineered DCs, immune cells
other than DC or TSCs are administered once. In another embodiment,
the DCs, immune cells other than DC or TSCs are administered more
than once if the single administration is proved to be safe and
well tolerated and additional injection(s) would benefit the
patient. The retreatment criteria is that the subject's disease is
stable or showing clinical (i.e., CT scans (regression of
tumor(s)), serum chemistry, urinanalysis, hematology, vital signs,
decrease in tumor diameter, etc.) or subjective signs (i.e.,
improved ECOG status, etc.) of improvement. The retreatment may be
initiated at 1, 2, 3, or 4 weeks, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, or 12 months, or 1, 2, 3, 4, or 5 years of the first
treatment.
[0255] The efficacy and safety of multiple doses of the transgene
may evaluated by fine needle aspiration biopsies of the tumor and
associated draining lymph nodes. These may be collected on day -12
to -7 and day 14 of the retreatment period for in vivo assessment
of transgene expression of hIL-1 2 and cellular immune response.
Biopsies may be evaluated by standard light microscopy and
immunohistochemistry to assess cellular infiltration of T cells
into the tumor and draining lymph nodes, RT-PCR on RNA may be used
with appropriately designed primers. Blood may be drawn for a serum
cytokine profile on day -12 to -7, day 8 and day 14 of the
retreatment period. A serum cytokine profile may be obtained to
determine if the expression of other cytokines is affected by
treatment with the hIL-12 transgene. Multiplex cytokine testing may
be done in the serum by Luminex for IL-12, INF-gamma, IP-10, and
other Th1/Th2 cytokine such as IL-1, TNF-alpha, IL-4, IL-5, and
IL-10.
[0256] In one embodiment, the activating ligand is administered at
substantially the same time as the in vitro engineered DC, immune
cells other than DC, or TSC, e.g., within one hour before or after
administration of the cells. In another embodiment, the activating
ligand is administered at or less than about 24 hours after
administration of the in vitro engineered DC, immune cells other
than DC, or TSC. In still another embodiment, the activating ligand
is administered at or less than about 48 hours after the in vitro
engineered DC, immune cells other than DC, or TSC. In another
embodiment, the ligand is RG-115932. In another embodiment, the
ligand is administered at a dose of about 1 to 50 mg/kg/day. In
another embodiment, the ligand is administered at a dose of about
30 mg/kg/day. In another embodiment, the ligand is administered
daily for a period of 5 to 28 days. In another embodiment, the
ligand is administered daily for a period of 14 days. In another
embodiment, about 1.times.10.sup.6 to 1.times.10.sup.8 cells are
administered. In another embodiment, about 5.times.10.sup.7 cells
are administered.
[0257] To demonstrate an effective IL-12-mediated gene therapy, a
conditional IL-12 cDNA expression system is used that allows one to
turn on IL-12 production by DC.RheoIL12 cells at various time
points post-intratumoral injection. Based on the results in the
aggressive B16 melanoma model in C57BL/6 mice, the following
conclusions were made: 1) elevated levels of IL-12 are secreted
from DC.RheoIL12 in the presence of the activating ligand RG-115830
but not in the absence of the ligand; 2) intratumoral
DC.RheoIL12-based therapy is as effective as intratumoral
DC.cIL12-based therapy as long as RG-115830 is administered to
treated animals within 24 h of DC injection (and at later time
points of ligand provision, RG-115830 therapy fails); 3) IL-12
expression in DC appears to prolong the survival of these cells in
the tumor microenvironment and is associated with higher numbers of
intratumorally-injected DC that migrate to tumor-draining lymph
nodes; and 4) the strongest immune correlate to therapy outcome is
the level of tumor-specific CD8.sup.+ T cells cross-primed by the
therapy and not the number of injected DC sustained in the tumor
microenvironment. Overall, these data suggest that DC.IL12-based
therapies likely succeed based on their positive influence on the
afferent (cross-priming) of Type-1 CD8.sup.+ T cell effectors and
not on later efferent events, such as injected DC-mediated
recruitment of anti-tumor T cells into the tumor microenvironment,
etc.
[0258] Prior to intratumoral injection, the cells (immune cells or
TSC) may be treated with a factor to stimulate the activity of the
cells. For example, the cells may be treated with a co-stimulatory
molecule such as positive co-stimulatory molecule including OX40L,
4-1BBL, CD40, CD40L, GITRL, CD70, LIGHT or ICOS-L or a negative
co-stimulatory molecule such as anti-CTLA4, anti-PD-L1 or
anti-PD-L2 antibodies. For example, the cells (e.g., DC, immune
cells or TSC) may be incubated with a cell expressing one or more
co-stimulatory molecule, e.g., J588 lymphoma cells expressing CD40
ligand molecule. In another embodiment, the cells (immune cells or
TSC) may be treated with a counter immune suppressant molecule
(tolerance inhibitor) such as anti-TGF-beta antibodies (for
inhibiting TGF signaling within the microenvironment), anti-IL10
antibodies, TGF-beta RU DN (to inhibit TGF signaling within gene
modified cells), IL-10R DN, dnFADD (to inhibit cell death pathways
within the cells), anti-SOCS1 antibodies, siRNA or decoy (to
inhibit suppressive cytokine signaling within the cells), or
anti-TGF-alpha antibodies.
[0259] IL-12 production from DC and other antigen presenting cells
acts on CD4.sup.+ and CD8.sup.+ T cells to skew them into a Th1 or
Tc1 type phenotype, respectively. Therefore, it is possible to
measure the effect of IL-12 on a population of cells by measuring
the level of expression or activity of the Th1/Tc1 type cytokine,
IFN-.gamma. in a biological sample from a patient.
[0260] For the purposes of the invention, the invention provides a
method for determining the efficacy of an in vitro engineered DC-,
immune cells other than DC- or TSC-based therapeutic regimen in a
cancer patient, comprising:
[0261] a. measuring the level of expression or the level of
activity or both of interferon-gamma (IFN-.gamma.) in a first
biological sample obtained from a human patient before
administration of in vitro engineered DC, immune cells other than
DCs, or TSC, thereby generating a control level;
[0262] b. administering intratumorally to said patient the in vitro
engineered DC, immune cells other than DCs, or TSC;
[0263] c. administering to said patient an effective amount of
activating ligand;
[0264] d. measuring the level of expression or the level of
activity or both of IFN-.gamma. in a second biological sample
obtained from said patient at a time following administration of
said activating ligand, thereby generating data for a test level;
and
[0265] e. comparing the control level to the test level of
IFN-.gamma., wherein data showing an increase in the level of
expression, activity, or both of IFN-.gamma. in the test level
relative to the control level indicates that the therapeutic
treatment regimen is effective in said patient.
[0266] In one embodiment, the invention provides a method for
determining the efficacy of an in vitro engineered immune cells
other than DC or TSC-based therapeutic regimen in a cancer patient,
comprising:
[0267] a. measuring the level of expression or the level of
activity or both of interferon-gamma (IFN-.gamma.) in a first
biological sample obtained from a human patient before
administration of in vitro engineered immune cells other than DC or
TSC, thereby generating a control level;
[0268] b. administering intratumorally to said patient in vitro
engineered immune cells other than DC or TSC;
[0269] c. administering to said patient an effective amount of
activating ligand;
[0270] d. measuring the level of expression or the level of
activity or both of IFN-.gamma. in a second biological sample
obtained from said patient at a time following administration of
said activating ligand, thereby generating data for a test level;
and
[0271] e. comparing the control level to the test level of
IFN-.gamma., wherein data showing an increase in the level of
expression, activity, or both of IFN-.gamma. in the test level
relative to the control level indicates that the therapeutic
treatment regimen is effective in said patient.
[0272] The term "subject" means an intact insect, plant or animal.
It is also anticipated that the ligands will work equally well when
the subject is a fungus or yeast. The term "subject" is meant any
subject, particularly a mammalian subject, for whom diagnosis,
prognosis, or therapy is desired. Mammal include, but are not
limited to, humans, domestic animals, farm animals, zoo animals
such as bears, sport animals, pet animals such as dogs, cats,
guinea pigs, rabbits, rats, mice, horses, cattle, bears, cows;
primates such as apes, monkeys, orangutans, and chimpanzees; canids
such as dogs and wolves; felids such as cats, lions, and tigers;
equids such as horses, donkeys, and zebras; food animals such as
cows, pigs, and sheep; ungulates such as deer and giraffes; rodents
such as mice, rats, hamsters and guinea pigs; and so on. In certain
embodiments, the animal is a human subject.
[0273] The term "animal" is intended to encompass a singular
"animal" as well as plural "animals" and comprises mammals and
birds, as well as fish, reptiles, and amphibians. The term animal
also encompasses model animals, e.g., disease model animals. In
some embodiments, the term animal includes valuable animals, either
economically or otherwise, e.g., economically important breeding
stock, racing animals, show animals, heirloom animals, rare or
endangered animals, or companion animals. In particular, the mammal
can be a human subject, a food animal or a companion animal.
[0274] As used herein, an "mammal in need thereof' refers to a
mammal for whom it is desirable to treat, i.e., to reduce the size
of a tumor or eliminate a tumor.
[0275] The method of the invention depends on the tumor antigen
capture by the intratumorally injected dendritic cells from the
tumor environment and priming the T cells in the draining lymph
nodes to develop a tumor-specific T cell response. Therefore, the
DC should be at a state of high endocytotic activity at the time of
intratumoral injection for optimal therapeutic benefit. It has been
well established that the immature DCs prepared from CD14+
monocytes by treatment with GM-CSF and IL-4 for about 6-7 days are
of immature phenotype and show high rate of endocytosis (Cella et
al. 1999; Gilboan, 2007). Maturation of DCs is associated with
suppression of the endocytic activity. IL-12 has been shown to act
on the immature DCs and signal the expression of maturation
inducing factors (Nagayama et al. 2000). Therefore, by using the
RTS, it is possible to optimize the human therapeutic outcome by
delaying the expression of IL-12 in the trasduced DC till they are
injected into the tumor. Since a constitutive expression system
lacks this ability to temporally control the expression, the
autocrine action of the IL-12 produced and the resultant course of
maturation cannot be controlled (Mazzolini et al. 2005). In
addition, the invention which will test the performance of a
regulated gene expression system in human subjects can find the
application of the system in other human gene therapy areas.
[0276] Without wishing to be bound by theory, it is expected that
the invention will support the use of intratumorally administered
in vitro engineered DC-, immune cells other than DC- or TSC-based
gene therapy in the clinical setting, focusing on the objective
clinical response as a primary study endpoint, and cross-primed
anti-tumor CD8.sup.+ T cells (producing IFN-.gamma.) as a secondary
study endpoint. Data reveals that the ability to turn the M-12
expression on and off in vivo adds an element of safety and
therapeutic control to the treatment in that both the timing and
level of IL-12 expression may be controlled by the administration
of ligand, and further that the timing of IL-12 expression is
expected to be critical to the therapeutic effectiveness of the
method.
[0277] The invention further supports the therapeutic applications
of in vitro engineered cells with conditionally expressed genes of
interest as innovative approaches for the effective and efficient
treatment of human diseases.
[0278] In the event of conflict between any teaching or suggestion
of any reference cited herein and the specification, the latter
shall prevail, for purposes of the invention.
[0279] Specific embodiments according to the methods of the
invention will now be described in the following examples, which
are provided for the purposes of illustration and are not intended
to limit the scope of the prevent invention.
EXAMPLES
Example 1
DC.RheoIL12 Conditionally Produce High Levels of IL-12p70 in
Response to Ligand RG-115830 In Vitro
1.1 Materials and Methods
[0280] 1.1.1 Mice
[0281] Female 6-8 week old C57BL/6 wild-type and
C5713L/6-TgN(ACTbEGFP)1Osb/J EGFP Tg mice were purchased from the
Jackson Laboratory (Bar Harbor, Me.) and maintained in
micro-isolator cages. Animals were handled in accordance with
recommendations for the proper care and use of laboratory
animals.
[0282] 1.1.2 Cell Lines
[0283] The B16 melanoma and EL-4 thymoma H-2b cell lines, syngenic
to C57BL/6 mice have been described previously (Itoh et al., 1994).
Cell lines were maintained in CM (RPMI 1640 supplemented with 10%
heat-inactivated fetal bovine serum, 100 units/ml penicillin, 100
.mu.g/ml streptomycin, and 10 mM L-glutamine; all reagents from
Invitrogen, Carlsbad, Calif.) in a humidified incubator at 5%
CO.sub.2 and 37.degree. C.
[0284] 1.1.3 Generation of Dendritic Cells (DC)
[0285] DCs were generated from murine bone marrow as previously
described (Tatsumi et al., 2003). Briefly, wild-type or EGFP Tg
mouse BM was cultured in CM supplemented with 1000 units/ml
recombinant murine granulocyte/macrophage colony-stimulating factor
and recombinant mIL-4 (Peprotech, Rocky Hill, N.J.) at 37.degree.
C. in a humidified, 5% CO.sub.2 incubator for 7 days. CD11c.sup.+
DC were then isolated using specific MACSTM beads, per the
manufacturer's protocol (Miltenyi Biotec, Auburn, Calif.).
CD11c.sup.+ DC produced in this manner were >95% pure based on
morphology and co-expression of the CD11b, CD40, CD80, and class I
and class II MHC antigens.
[0286] 1.1.4 Viral Vectors
[0287] The control adenoviral vector rAd..psi.5 and rAd.cIL12,
encoding mIL-12 driven off a CMV promoter (Tatsumi et al., 2003),
were produced and provided by the University of Pittsburgh Cancer
Institute's Vector Core Facility.
[0288] The rAd.RheoIL12 vector was produced in the following
manner. The coding sequences for VP16-RXR and Gal4-EcR separated by
the EMCV internal ribosome entry site (IRES) sequence were inserted
into the adenoviral shuttle vector under the control of the human
ubiquitin C promoter. Subsequently, the coding sequences for the
p40 and p35 subunits of IL12 separated by an IRES sequence, and
placed under the control of a synthetic inducible promoter, were
inserted upstream of the ubiquitin C promoter (See FIG. 1). The
performance of the system by expressing the two fusion proteins
(VP-16 RXR v. Gal4-EcR) under separate promoters of varying
strengths showed that a higher VP16-RXR to Gal4-EcR ration gave the
best performance. Thus, VP-16 RXR upstream of the IRES and Gal-4
EcR downstream gave optimal performance than the converse
design.
[0289] The shuttle vector carrying these transcription units for
the two fusion proteins and inducible IL12 subunits was recombined
with the Adenoviral backbone (AdEasy1, stratagene, La Jolla,
Calif.) in E. coli BJ5183 cells. After verifying the recombinant
clone, the plasmid carrying the rAd.RheoIL12 genome was grown in
and purified from XL10-Gold cells, digested off the plasmid
backbone and packaged by transfection into HEK 293 cells.
[0290] The resulting primary viral stock was amplified by
re-infection of HEK 293 cells and was purified by CsCl
density-gradient centrifugation.
[0291] 1.1.5 ELISA
[0292] Day 7 cultured DC were untreated, were infected with
recombinant Ads encoding murine IL-12p70 driven off a constitutive
(rAd.cIL12) or inducible (rAd.RheoIL12) promoter, or were infected
with mock, control vector rAd.psi.5, over a range of MOIs. At
various time points after this (0-48 h), DC were then cultured in
the absence or presence of an activating ligand (10-200 .mu.g/ml)
for an additional 24 h prior to analysis of IL-12p70 secretion
using a specific ELISA kit (BDPharMingen, San Diego, Calif.; lower
level of detection=62.5 pg/ml). In some cases, to discern the
stringency of conditional cytokine production, DC infected with
rAd.RheoIL12 (i.e. DC.RheoIL12), that had been pretreated with the
activating ligand, were washed free of ligand and cultured in
control media for an additional 24 h prior to analysis of IL-12p70
secretion. Alternatively, after 48 h, infected DC were harvested
and analyzed for phenotype and for production of IL-12p70 using the
ELISA kit (BD-PharMingen, San Diego, Calif.), with a lower level of
detection of 62.5 pg/ml.
[0293] 1.1.6 Flow Cytometry
[0294] For phenotypic analysis of adenovirus infected DC, PE- or
FITC-conjugated mAbs against mouse cell surface molecules (CD11b,
CD11c, CD40, CD54, CD80, CD86, H-2Kd, I-Ad (all from
BD-PharMingen)) and appropriate isotype controls were used, and
flow cytometric analysis was performed using a FACscan (Becton
Dickinson, San Jose, Calif.) flow cytometer.
1.2 Results
[0295] 1.2.1 Murine BM-Derived DC Infected with Rheo-IL12
Conditionally Produce High Levels of IL-12p70 When Treated with
Ligand In Vitro.
[0296] DC cultured from C57BL/6 (B6) mouse BM for 7 days in the
presence of rmIL-4 and rmGM-CSF were left untreated, or infected at
various MOIs with control rAd..psi.5, rAd.cIL-12 (encoding
mIL-12p70 under a constitutive CMV promoter) or rAd.RheoIL12
(encoding IL-12 p70 under a conditional promoter responsive to the
small molecule ligand RG-115830). Forty-eight hours after
infection, DC were cultured in the absence or presence of RG-115830
for an additional 24 h, at which time, culture supernatants were
harvested for quantitation of IL-12p70 production by ELISA. As
shown in FIG. 2A, control uninfected DC or DC infected with
Ad..psi.5 in the absence or presence of exogenous drug failed to
produce elevated levels of IL-12p70 when compared with DC infected
with rAd.cIL12 (DC.cIL12). DC infected with rAd.RheoIL-12
(DC.RheoIL12) only produced IL-12p70 when treated with RG-115830
(See FIGS. 2A and 2B). Based on the results of "criss-cross"
experiments, optimal infected DC production of IL-12p70 occurred
using an MOI of 100, with cells treated with 50-200 .mu.g/ml
RG-115830 (FIG. 2A). Delayed provision of RG-115830 to DC.RheoIL12
for up to 48 h did not result in any significantly reduced
production of IL-12p70 when compared to addition of this ligand at
the 0 h timepoint in vitro (FIG. 2B). Finally, removal of ligand
acutely silenced the ability of DC.RheoIL12 (previously activated
by ligand) to continue to produce elevated levels of IL-12p70 in
vitro (FIG. 2C).
Example 2
Intratumoral Administration of In Vitro Engineered Dendritic Cells
to Animals
2.1 Methods and Materials
[0297] 2.1.1 B16 Tumor Model
[0298] B6 mice received subcutaneous injection of 1.times.10.sup.5
B16 melanoma cells in the right flank on day 0. On day 7, tumors
reached a size of approximately 20-30 mm.sup.2 and mice were
treated with i.t. injections of PBS or 1.times.10.sup.6 control vs.
adenoviral transduced (MOI=100) DC in a total volume of 50 .mu.l of
PBS. Mice also received i.p. injections of 200 .mu.g RG-115830 (in
50 .mu.l DMSO) vs. DMSO carrier control that were initiated at 0 h,
24 h or 48 h post-DC administration, as indicated. After
initiation, mice received a total of 5 consecutive daily
intraperitoneal injections of RG-115830 at this dose. In additional
experiments, ligand was administered beginning on the day of DC
injection and then terminated 1, 3 or 5 days post-DC injection to
discern whether early cessation of IL-12p70 transgene promotion
reduced the therapeutic benefits of this approach. In all cases,
tumor size was assessed every 3 or 4 days and recorded in mm.sup.2
by determining the product of the largest perpendicular diameters
measured by vernier calipers. Data are reported as the average
tumor area.+-.SD. All animal cohorts contained 5 mice/group.
[0299] In indicated experiments, animals rendered tumor-free (45
days) post-therapy were rechallenged with the B16 melanoma
(10.sup.5 cells injected on the left flank, i.e. contralateral to
the original B16 challenge site) and MC38 colon carcinoma (10.sup.5
cells on the right flank) cells in order to discern the presence
and specificity of memory immunity in these mice. All data are
reported as the average tumor area.+-.SD. All animal cohorts
contained 5 mice/group.
[0300] To assess the fate and function of injected DC, day 7
BM-derived, CD11c.sup.+ DC were generated from
C57BL/6-TgN(ACTbEGFP)1Osb/J EGFP Tg mice. EGFP.sup.+ CD11c.sup.+ DC
were left uninfected or were infected with rAd viruses, as
indicated above. Forty-eight hours after infection,
1.times.10.sup.6 control or virally-infected DC were harvested,
washed in PBS, and injected into day 7 B16 tumor lesions
established in syngenic B6 mice. Three days after DC injection,
tumors and draining inguinal lymph nodes (LN) were resected, fixed
for 1 h in 2% paraformaldehyde (in PBS), and then cryoprotected in
30% sucrose in PBS before being shock frozen in liquid
nitrogen-cooled isopentane. Five micron frozen sections were then
generated and counterstained with 2 mg/ml Hoechst 33258
(Sigma-Aldrich, St. Louis, Mo.) for 3 min. The washed sections were
then mounted in Gelvatol (Monsanto Chemical Co., St. Louis, Mo.)
and observed using an Olympus BX51 microscope equipped with a
cooled charge-coupled device color camera.
[0301] 2.1.2 Assessment of Specific CM.sup.+ T Cell Responses
Against B16 Melanoma
[0302] Pooled CD8.sup.+ T cells were isolated to a purity of
>95% from the spleens of 2 treated mice/group 25 days after
tumor inoculation using magnetic bead cell sorting (MACSTM;
Miltenyi Biotec) and then co-cultured (1.times.10.sup.5/well) with
1.times.10.sup.4 irradiated (10,000 rads) B16 or EL-4 tumor cells.
After 48 h incubation, culture supernatants were collected and
analyzed for IFN-.gamma. release using a commercial ELISA
(BD-PharMingen) with a lower limit of detection of 31.5 pg/ml. Data
are reported as the mean SD of triplicate determinations.
[0303] 2.1.3 Statistical Analysis
[0304] All experiments with three or more groups in which treatment
was applied as a completely randomly design were first analyzed by
a one-way or two-way factorial ANOVA. If the resulting P was
<0.05, specific pairwise contrasts were tested with a T test
with Welch's correction for unequal variance as needed. Data were
checked for distributional properties, and appropriate
transformations were applied. Analyses of IFN-.gamma. production
from splenocyte-derived T cells were conducted with the exact
Kruskal-Wallis test. If the P for the Kruskal-Wallis test was
<0.05, priori contrasts were evaluated with the Wilcoxon test.
The analysis of therapeutic single tumor inoculation murine
treatment models was conducted with mixed linear models. Data were
log transformed, within-mouse covariance was estimated, and fixed
effects of treatment were adjusted for random mouse effects. Raw Ps
for comparing pairs of groups at a single time were adjusted by
bootstrap re-sampling. Tumor rejection rates were fit to a
generalized linear model (with binomial link) that incorporated
treatment group, day of observation, and their interaction.
2.2 Results
[0305] 2.2.1 Intratumoral Administration of DC.cIL12 Alone or
DC.RheoIL12 Combined with i.p. Administration of RG-115830 Promotes
the Regression of Established s.c. B16 Melanoma Lesions
[0306] B16 melanoma cells (1.times.10.sup.5) were injected s.c. in
the left flank of syngenic H-2.sup.b B6 mice and allowed to
establish. On day 7, mice were randomized into cohorts of 5 animals
each, with a mean cohort tumor size of approximately 20-30
mm.sup.2. Mice then received intratumoral injections of PBS or
10.sup.6 DC (pre-infected in vitro for 48 h with rAd..psi.5,
rAd.cIL12 or rAd.RheoIL12) in a total volume of 50 .mu.l PBS.
Animals also received DMSO or RG-115830 (in DMSO) intraperitoneal
injections at the time of DC administration (i.e., day 1 of
treatment) or at 24 h or 48 h post-DC administration (i.e., day 2
of treatment). As depicted in FIGS. 3A and 3B, the treatment of
mice with RG-115830 alone or DC.RheoIL12 in the absence of
RG-115830 failed to yield any therapeutic benefit. In marked
contrast, tumors treated with DC.cIL-12 or DC.RheoIL-12+RG-115830
(provided within 24 h of DC injection in concert with a 5 day
course of ligand administration) regressed in size over the
following 3 weeks. These therapies were statistically
indistinguishable based on tumor size measurements, and yielded
100% (5/5 mice) tumor regression rates in each of these instances.
Interestingly, if RG-115830 administration was delayed until 48 h
after intratumoral DC injection (at a time point when this agent
can efficiently promote IL-12p70 production from DC.RheoIL-12 in
vitro, See FIG. 2B), DC.RheoIL12-based therapy resulted in a
slightly inhibited tumor growth rate (p<0.05 for all time points
after day 10), but all animals progressed and required sacrifice by
day 30 (FIG. 3A). This suggests that therapeutic benefit of
intratumoral DC.IL12 treatment is critically dependent upon
IL-12p70 production at predominantly early time points (occurring
presumably within the tumor lesion and/or draining lymph
nodes).
[0307] Additional experiments were performed in which activating
ligand (RG-115830) was administered to DC.RheoIL-12 injected mice
for 1, 3 or 5 days post-DC injection (FIGS. 3B-3C). The results of
these studies suggest that early termination of ligand
administration impacts the anti-tumor efficacy of i.t. delivered
DC.RheoIL-12, with inhibition of tumor growth limited or ablated if
ligand is not provided to mice for approximately 5 or more days
after provision of gene-modified DC. These findings are consistent
with data provided in FIGS. 2B and 2C, and support the tight
(ligand-dependent) regulation of therapeutic impact resulting from
injected DC.RheoIL-12 in this model. Furthermore, when taken
together, the results depicted in FIG. 3 strongly suggest that
optimal anti-melanoma efficacy associated with i.t. delivery of
DC.RheoIL12 results from provision of the ligand during the d1-d5
period post-DC injection into B16 tumors.
[0308] 2.2.2 Delayed Activation of Conditional DC.RheoIL12 Therapy
is Ineffective Due to the Apparent Failure of Injected DC to
Survive In Vivo.
[0309] Our previous report (Tatsumi et al., 2003) suggested that
IL-12 gene insertion into DC promotes the enhanced survival of
these cells after injection into the tumor microenvironment and the
consequent capacity of these cells to cross-prime anti-tumor
CD8.sup.+ T cells and conceivably recruit circulating effector T
cells into the tumor microenvironment in vivo. Hence, the next
attempt was to discriminate whether the failure of DC-RheoIL12
therapy initiated (by i.p. RG-115830 administration) 48 h after DC
injection was due to the inability of DC to persist in the tumor
microenvironment, the inability of these cells to traffic to
tumor-draining LN and/or the inability of specific CD8.sup.+ T
cells to be cross-primed as a result of treatment. Experiments as
outlined in FIG. 3A were recapitulated, with 2 mice per cohort
sacrificed 72 h after intratumoral injection of DC, with the
exception that EGFP Tg (H-2.sup.b) mice were used as the source of
bone marrow for DC generation. Tumor and LN were resected and
tissue sections prepared for analysis of EGFP.sup.+ DC by
fluorescence microscopy. The remaining 3 animals/cohort were
followed until day 25, when they were sacrificed and pooled
splenocytes isolated for analysis of B16-specific CD8.sup.+ T cell
responses.
[0310] As depicted in FIG. 4, the ability to resolve EGFP.sup.+ DC
in tumor or LN 72 h after i.t. injection was strictly dependent on
the activation of the IL-12 transgene within 24-48 h of in vivo
administration of these cells. EGFP.sup.+ DC.cIL12 and DC.RheoIL12
could be readily observed in B16 lesions and were seen more rarely
within draining LNs in mice injected i.t. with DC.cIL12 or
DC.RheoIL12 (if RG-115830 was provided i.p. at 0 h or 24 h post-DC
administration). Very few or no EGFP.sup.+ DC were detectable in
tissues harvested from mice treated with control (uninfected) DC or
DC.RheoIL12 (where RG-115830 administration was delayed for 48 h
post-DC injection). When comparing the tissues isolated from mice
treated with DC.RheoIL12 and RG-115830 provided at 0 h vs. 24 h,
there were more EGFP.sup.+ DC in both the tumor (p=0.001) and
draining LN (p=0.02) when the activating drug was provided
earlier.
[0311] 2.2.3 Therapeutic Benefits of DC.RheoIL12 Administration are
Associated with the Induction of Specific CD8+ T Cells and Durable
Anti-Tumor Immunity.
[0312] Given the apparent dependency of injected DC vitality on the
timing of ligand injection, we would have predicted a superior
degree of specific CD8.sup.4 T cell cross-priming in the case of
mice receiving DC.RheoIL12 activated at 0 h vs. later time points
by RG-115830. Interestingly, while this was certainly observed for
the 0 h (DC.RheoIL-12, d1-5) vs. 48 h (DC.RheoIL-12, d3-d7)
DC.RheoIL-12 cohorts, it was not the case when comparing the 0 h
vs. 24 h (DC.RheoIL-12+L,d2-d6) DC.RheoIL12 groups (FIG. 5A).
Indeed, the in vitro splenic CD8.sup.+ T cell responses
(IFN-.gamma. secretion) against relevant B16 vs. irrelevant EL-4
tumor targets was comparable for both of these cohorts, and these
each approximated that detected in mice treated with DC.cIL12.
Overall, these CD8.sup.+ T cell response profiles appeared to
directly correlate with the therapy outcome (FIG. 3A). Similar to
FIG. 5A, splenic T cells response to the specific B16 tumor cells
vs. unrelated MC38 cells by IFN-gamma production was correlated to
the treatment outcome.
[0313] To address whether effective DC.RheoIL12-based therapy was
associated with the development of durable anti-tumor immunity,
tumor-free animals were (re)challenged with relevant B16 melanoma
cells or irrelevant MC38 colon carcinoma cells on day 45
(post-initial B16 challenge). As shown in FIG. 5B, all mice
previously cured of their melanomas exhibited specific protection
against B16 tumor cells, whereas MC38 tumor lesions grew
progressively. It shows that the dendritic cells of the invention
have additional safety and potential therapeutic control to the
treatment modality (in that both the timing and level of IL-12
expression may be controlled by the administration of ligand).
Example 3
Comparison or Ligand Route/Dose of Administration on Therapeutic
Effect
3.1 Methods and Materials
[0314] B16 melanomas were established s.c. for 7 days in the right
flanks of syngenic B6 mice. On day 7, 10.sup.6 DC.SP1-IL12 (optimal
switch identified in FIG. 10 comparison) were injected
intratumorally (i.t.). Activating ligand (RG-115932) was provided
either as an injection i.p., via oral gavage in Labrasol, or via
drug-containing chow provided ad libitum beginning on the day
preceding DC injection (and daily thereafter for 6 days). Each
cohort contained 5 animals, with tumor growth monitored every 3-4
days and reported as mean size (mm.sup.2 based on the product of
orthogonal measurements). The treatment for each cohort is
described below.
TABLE-US-00001 Cohort # Treatment Description 1 Control, No DC, No
ligand 2 Control, No DC, Ligand i.p., 50 mg/kg/day (Max) 3 Control,
No DC, chow, ad libitum (45 mg/kg/day) 4 Control, No DC, oral
gavage, 30 mg/kg/day 5 DC.SP1-IL12, No ligand 6 DC.SP1-IL12, i.p.,
1 mg/kg/day 7 DC.SP1-IL12, i.p., 3 mg/kg/day 8 DC.SP1-IL12, i.p.,
10 mg/kg/day 9 DC.SP1-IL12, i.p., 30 mg/kg/day 10 DC.SP1-IL12,
i.p., 50 mg/kg/day 11 DC.SP1-IL12, chow, ad libitum (45 mg/kg/day)
12 DC.SP1-IL12, oral gavage, 30 mg/kg/day
3.2 Results
[0315] The results show that administration of ligand alone at any
dose or via any route had no impact on B16 tumor growth (FIG. 6).
DC-SP1 i.t. therapy is effective in controlling B16 growth, but
only if ligand is applied, with all routes of ligand administration
yielding some degree of efficacy. DC-SP1 i.t. therapy using i.p.
administered ligand yielded a clear ligand dose-tumor inhibition
response pattern, with optimal anti-tumor effects at ligand doses
>30 mg/kg/day. Ligand applied to DC-SP1 i.t. therapy at a dose
of 30 mg/kg/day was equally effective if ligand is administered by
i.p. injection, oral gavage, or diet admix. An even higher dose of
ligand provided in chow was somewhat less effective. Since only the
R enantiomer (RG-115932) is capable of activating the RTS, chow
containing the racemic mixture provides only c.a. 20-22.5 mg/kg/day
of the active enantiomer. In this regard, tumor regression observed
in animals receiving racemic mixture AD via chow (i.e.,
.about.20-22.5 mg/kg/day of the active enantiomer RG-115932) was
consistent with the i.p. dose response seen with pure RG-115932 in
that the anti-tumor effect in the cohort on chow fell between that
observed with the 10 and 30 mg/kg/day i.p. RG-115932 dose groups.
These data suggest that oral administration of the ligand is
effective for inducing a therapeutic effect. The availability of
oral administration of the ligand would ease the burden of the
treatment on patients.
[0316] This Activator Drug dependent effect was associated with (1)
transgene expression in the tumor and DLN, (2) prolonged Ad-DCs
survival in the tumor microenvironment, (3) migration and
persistence of AdDCs in the DLN, and (4) induction of anti B16 CD8+
T cells.
[0317] FIG. 10 shows a comparison of the effects of different
IL-12-containing adenoviral vectors. The SP1-RheoIL-12 variant was
the most effective of the Rheoswitch-containing variants.
SP1-RheoIL-12 differs from oldRheoIL-12 in the vector backbone
(AdEasy1 vector for the oldRheoIL-12 and RAPAd vector of ViraQuest
on the Sp1-RheoIL-12). TTR-RheoIL-12 differs from oldRheoIL-12 in
that it contains a TTR minimal promoter downstream of the Gal4
response element. As FIG. 10 illustrates, SP1-RheoIL-12 was more
effective than TTR-RheoIL-12 in reducing B16 melanoma tumor
size.
[0318] FIG. 11 shows lack of B16 melanoma tumor formation after
rechallenge of mice previously treated with dendritic cells
containing recombinant adenoviral Rheoswitch inducible IL-12
(DC-SP1-RheoIL-12). This shows that B16 melanoma tumors were
prevented from growing for up to 25 days when B16 immune mice were
re-inoculated 45 days after the first inoculation with B16 cells.
Murine dendritic cells were generated from bone marrow of B6 mice
by 7 day culture in complete media (RPMI-1640, 10% FBS) containing
rmIL-4 plus rmGM-CSF. CD11c positive dendritic cells were then
isolated using specific MACS beads per manufacturer's protocol
(Miltenyi Biotech) and infected at MOI of 100 using rAd.IL-12
(RheoIL-12 vs. SP1 vs. TTR) for 24 hours prior to injection of 10E6
DC into established day 9 s.c. B16 melanoma tumors (5 mice per
group, tumor on right flank). Mice were treated or not with daily
i.p. injections of the activating ligand RG-115830 (30 mg/kg in 50
microliter DMSO) on days 0-4 post DC injection. Tumor size was
monitored every 3-4 days and is reported in mm squared as product
of orthogonal diameters. To evaluate the specificity of
therapy-associated protection, all tumor-free animals were
rechallenged with 10E5 B16 melanoma cells on the left flank versus
MC38 colon carcinoma cells on the right flank on day 45 post
initial B16 tumor challenge. MC38 tumors formed but B16 tumors did
not form.
[0319] FIG. 12 shows a comparison among numbers of dendritic cells
(DC-SP1-RheoIL-12) injected into the B16 tumor (10.sup.5, 10.sup.6,
10.sup.7) and length of time of ligand administration and tumor
regression in B16 melanoma tumor mouse model (6 days, 13 days).
FIG. 12 shows the dependence of the dose of transduced DCs injected
into the tumor and the duration of AD administration (i.p.
injection, 30 mg/kg/day) on the inhibition of tumor growth. Tumor
bearing mice were given a single intratumoral injection of AdDCs at
doses of 10.sup.5, 10.sup.6, and 10.sup.7 cells and daily i.p.
injection of Activating Ligand at a single dose of 30 mg/kg/day for
6 days or 13 days beginning on the day of the injection of 10.sup.7
cells. Also, a significantly more robust suppression of tumor
growth was observed if the Activating Ligand were provided for 13
days instead of 6 days. Ligand (RG-115932) administered for 13 days
in a row in combination with 10.sup.7 dendritic cells was effective
in causing tumor regression over a 25 day period. This suggests
that in contrast to the belief that ex vivo transduced DCs survive
for only a few days after injection into tumors, the AdDCs
expressing IL-12 under the control of the RTS are likely still
intact for more than 1 week after intratumoral injection and may
even remain alive and responsive to Activating Ligand for as much
as 13 days after injection. There was no effect of the Activating
Ligand alone (without AdDCs) on tumor growth.
[0320] In a similar experiment to FIG. 12, the Activating Ligand
was administered by oral gavage for 9 and 12 days. The anti-tumor
dose response with an oral formulation of the Activating Ligand at
different doses in Labrasol was assessed. A dose dependent
anti-tumor effect was observed and the greatest responses seen in
animals receiving 50 mg/kg/day orally for 12 days. Indeed, at all
doses tested, 13 days of the Activating Ligand treatment was
superior to 9 days of the Activating Drug treatment. This suggests
that DCs which survive and sustain IL-12 production for at least 9
to 12 days in vivo (either in the tumor microenvironment` or
lymphoid organs) are important for optimal treatment efficacy.
[0321] FIG. 13 shows that the therapy described herein was not
associated with untoward loss in animal weight due to wasting.
Wasting and weight loss is often associated with high levels of
interferon-gamma and TNF-alpha which are known to be upregulated in
response to IL-12.
[0322] B16 melanomas were established s.c. for 7 days in the right
flanks of 5 syngeneic B6 mice. On day 7, DC.SP1-IL-12 (bone marrow
derived DC infected at an MOI of 100 using the SP1 optimal switch)
were injected intratumorally (i.t.) at doses of 10E5, 10E6 or 10E7.
RG-115932 was provided by i.p. injection beginning on the day of DC
injection (and daily thereafter for either 6 days or 13 days). Each
cohort contained 5 animals, with tumor growth monitored every 3-4
days and reported as mean size (mm squared based on the product of
orthogonal measurements). Individual animal weights were also
assessed at the time of tumor measurements (FIG. 13). All animals
rendered free of disease by any therapy were rechallenged on day 50
(post-initial B16 tumor inoculation) with 10E5 B16 melanoma cells
on the opposite flank (left flank) of the original tumor and with
10E5 MC38 colon carcinoma cells on the right flank. Tumor growth
was monitored every 3-4 days and compared against growth observed
in naive (untreated) animals (see FIG. 12).
[0323] FIG. 14 shows lack of B16 melanoma tumor formation after
rechallenge of mice previously treated with dendritic cells
containing recombinant adenoviral Rheoswitch inducible IL-12 and
activator ligand RG-115932. FIG. 14 therefore shows that B16
melanoma tumors were prevented from growing for up to 24 days when
B16 immune mice were re-inoculated with B16 cells. FIG. 14 also
illustrates that B16 naive mice were not protected from tumor
formation, as were MC38 immune mice and MC38 naive mice. MC38 is a
colon carcinoma known in the art. This demonstrates the specificity
of immunization caused by the original B16 tumor injection with
dendritic cells containing recombinant adenoviral Rheoswitch
inducible IL-12.
[0324] DCs produced by differentiation of CD14+ cells are of
immature phenotype and do not produce detectable levels of IL-12
(Cella et al. 1999). FIG. 15 indicates that the murine DCs produced
by treatment of bone marrow cells with GM-CSF and IL-4 for 7 days
followed by CD11c+ selection also showed absence of detectable
IL-12 expression after transeduction with the adenoviral vector
harboring IL-12 under control of the RTS. Treatment of the
transduced cells with varying doses of the Activator Drug
(RG-115932) produced IL-12 in a dose dependent manner.
[0325] Adenoviral transduction has been reported to induce some
degree of maturation in the DCs by penton-integrin interaction that
leads to TNF-alpha production through the NFkB activation pathway.
The autocrine action of TNF-alpha is reported to be the
maturation-inducing signal for DCs in this case (Philpott et al.
2004). The short adenoviral transduction (2-3 hours) used and
choosing the MOI to the minimal required levels are expected to
limit this early maturation effect.
Example 4
[0326] In this example, dendritic cells were isolated from bone
marrow, transduced with the Adenoviral constructs depicted in FIG.
7, and mice bearing syngeneic intracranial GL261 gliomas were
intratumorally injected with engineered dendritic cells; and
RG-115830 was injected intra-peritoneal. FIG. 7 shows the results
of intratumoral injection of mouse intracranial glioma GL261 with
dendritic cells transduced with polynucleotides encoding IL-12
and/or IFN-alpha under the control of RTS or lacking RTS. The data
reveal that ligand induced expression of IFN-alpha and IL-12
through activation of the RTS with RG-115830 ligand promoted 75
percent survival at 50 days of GL261 glioma mice; as compared to
IFN-alpha expression alone. Furthermore, the control provided by
the RheoSwitch and ligand promoted enhanced survival.
Example 5
[0327] The safety, tolerance, transgene function, and immunological
effects of intratumoral injection(s) of adenoviral transduced
autologous dendritic cells engineered to express hIL-12 under
control of the RTS in subjects with stage III and IV melanoma will
be evaluated through procedures such as those described below.
[0328] A study involving study subjects with stage III and IV
melanoma will be conducted in 4 cohorts (groups) of subjects each
subject receiving a single intratumoral injection (into a melanoma
tumor) of adenoviral transduced autologous (reinserted into the
same subject that they came from) dendritic cells (DCs) engineered
to express human interleukin-12 (hIL-12) at a dose of
5.times.10.sup.7 in combination with daily oral doses of activator
drug (activating ligand). The study will use injections of
dendritic cells transduced ex vivo (after the cells are removed
from the subjects) with adenoviral vector for inducible expression
of human IL-12. The IL-12 production is "turned on" (induced) from
the injected DCs through the activation of the RTS by the oral
administration of the activator drug (RG-115932). Safety and
tolerance will be assessed through physical examinations (including
ECOG performance status), vital signs measurements, serum
chemistry, urinalysis, hematology, adverse events "side-effects",
and antibodies and cellular immune response to the adenovirus,
components of RTS, and the Activator Drug. To evaluate progress,
single dose and steady-state pharmacokinetics/ADME of oral
Activator Drug and its major metabolites, analysis of hIL-12 levels
and cellular immune response (T cells) in biopsies of the target
tumors, draining lymph nodes, and peripheral circulation, as well
as a serum cytokine profile will be measured.
[0329] For instance, 16 subjects with stage III and IV melanoma are
divided into four cohorts with cohorts 1 and 2 containing three
subjects and cohorts 3 and 4 containing 5 subjects. All subjects
will receive a single intratumoral injection of 5.times.10.sup.7
autologous DC transduced with adenoviral vector encoding human
IL-12 under the RTS control. The subjects will receive a single
daily oral dose of activator drug (cohort 1: 0.01 mg/kg, cohort 2:
0.1 mg/kg, cohort 3: 1.0 mg/kg or cohort 4: 3 mg/kg) the first does
starting approximately 3 hours prior to the DC injection on day 1
and continuing for 13 more consecutive days. Additional
injection(s) of adenovirally transduced autologous dendritic cells
in combination with 14 single (once) daily oral doses of activator
drug may be administered to eligible subjects who meet the criteria
for retreatment. Safety, tolerance, and dendritic cell function are
assessed for all subjects in each group of cohort 1 for up to one
month after injection of the in vitro engineered dendritic cells
before enrolling subjects to receive the next highest dose of the
activator drug. The safety assessment will continue in all subjects
for 3 months after the initial injection of the engineered
dendritic cells with the possibility of extending the follow-up
period to a total of six months to monitor subject safety if
toxicity is observed or the subject receives additional
injection(s) of the dendritic cells.
[0330] Such a study demonstrates the safety and tolerance of a
single or multiple intratumoral injection(s) of adenoviral
transduced autologous dendritic cells in combination with an oral
activator drug in subjects with melanoma. The study provides
steady-state pharmacokinetics/ADME of the oral activator drug. The
study demonstrates functionality of the RTS in subjects by
measuring hIL-12 expression of adenovirus transduced autologous
dendritic cells in target tumor and/or draining lymph nodes in
response to the activation of the RTS by the oral administration of
the activator drug. Furthermore, the study demonstrates the
immunological effects of the adenoviral transduced autologous
dendritic cells in terms of the cellular immune response in the
target tumor, draining lymph nodes, and peripheral circulation
following oral administration of the activator drug.
[0331] Melanoma is selected as an exemplary cancer (for use with
the RTS) because stage III and IV patients have no viable therapies
available, melanoma in particular among solid tumors has been shown
to respond to immunotherapy approaches, and melanoma tumors are
readily accessible for intratumoral injection and biopsy. The
subjects included in the study have unresectable stage III or IV
melanoma, which has at least 0.5 cm in diameter, any tumor
thickness, any number of lymph node involvement, in-transit
metastases, or distant metastases.
5.1. Preparation of Adenovirus Harboring the RheoSwitch Therapeutic
System and hIL-12
[0332] The recombinant DNA is transferred to dendritic cells (DC)
by ex vivo adenoviral vector transduction. The recombinant DNA is
used to express human IL-12(p70) from intratumorally injected
immature dendritic cells which confers survival and stimulates
maturation of DC in the tumor environment resulting in their
subsequent migration to the draining lymph nodes. This leads to a
bias toward the differentiation of T helper cells to Th1 type and
also activation of tumor-specific cytotoxic T cells by cross
priming with the tumor antigens.
[0333] The recombinant DNA used as the recombinant adenoviral
vector allows the expression of human IL-12 under the control of
the RheoSwitch.RTM. Therapeutic System (RTS). The RTS comprises a
bicistronic message expressed from the human Ubiquitin C promoter
and codes for two fusion proteins: Gal4-EcR and VP16-RXR. Gal4-EcR
is a fusion between the DNA binding domain (amino acids 1-147) of
yeast Gal4 and the DEF domains of the ecdysone receptor from the
insect Choristoneura fumiferana. In another embodiment, the RTS
consists of a bicistronic message expressed from the human
Ubiquitin C promoter and codes for two fusion proteins: Gal4-EcR
and VP16-RXR. Gal4-EcR is a fusion between the DNA binding domain
(amino acids 1-147) of yeast Gal4 and the DEF domains of the
ecdysone receptor from the insect Choristoneura fumiferana.
VP16-RXR is a fusion between the transcription activation domain of
HSV-VP16 and the EF domains of a chimeric RXR derived from human
and locust sequences. These Gal4-EcR and VP16-RXR sequences are
separated by an internal ribosome entry site (IRES) from EMCV.
These two fusion proteins dimerize when Gal4-EcR binds to a small
molecule drug (RG-115932) and activate transcription of hIL-12 from
a Gal4-responsive promoter that contains six Gal4-binding sites and
a synthetic minimal promoter. The RTS transcription unit described
above is placed downstream of the hIL-12 transcription unit. This
whole RTS-hIL12 cassette is incorporated into the adenovirus 5
genome at the site where the E1 region has been deleted. The
adenoviral backbone also lacks the E3 gene. A map for the
adenoviral vector Ad-RTS-hIL-12 is shown in FIG. 8.
[0334] The recombinant adenoviral vector used in this study
contains the following exemplary regulatory elements in addition to
the viral vector sequences: Human Ubiquitin C promoter, Internal
ribosome entry site derived from EMCV, an inducible promoter
containing 6 copies of Gal4-binding site, 3 copies of SP-1 binding
sites, and a synthetic minimal promoter sequence, SV40
polyadenylation sites, and a transcription termination sequence
derived from human alpha-globin gene. It should be understood that
other regulatory elements could be utilized as alternatives.
[0335] An exemplary recombinant adenoviral vector Ad-RTS-hIL-12 has
been produced in the following manner. The coding sequences for the
receptor fusion proteins, VP16-RXR and Gal4-EcR separated by the
EMCV-IRES (internal ribosome entry site), are inserted into the
adenoviral shuttle vector under the control of the human ubiquitin
C promoter (constitutive promoter). Subsequently, the coding
sequences for the p40 and p35 subunits of hIL-12 separated by IRES,
placed under the control of a synthetic inducible promoter
containing 6 copies of Gal4-binding site are inserted upstream of
the ubiquitin C promoter and the receptor sequences. The shuttle
vector contains the adenovirus serotype 5 sequences from the left
end to map unit 16 (mu16), from which the E1 sequences are deleted
and replaced by the RTS and IL-12 sequences (RTS-h[L-12). The
shuttle vector carrying the RTS-hIL-12 is tested by transient
transfection in HT-1080 cells for Activator Drug-dependent IL-12
expression. The shuttle vector is then recombined with the
adenoviral backbone by cotransfection into HEK 293 cells to obtain
recombinant adenovirus Ad-RTS-hIL-12. The adenoviral backbone
contains sequence deletions of mu 0 to 9.2 at the left end of the
genome and the E3 gene. The shuttle vector and the adenoviral
backbone contain the overlapping sequence from mu9.2 to mu16 that
allows the recombination between them and production of the
recombinant adenoviral vector. Since the recombinant adenoviral
vector is deficient in the E1 and E3 regions, the virus is
replication-deficient in normal mammalian cells. However, the virus
can replicate in HEK 293 cells that harbor the adenovirus-5 E1
region and hence provide the E1 function in trans.
[0336] An exemplary recombinant adenoviral vector has been produced
in the following manner: The linearized shuttle vector carrying the
DNA elements for inducible expression of human IL12, and the
adenoviral backbone are co-transfected into HEK293 cells.
Recombination between the overlapping sequences on the shuttle
vector and the viral backbone results in the production of
recombinant adenovirus and is packaged into viral particles in the
HEK293 cells. The HEK293 cells are grown in DMEM containing fetal
bovine serum.
[0337] The virus used for the proposed study was purified by CsCl
density gradient centrifugation. The recombinant adenovirus
undergoes two rounds of plaque purification and the resulting seed
stock is used to produce a master viral bank (MVB) by amplification
in HEK293 cells from a fully characterized master cell bank. The
MVB undergoes extensive cGMP/GLP release tests including
replication competent adenovirus (RCA), sterility, mycoplasma,
adventitious viruses, retrovirus, human viruses HIV1/2, HTLV1/2,
HAV, HBV, HCV, EBV, B19, CMV, 7 and 8, bovine and porcine virus,
complete vector sequencing and functional testing by AD-induced
IL12 expression in human cell lines.
[0338] The virus from MVB may be used for production of the
purified virus in a cGMP facility and may again undergo release
tests including identity, RCA, sterility, mycoplasma, adventitious
viruses, viral particle-to-infectious units ratio, contamination of
host cell DNA, endotoxin and proteins and functional testing by
AD-induced IL12 expression in human cell lines.
[0339] 6.2. Transduction of Autologous Dendritic Cells by
Adenovirus Containing hIL-12 Transgene and RheoSwitch.RTM.
Therapeutic System (RTS)
[0340] Dendritic cells derived from the human subjects are
transduced ex vivo and injected into the tumor. The DC will be
characterized before viral transduction for viability, purity
(typically >80% cells showing DC phenotype), sterility,
mycoplasma and endotoxin. After viral transduction, the cells are
washed repeatedly to remove any unabsorbed virus. Supematant from
the last wash will be tested for the content of residual virus by
PCR. Since the DCs are transduced ex vivo by adenoviral vector
(non-integrating virus) and the life span of DCs after intratumoral
injection and the subsequent migration to draining lymph nodes is
short, it is not expected that the viral DNA will be incorporated
into any non-target cells. The protocol used for adenoviral
transduction of DCs is expected to yield 80-90% transduction and is
considered very efficient.
[0341] Harvesting of PBMC by leukapheresis: Subjects undergo a
standard 90 to 120 minutes leukapheresis at the Apheresis Unit of
the UPCI Outpatient. The leukapheresis procedure involves the
removal of blood from a vein in one arm; the passage of blood
through a centrifuge (cell separator), where its components are
separated and one or more components are removed; and the return of
the remaining components to the subject's vein in the same or other
arm. No more than 15% of the subject's total blood volume is
withdrawn at any one time as blood is processed through the cell
separator device. In the cell separator, blood is separated into
plasma, platelets, white cells and red blood cells. White blood
cells (WBC) are removed and all the other components are returned
into the subject's circulation. Every attempt is made to use two
peripheral IV lines for this procedure. If that is not possible, a
central line may be necessary. The subject has to be cleared by
physician to undergo leukapheresis, and is routinely screened for
vital signs (including blood pressure) prior to the procedure.
[0342] Processing: After collection, the leukapack is delivered by
hand to the CPL, and is immediately processed by centrifugal
elutriation in ELUTRA.TM.. This is a closed system validated for
clinical use. The monocyte fraction is recovered, and after the
recovery and viability of cells are established, they are
transferred to an Aastrom cartridge for 6-day culture in the
presence of IL-4 and GM-CSF. All processing and washing procedures
are performed under sterile conditions.
[0343] Initial plating: Monocytes recovered from a single leukapack
are counted in the presence of a trypan blue dye to determine the
number of viable cells. Monocytes are evaluated for purity by flow
cytometry. Monocytes are resuspended at 5 to 10.times.10.sup.6
cells/mL in serum-free and antibiotic-free CellGenix medium,
containing 1,000 TU/mL of IL-4 and 1,000 IU/mL of GM-CSF per
SOP-CPL-0166, and placed in an Aastrom cartridge. A minimum loading
volume of 50 ml and a minimum cell number are required for cassette
inoculation.
[0344] Culture: The Aastrom cartridge is placed in the incubator in
the Replicell System, a fully closed, cGMP-compatible automated
culture device for immature DC generation.
[0345] Immature DC harvest: On day 6, the Aastrom cartridge is
removed from the incubator and immature DCs are harvested. The
cells are recovered by centrifugation at 1,500 rpm, washed in
CellGenix medium, counted in the presence of a trypan blue dye and
checked for morphologic and phenotypic characteristics.
[0346] Viability: This is determined by performing hemocytometer
cell counts in the presence of trypan blue. Generally, >95% of
harvested cells are viable, i.e., exclude a trypan blue dye. If
viability is less than 70% the immature DCs will be discarded.
[0347] Phenotyping: The cells generated in culture are counted by
microscopic observation on a hemocytometer, and a preliminary
differential count (DC vs. lymphocytes) is obtained using a trypan
blue dye. Confirmation of the differential count is made by flow
cytometry, gating on DC vs. lymphocytes and using high forward and
side scatter properties of immature DC as the criterion for their
identification. Immature DCs routinely contain >80% of cells
with dendritic cell morphology and have DC phenotype.
[0348] IL-12p70 potency assay: It has been established that mature
DCs (mDCs) have the ability to produce IL-12p70 spontaneously or
upon activation with CD40L with or without addition of innate
immunity signals (e.g., LPS). A standardized IL-12p70 production
assay was recently established and is applicable to small samples
or large lots of DC vaccines generated under a variety of
conditions. The current potency assay consists of two distinct
steps, the first involving co-incubation of responder DCs with J588
lymphoma cells stably transfected with the human CD40 ligand gene
as stimulators. The second step involves testing of supernatants
from these co-cultures for levels of IL-12p'70 secreted by DCs
stimulated with J558/CD40L+/-LPS in the Luminex system. This
potency assay has an inter-assay CV of 18.5% (n=30) and a broad
dynamic range, which facilitates evaluation of various DC products
characterized by vastly different levels of IL-12p70 production.
The normal range for the assay established using DC products
generated from monocytes of 13 normal donors was 8-999 pg/mL, with
a mean of 270 pg/mL
[0349] Production and Release Criteria for Dendritic Cells
[0350] Each lot of the in vitro generated dendritic cells is tested
for the presence of microbial contaminants (aerobic and anaerobic
bacteria, fungi and mycoplasma), as well as endotoxin and are
phenotypically and functionally characterized. All dendritic cells
to be injected into subjects will be fresh and will not undergo
croypreservation.
[0351] Quality assurance testing of DC: DC generated as described
above are evaluated for sterility, viability, purity, potency and
stability. Criteria for release of the cellular product are
established and rigorously followed.
[0352] Viability: The cells generated in culture are counted by
microscopic observation on a hemacytometer, and a differential
count (DC vs. lymphocytes) is obtained using a trypan blue dye.
This count provides the percentage of viable cells in the tested
culture. More than 70% cell viability by trypan blue exclusion and
minimum 70% cells expressing HLA-DR and CD86 as the
monocyte-derived DC markers are required for passing the release
criteria. Additional markers may be included for exploratory
analysis such as CD83 and CCR7 for assessing the DC maturation
status, and CD3 and CD19 to assess the lymphocytes
contamination.
[0353] Purity: Two-color flow cytometry analysis of cells stained
with FITC- and PE-conjugated mAbs is used to determine that the DC
population identified morphologicallly expresses the surface
antigens defined for DC and lack the monocyte and T and B cell
lineage antigens. For vaccine preparation, the DC generated must
express HLA-DR and CD86 and must not express CD3, CD19, or CD14. To
be considered as mDC, the cells must express CD83+ and CCR7+.
[0354] Potency: To define a measure of potency for the DC, we
determined their ability to produce IL-12p70 as described
above.
[0355] Sterility: DC are tested by bacterial (Aerobic and
anaerobic) and fungal cultures using the BD Bactec system (Becton
Dickinson Co., Sparks, Md.) at the University of Pittsburgh Medical
Center Microbiology Laboratory. Final results of the microbial
cultures are available in 14 days. Prior to release of the DC for
vaccine use, a gram stain is performed and must be negative for the
presence of microorganisms.
[0356] The IMCPL tests for mycoplasma by the use of the Gen-Probe
Mycoplasma Tissue Culture Rapid Detection System (Gen-Probe, Inc.
San Diego, Calif.), which is based on nucleic acid hybridization
technology. Endotoxin testing is performed using the Limulus
Amoebocyte Lysate Pyrogen Plus assay (Bio Whittaker, Inc.,
Walkerville, Md.). Endotoxin testing is performed on the cell
culture at the time of harvest and prior to release of the final
product. The acceptable endotoxin level is <5 EU/kg body weight.
Untransduced and transduced dendritic cells will be cryopreserved
for future analysis.
[0357] It is expected that all the transduced cells will express
the transgene. More than 80% of the DCs are expected to be
transduced. The product will be biologically active since the
native coding sequence is maintained in the transgene. The
viral-transduced DCs injected into the tumor are of immature DC
phenotype and do not express IL-12 till they undergo maturation,
and hence at this stage, the IL-12 expression is mostly from the
transgene. Since the expression of the IL-12 transgene is induced
by the small molecule activator drug RG-115932 in a dose dependent
way, we can control the level of transgene expression in the
transduced DCs to the desired levels. A small portion of the
transduced DCs prepared for administration to the human subjects
may be tested in vitro for the activator drug-dependent induction
of IL12 expression. IL-12 expression may be assayed by ELISA with a
sensitivity of 4 ng/ml.
[0358] The in vivo mouse tumor model is similar to the human
studies in that mice bearing subcutaneous melanoma (B16) tumor were
treated in the same way as the proposed human study by the
injection of adenoviral transduced DCs and induction of murine IL12
transgene. After tumor regression was observed, rechallenge with
the same tumor cells did not result in tumor growth, indicating
systemic tumor immunity.
[0359] It is expected that in vitro induction of IL-12 from cells
transduced by the vector used in the proposed study yields about
500 ng IL-12 per 10.sup.6 cells in 24 hours, determined by ELISA.
In preclinical studies using mouse model of melanoma, intratumoral
injection of 10.sup.6 or more transduced DCs showed efficacy.
However, it is expected that the required intratumoral injection
may show efficacy at levels below this amount and therefore
injections of 5.times.10.sup.7 transduced DCs may be utilized as a
starting point to determine if less or greater amounts are
required.
[0360] For instance, in vitro, human and mouse cell lines and
primary dendritic cells transduced with recombinant adenoviral
vector carrying the genes for IL12 show induction of IL12
expression in response to the activator drug in a dose dependent
way.
[0361] Adenoviral transduction of human DCs at different MOI and
for different duration of viral adsorption showed efficient
transduction of these cells by 3 hour viral adsorption at MOI of
500. The activator drug induced IL-12 expression in these
transduced human DCs (FIG. 9).
[0362] For in vivo experiments on a mouse melanoma model described
above, C57/BL6 mice were given subcutaneous injections of B16 cells
to form tumors. Intratumoral injection of DCs transduced with
adenoviral vector carrying the murine IL-12 genes under the control
of the RTS, along with administration of the activator drug
resulted in systemic immunity specific to the tumor. The treatment
resulted in tumor regression. Rechallenge of the cured mice after
50 days with B16 cells showed that the B16 cells did not form
tumors. This induction of tumor immunity was dependent on the
administration of the activator drug, and hence IL-12 expression,
in the transduced DCs injected. The activator drug was effective in
intraperitoneal and oral routes. See FIG. 11 and FIG. 14.
6.3. Formulation of Activator Drug
[0363] The activator drug used herein is formulated in any one of
the following formulations: [0364] (1) 100% Labrasol; [0365] (2)
Listerine flavored Labrasol (Latitude Pharmaceuticals Inc., USA)
comprising (a) menthol, (b) thymol, (c) eucalyptol, (d) aspartame,
(e) sodium saccharine, (f) citric acid, (g) peppermint flavor, (h)
cream flavor, (i) labrasol; [0366] (3) Miglyol 812 and phospholipon
90G (Latitude Pharmaceuticals Inc., USA); or [0367] (4) Miglyol
812, phospholipon 90G and Vitamin E tocopheryl polyethylene glycol
succinate (Latitude Pharmaceuticals Inc., USA).
6.4. Delivery
[0368] While a variety of concentrations and specific protocols may
be imagined, one example for treating patients would include
patients receiving intratumoral injection(s) of transduced
autologous dendritic cells (AdDCs) at a concentration of
5.times.10.sup.7 suspended in sterile saline engineered to express
hIL-12 (human interleukin 12) under control of the RTS in
combination with the oral activator drug (RG-115932).
[0369] 6.4.1. Initial Treatment
[0370] Day 1 Inpatient Visit: On day 1, a baseline physical
examination (including vital signs, weight, and ECOG status) is
performed. Urine is collected and blood drawn for baseline serum
chemistry, urinanalysis, and hematology (safety profile).
Approximately 3 hours before the intratumoral injection of the in
vitro engineered dendritic cells, each subject is dosed with an
activator drug (cohort 1-0.01 mg/kg, 0.3 mg/kg, 1.0 mg/kg, and 3
mg/kg) immediately after a meal. Blood is drawn at specified time
intervals (predose, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, and 24 hours
after the AD dose) on day 1 for evaluation of single dose
pharmacokinetics of the activator drug and its major metabolites.
Each subject receives a single intratumoral injection of adenoviral
transduced autologous dendritic cells at a concentration of
5.times.10.sup.7 cells, engineered to express hIL-12 under the
control of the RTS. The subjects are carefully monitored for local
injection site reactions and/or hypersensitivity reactions. Day 2
through 14 Inpatient Visit: On days 2 through 14, each subject is
dosed with the activator drug immediately after a meal. Vital signs
and adverse events are collected daily on days 2 through 14. On day
4.+-.24 hours, biopsies of the tumor and/or draining lymph nodes
are removed from approximately 50% of the subjects for measurement
of hIL-12 and cellular immune response. On day 8, weight is
measured. On day 8.+-.24 hours, biopsies of the tumor and/or
draining lymph nodes are removed from subjects who did not have a
biopsy performed on day 4 for measurement of hIL-12 and cellular
immune response. Blood is drawn on day 4.+-.24 hours and day
8.+-.24 hours for assay of potential antibodies and cellular immune
response against the adenovirus and/or the RTS components. A serum
cytokine profile is also obtained to determine if the expression of
other cytokines is affected by treatment with the hIL-12 transgene.
On day 8, urine is collected and blood is drawn for baseline serum
chemistry, urine analysis, and hematology (safety profile). On Day
8, blood is drawn at specified time intervals (predose, 0.5, 1, 2,
4, 6, 8, 12, 16, and 24 hours after the AD dose) for evaluation of
steady-state pharmacokinetics/ADME of the activator drug and its
major metabolites.
[0371] Day 14 Inpatient Visit: On day 14, each subject is dosed
with the Activator Drug immediately after a meal. Each subject
receives a physical examination (including vital signs, height,
weight and ECOG status). Urine is collected and blood is drawn for
serum chemistry, urinalysis, and hematology (safety profile). Blood
is drawn on day 14.+-.24 hours for assay of potential antibodies
and cellular immune response against the adenovirus and/or the RTS
components. A serum cytokine profile is also obtained to determine
if the expression of other cytokines is affected.
[0372] Blood is collected from the subjects at specified inpatient
and outpatient visits to measure potential antibodies and cellular
immune response to the adenovirus and components of the RTS. Blood
is obtained for a baseline serum cytokine profile. The AdVeGFP
infectivity blocking type assay is used to detect an antibody
response to the adenoviral vector (Gambotto, Robins et al. 2004).
Antibody response to the RTS components will be assessed by western
blot and/or ELISA using serum from the patient and the RTS proteins
produced from an expression vector. In addition, multiplex cytokine
testing will be done in the serum by Luminex for IL-12, IFN-gamma,
IP-10, and other Th1/Th2 cytokines such as IL-2, TNF-alpha, IL-4,
IL-5, and IL-10. These antibody and cytokine assays will need about
10 ml of blood.
[0373] The cellular immune response assays use about 50-60 ml blood
and CD4 and CD8 T cell subsets are separated from it. The separated
T cells are mixed with autologous DCs transduced with empty AdV
vector, AdV-RTS, or AdV-RTS-hIL12 vectors in an ELISPOT assay for
INF-gamma production by the T cells activated by the AdV- and
RTS-derived antigens, if any. Similar assays are performed using
the tumor cells as such and/or DCs expressing shared melanoma
antigens to assess the early response to the tumor. Additional
assays are also performed as necessary.
[0374] On day 14.+-.24 hours, biopsies of the tumor and/or draining
lymph nodes are removed from all subjects that have tissue
available for measurement of hIL-12 and cellular immune response.
Adverse events are recorded. After completion of the day 14
procedures, each subject are discharged from the inpatient clinic
and asked to return in approximately 3 weeks for the 1 month
follow-up outpatient visit.
[0375] Early Termination Visit: If a subject cannot complete the
inpatient treatment phase, the following early termination visit
procedures will be conducted prior to discharge from the clinic.
Each subject receives a physical examination (including vital
signs, height, weight and ECOG status). Urine is collected and
blood is drawn for serum chemistry, urinalysis, and hematology
(safety profile). Blood will be drawn for assay of potential
antibodies and cellular immune response against the adenovirus
and/or the RTS components as described above. A serum cytokine
profile will also be obtained as described above to determine if
the expression of other cytokines is affected. Biopsies of the
tumor and/or draining lymph nodes are removed from all subjects
that have tissue available for measurement of hIL-12 and cellular
immune response. Adverse events are recorded. After completion of
the early termination visit procedures, each subject is discharged
from the inpatient clinic and asked to return in approximately 3
weeks for the 1 month follow-up outpatient visit.
[0376] Month 1 Through 4 Follow-Up Visits: Adverse events will be
collected during the month 1 through 4 follow-up period. At the
month 1, 2, and 3 visits, follow-up physical examinations
(including vital signs, weight and ECOG status) will be conducted.
Urine and blood will be collected at the month 1, 2, and 3 visits
for serum chemistry, urinalysis, and hematology (safety profile).
Blood will be collected at the month 1 and 3 visit for assay of
potential antibodies and cellular immune response against the
adenovirus and/or the RTS components. Blood will be obtained at the
month 1 visit for serum cytokine profiling. At the month 1 visit,
biopsies of the tumor and/or draining lymph nodes will be performed
on subjects with available tissue, for measurement of hIL-12 and
cellular immune response. CT/PET scans will be performed at the
month 2 and 4 visits to assess overall disease progression or
regression.
[0377] Month 5 Through 6 Follow-Up Visits: If drug-related toxicity
is observed at month 3 or month 4, month 5 through month 6
follow-up visits will be conducted. Adverse events will be
collected during the month 5 through month 6 follow-up period.
Subject safety will be monitored by a telephone call from clinic
personnel to each subject during month 5 and through an outpatient
visit at month 6. Should a drug-related toxicity occur or continue
through month 5 and be deemed by the investigator to be serious,
not stabilized, or warrant further evaluation; clinic personnel
will not only telephone the subject but will ask that the subject
visit the clinic. At the month 6 visit, follow-up physical
examinations (including vital signs, weight and ECOG status) will
be conducted. Urine and blood will be collected for serum
chemistry, urinalysis, and hematology (safety profile). Blood will
also be collected at the month 6 visit for assay of potential
antibodies and cellular immune response against the adenovirus
and/or the RTS components. CT/PET scans will be performed at the
month 6 visit to assess overall disease progression or
regression.
[0378] Activator Drug Dosing & Stopping Criteria: If a
dose-limiting toxicity (DLTs; i.e., a total of >2 of 3 subjects
enrolled in a group in cohort 1 experience a >grade 3 toxicity
according to CTCAE v3.0) is determined, at a given dose level the
next group of 3 subjects will be administered the same dose level
of Activator Drug. If DLT are observed in 1 or more subjects in the
additional dose group, dose escalation will be discontinued and the
next lower dose is considered maximum tolerated dose (MTD),
otherwise dose escalation resumes until MTD is reached or to a
maximum dose of 10 mg/kg (whichever occurs first).
[0379] Safety, tolerance, and transgene function will be assessed
for all subjects in each group of cohort 1 up to one month after
injection of AdDCs before enrolling subjects to receive the next
highest dose of Activator Drug.
[0380] If a subject experiences a >grade 3 toxicity according to
CTCAE v3.0 that is deemed possibly, probably or definitely related
to study drug treatment, dosing with the Activator Drug will be
discontinued for that subject and the subject will undergo early
termination visit procedures.
[0381] Study Stopping Criteria: If >70% of subjects in a cohort
experience a >grade 3 toxicity according to CTCAE v3.0 that is
deemed probably or definitely related to study drug treatment,
dosing with the Activator Drug will be discontinued for all
subjects and all subjects will undergo early termination visit
procedures.
[0382] Investigational Trial Medication: A combination of two
investigational medications will be evaluated for safety,
tolerance, transgene function, and immunological effects in this
trial. The small molecule Activator Drug will be administered as an
oral solution at doses of 0.01 mg/kg, 0.1 mg/kg, 1.0 mg/kg and 10.0
mg/kg to subjects with Stage III or IV melanoma once daily for
fourteen consecutive days in combination with a single intratumoral
injection of adenoviral transduced autologous dendritic cells at a
concentration of 5.times.10.sup.7, engineered to express hIL-12.
There is an option for subjects to receive an additional
intratumoral injection of AdDCs in combination with treatment for
14 consecutive days with the Activator Drug.
[0383] 6.5. Assessment of Safety to Assessment of Transgene
Function and Immunological Effects
[0384] Endotoxin testing is performed on the cell culture at the
time of harvest and prior to release of the final product. The
acceptable endotoxin level is <5 EU/kg body weight. Untransduced
and transduced dendritic cells will be cryopreserved for future
analysis.
[0385] Assessment of Safety: The safety of a single intratumoral
injection of adenoviral transduced autologous dendritic cells in
combination with the oral Activator Drug will be evaluated by
physical examinations, vital signs, serum chemistry, urinalysis,
hematology, adverse events, and antibodies and cellular immune
response to adenovirus and components of the RTS during the trial
and 12 month follow-up. Pregnancy tests will be done on females of
childbearing potential at screening. A list of concurrent
medications will also be obtained from subjects at screening and
day 0 of the retreatment period to determine if there is any
relationship between concurrent medications and potential adverse
events. An approximate total of 89 teaspoons (439 ml) of blood plus
leukapheresis will be collected from subjects during the screening
phase and initial inpatient treatment phase (26 day period). An
approximate total of 75 teaspoons (370 ml) of blood plus
leukapheresis will be collected from subjects during the inpatient
retreatment phase (26 day period, 5-6 weeks from previous inpatient
visits). An approximate total of 46 teaspoons (227 ml) of blood
will be collected from subjects during the outpatient follow-up
phase (Month 1-6).
[0386] Physical Examinations: Complete physical examinations
(including ECOG performance status) will be conducted at specified
inpatient and outpatient visits. Medical history and demographics
of each subject will also be recorded at the screening visit.
[0387] Vital Signs: Vital signs of each subject will be included in
each scheduled physical examination but will also be taken at all
inpatient and outpatient visits. Vital signs will include blood
pressure, pulse, temperature, and respirations. Weight and height
will also he recorded at specified visits. Vital signs (minus
height and weight) will be recorded every hour for the first two
hours and then every 8 hours after dosing of the Activator Drug has
occurred.
[0388] Blood Chemistry: A random, non-fasted blood sample will be
drawn from each subject at specified inpatient and outpatient
visits and serum harvested for chemistry. The following serum tests
will be performed: AST (aspartate transaminase), ALT (alanine
transaminase), GGT (gammaglutamyl transpeptidase), LDH (lactic
dehydrogenase), LAP (leucine aminopeptidase), alkaline phosphatase,
creatinine, total bilirubin, total protein, albumin, blood urea
nitrogen, total cholesterol, glucose, and electrolytes.
[0389] Urinanalysis: A random, mid-stream urine sample will be
collected from each subject at specified inpatient and outpatient
visits and is analyzed. The following tests will be performed:
description of color and appearance, specific gravity, pH, glucose,
ketone bodies, protein, red and white blood cell number, and
pyroluria.
[0390] Hematology: A random, non-fasted blood sample will be drawn
from each subject at specified inpatient and outpatient visits and
hematologic tests will be run as follows: complete blood count
(CBC) including white blood cell count, differential white blood
cell count, red blood cell count, hematocrit, hemoglobin, red blood
cell indices, and platelet count. PTT (partial thromboplastin time)
and PT (prothrombin time) will also be evaluated.
[0391] Adverse Events: The NCI Common Terminology Criteria for
Adverse Events (CTCAE version 3.0) will be utilized to evaluate
toxicity in the trial and 3-6 month follow-up period. An adverse
event/experience is any reaction, side effect, or other untoward
event (signs, symptoms, changes in laboratory data) associated with
the use of a test article (drug, biologic, or device), whether or
not the event is considered related to the test article. A serious
adverse event/experience is any adverse event or experience that
results in any of the following outcomes: death, a life-threatening
adverse experience, a congenital anomaly/birth defect, inpatient
hospitalization or prolongation of existing hospitalization, or a
persistent or significant disability/incapacity. Important medical
events/experiences that may not result in death, be
life-threatening, or require hospitalization may be considered
serious adverse experiences when, based upon appropriate medical
judgment, they may jeopardize the subject or may require medical or
surgical intervention to prevent one of the outcomes listed in this
definition.
[0392] The severity of adverse events will be graded as follows:
Grade 1 (mild), Grade 2 (moderate), Grade 3 (severe), Grade 4
(life-threatening or disabling), Grade 5 (death related to adverse
event). The relationship between an adverse event and study
medication will be determined on the basis of clinical judgment and
the following definitions: [0393] a. definitely related is an
adverse event that follows a reasonable temporal sequence from
administration of the study medication, follows a known response
pattern to the study medication, and, when appropriate to the
protocol, is confirmed by improvement after stopping the study
medication (positive dechallenge) and by reappearance of the
reaction after repeat exposure (positive rechallenge) and cannot be
reasonably explained by known characteristics of the subject's
clinical state or by other therapies, [0394] b. probably related is
an adverse event that follows a temporal sequence from
administration of the study medication, follows a known response
pattern to the study medication and, when appropriate to the
protocol, is confirmed by improvement after dechallenge, and cannot
be reasonably explained by the known characteristics of the
subject's clinical state or by other therapies, [0395] c. possibly
related is an adverse event that follows a reasonable temporal
sequence from administration of the study medication and follows a
known response pattern to the study medication but could have been
produced by the subject's clinical state or by other therapies,
[0396] d. unrelated is an adverse event for which sufficient
information exists to indicate that the etiology is unrelated to
the study medication. Two or more of the following variables apply
to an unrelated adverse event: 1. The adverse event does not follow
a reasonable temporal sequence after administration of the study
medication, 2. The adverse event is readily explained by the
subject's clinical state or other therapies, 3. The adverse event
does not abate upon dose reduction or cessation of therapy
(assuming that it is reasonable to expect abatement of the adverse
event within the observed interval).
[0397] All observed or reported adverse events subsequent to
enrollment of subjects will be recorded. Any condition that is
present at time of enrollment that worsens will be recorded as an
adverse event. Adverse events will be determined on the basis of
volunteered symptoms and clinical observation and assessment at
trial visits. At each inpatient and outpatient visit, enrolled
subjects will be asked to volunteer information concerning adverse
events with non-leading questions such as "How are you feeling?"
Changes in laboratory values will be recorded as adverse events if
considered clinically significant and if clinical changes or action
are required such as initiating a treatment.
[0398] Potential Antibody and Cellular Immune Response to
Adenovirus and/or
[0399] Components of the RTS: Blood will be collected from the
subjects at specified inpatient and outpatient visits to evaluate
the potential antibody and cellular immune response to the
adenovirus and components of the RTS and tumor antigens. The
AdVeGFP infectivity blocking type assay will be used to detect an
antibody response to the adenoviral vector (Nwanegbo, et al. 2004).
Antibody response to the RTS components will be assessed by western
blot and/or ELISA using serum from the subjects and the RTS
proteins produced from an expression vector. In addition, multiplex
cytokine testing will be done in the serum by Luminex for IL-12,
IFN-gamma, IP-10, and other Th1/Th2 cytokines such as IL-2, TNFa,
IL-4, JL-5 and IL-10. These antibody and cytokine assays will need
about 10 ml of blood.
[0400] The cellular immune response assays use about 50-60 ml blood
and CD4 and CD8 T cell subsets will be separated from it. The
separated T cells will be mixed with autologous DCs transduced with
empty AdV vector, AdV-RTS, or AdV-RTS-hIL12 vectors in an ELISPOT
assay for IFN-gamma production by the T cells activated by the AdV-
and RTS-derived antigens, if any. Similar assays will be performed
using the tumor cells as such and/or DCs expressing shared melanoma
antigens to assess the early immune response to the tumor.
Additional assays may also be performed as necessary.
[0401] PREGNANCY TESTING: Females of childbearing potential is
administered a urine pregnancy test at the screening visit and
before the first inpatient visit of the retreatment phase. The
testing is performed at least 72, 48, 24, or 12 hours prior to the
administration of Activator Drug during both the initial treatment
and all retreatment periods. If the urine pregnancy test is
positive, then confirmation will be obtained with a serum pregnancy
test. If pregnancy is confirmed, the subject will not be allowed to
enter the trial or continue into the retreatment phase. The
pregnancy testing may be reperformed as many as necessary.
[0402] CONCOMITANT MEDICATION INQUIRY: At screening, and before the
first inpatient visit of the retreatment phase, each subject will
be asked to provide a list of concurrent medications to determine
any possible relationship to adverse events that occur during the
trial and follow-up phase.
[0403] RETREATMENT CRITERIA: If a subject has tolerated prior AdDC
inoculation without adverse reactions that are limiting, and has
shown no progression of disease or symptomatic decline at the time
of potential retreatment, they will be considered for retreatment.
If, in the opinion of the principal investigator, and treating
physician there is a potential clinical benefit for additional
intratumoral injection(s) of AdDCs in combination with Activator
Drug (maximum tolerated dose from cohort 1) for 14 consecutive
days, retreatment will be offered to the subject, provided the
following criteria are met: [0404] 1. There have been no limiting
toxicities, [0405] 2. The subject's disease is stable or showing
clinical or subjective signs of improvement, and [0406] 3. There is
no evidence of antibody or cellular immune response to adenovirus
components of RheoSwitch.RTM. Therapeutic System.
[0407] ASSESSMENT OF TRANSGENE FUNCTION AND IMMUNOLOGICAL EFFECTS:
Punch or excisional biopsies of the tumor and associated draining
lymph nodes will be collected during screening (day -12 to day -7),
day 4, day 8 and day 14 of the trial and at month 1 of the
follow-up (see Tables 3-5) for in vivo assessment of transgene
expression of hIL-12 and cellular immune response. Fine needle
aspiration biopsies of the tumor and associated draining lymph
nodes will be collected on day -12 to -7 and day 14 of the
retreatment period for in vivo assessment of transgene expression
of hIL-12 and cellular immune response. Biopsies will be evaluated
by standard light microscopy and immunohistochemistry to assess
cellular infiltration of T cells into the tumor and draining lymph
nodes. Biopsy sections will be read by a pathologist unaware of
study subject background. To distinguish between endogenous and
induced IL-12 expression by DCs in the tumor and draining lymph
nodes, RT-PCR on RNA will be used with appropriately designed
primers. Blood will be drawn for a serum cytokine profile at
screening, day 4, day 8 and day 14 of the trial, at month 1 of the
follow-up and on day -12 to -7, day 8 and day 14 of the retreatment
period (see Tables 3-5). A serum cytokine profile will be obtained
to determine if the expression of other cytokines is affected by
treatment with the hIL-12 transgene. Multiplex cytokine testing
will be done in the serum by Luminex for IL-12, IFN-gamma, IP-10,
and other Th1/Th2 cytokines such as IL-2, TNFa, IL-4, IL-5 and
IL-10. These antibody and cytokine assays will need about 10 ml of
blood.
[0408] SINGLE DOSE AND STEADY-STATE PHARMACOKINETICS OF ACTIVATOR
DRUG: Blood will be drawn at specified time intervals (predose,
0.5, 1, 1.5, 2, 4, 6, 8, 12, 16, and 24 hours after the morning
dose) on day 1 of the trial for evaluation of single dose
pharmacokinetics and on day 8 of the trial for measurement of
steady state pharmacokinetics/ADME of the Activator Drug and its
major metabolites. Plasma will be evaluated by HPLC to obtain the
following steady-state pharmacokinetic endpoints of the Activator
Drug and major metabolites: Cmax (maximum observed plasma
concentration), Tmax (time to maximum observed plasma
concentration), Ctrough (minimum observed plasma concentration
computed as the average of the concentrations at 0 and 24 hours),
C24 h (plasma concentration at 24 hours), AUC24 h (area under
plasma concentration-time curve from time 0 to 24 hours), Ke
(apparent elimination rate), and T112 (apparent half-life).
[0409] It is to be understood that the foregoing described
embodiments and exemplifications are not intended to be limiting in
any respect to the scope of the invention, and that the claims
presented herein are intended to encompass all embodiments and
exemplifications whether or not explicitly presented herein.
LITERATURE
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Sequence CWU 1
1
131648DNAMus sp.IL-12 p35 1atgtgtcaat cacgctacct cctctttttg
gccacccttg ccctcctaaa ccacctcagt 60ttggccaggg tcattccagt ctctggacct
gccaggtgtc ttagccagtc ccgaaacctg 120ctgaagacca cagatgacat
ggtgaagacg gccagagaaa aactgaaaca ttattcctgc 180actgctgaag
acatcgatca tgaagacatc acacgggacc aaaccagcac attgaagacc
240tgtttaccac tggaactaca caagaacgag agttgcctgg ctactagaga
gacttcttcc 300acaacaagag ggagctgcct gcccccacag aagacgtctt
tgatgatgac cctgtgcctt 360ggtagcatct atgaggactt gaagatgtac
cagacagagt tccaggccat caacgcagca 420cttcagaatc acaaccatca
gcagatcatt ctagacaagg gcatgctggt ggccatcgat 480gagctgatgc
agtctctgaa tcataatggc gagactctgc gccagaaacc tcctgtggga
540gaagcagacc cttacagagt gaaaatgaag ctctgcatcc tgcttcacgc
cttcagcacc 600cgcgtcgtga ccatcaacag ggtgatgggc tatctgagct ccgcctga
64821008DNAMus sp.IL-12 p40 2atgtgtcctc agaagctaac catctcctgg
tttgccatcg ttttgctggt gtctccactc 60atggccatgt gggagctgga gaaagacgtt
tatgttgtag aggtggactg gactcccgat 120gcccctggag aaacagtgaa
cctcacctgt gacacgcctg aagaagatga catcacctgg 180acctcagacc
agagacatgg agtcataggc tctggaaaga ccctgaccat cactgtcaaa
240gagtttctag atgctggcca gtacacctgc cacaaaggag gcgagactct
gagccactca 300catctgctgc tccacaagaa ggaaaatgga atttggtcca
ctgaaatttt aaaaaatttc 360aaaaacaaga ctttcctgaa gtgtgaagca
ccaaattact ccggacggtt cacgtgctca 420tggctggtgc aaagaaacat
ggacttgaag ttcaacatca agagcagtag cagttcccct 480gactctcggg
cagtgacatg tggaatggcg tctctgtctg cagagaaggt cacactggac
540caaagggact atgagaagta ttcagtgtcc tgccaggagg atgtcacctg
cccaactgcc 600gaggagaccc tgcccattga actggcgttg gaagcacggc
agcagaataa atatgagaac 660tacagcacca gcttcttcat cagggacatc
atcaaaccag acccgcccaa gaacttgcag 720atgaagcctt tgaagaactc
acaggtggag gtcagctggg agtaccctga ctcctggagc 780actccccatt
cctacttctc cctcaagttc tttgttcgaa tccagcgcaa gaaagaaaag
840atgaaggaga cagaggaggg gtgtaaccag aaaggtgcgt tcctcgtaga
gaagacatct 900accgaagtcc aatgcaaagg cgggaatgtc tgcgtgcaag
ctcaggatcg ctattacaat 960tcctcatgca gcaagtgggc atgtgttccc
tgcagggtcc gatcctag 10083660DNAHomo sapiensIL-12 p35 3atgtgtccag
cgcgcagcct cctccttgtg gctaccctgg tcctcctgga ccacctcagt 60ttggccagaa
acctccccgt ggccactcca gacccaggaa tgttcccatg ccttcaccac
120tcccaaaacc tgctgagggc cgtcagcaac atgctccaga aggccagaca
aactctagaa 180ttttaccctt gcacttctga agagattgat catgaagata
tcacaaaaga taaaaccagc 240acagtggagg cctgtttacc attggaatta
accaagaatg agagttgcct aaattccaga 300gagacctctt tcataactaa
tgggagttgc ctggcctcca gaaagacctc ttttatgatg 360gccctgtgcc
ttagtagtat ttatgaagac ttgaagatgt accaggtgga gttcaagacc
420atgaatgcaa agcttctgat ggatcctaag aggcagatct ttctagatca
aaacatgctg 480gcagttattg atgagctgat gcaggccctg aatttcaaca
gtgagactgt gccacaaaaa 540tcctcccttg aagaaccgga tttttataaa
actaaaatca agctctgcat acttcttcat 600gctttcagaa ttcgggcagt
gactattgac agagtgacga gctatctgaa tgcttcctaa 6604987DNAHomo
sapiensIL-12 p40 4atgtgtcacc agcagttggt catctcttgg ttttccctgg
tttttctggc atctcccctc 60gtggccatat gggaactgaa gaaagatgtt tatgtcgtag
aattggattg gtatccggat 120gcccctggag aaatggtggt cctcacctgt
gacacccctg aagaagatgg tatcacctgg 180accttggacc agagcagtga
ggtcttaggc tctggcaaaa ccctgaccat ccaagtcaaa 240gagtttggag
atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg
300ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt
aaaggaccag 360aaagaaccca aaaataagac ctttctaaga tgcgaggcca
agaattattc tggacgtttc 420acctgctggt ggctgacgac aatcagtact
gatttgacat tcagtgtcaa aagcagcaga 480ggctcttctg acccccaagg
ggtgacgtgc ggagctgcta cactctctgc agagagagtc 540agaggggaca
acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca
600gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa
gctcaagtat 660gaaaactaca ccagcagctt cttcatcagg gacatcatca
aacctgaccc acccaagaac 720ttgcagctga agccattaaa gaattctcgg
caggtggagg tcagctggga gtaccctgac 780acctggagta ctccacattc
ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840agcaagagag
aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc
900cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc
atcttggagc 960gaatgggcat ctgtgccctg cagttag 9875215PRTMus sp.IL-12
p35 5Met Cys Gln Ser Arg Tyr Leu Leu Phe Leu Ala Thr Leu Ala Leu
Leu 1 5 10 15 Asn His Leu Ser Leu Ala Arg Val Ile Pro Val Ser Gly
Pro Ala Arg 20 25 30 Cys Leu Ser Gln Ser Arg Asn Leu Leu Lys Thr
Thr Asp Asp Met Val 35 40 45 Lys Thr Ala Arg Glu Lys Leu Lys His
Tyr Ser Cys Thr Ala Glu Asp 50 55 60 Ile Asp His Glu Asp Ile Thr
Arg Asp Gln Thr Ser Thr Leu Lys Thr 65 70 75 80 Cys Leu Pro Leu Glu
Leu His Lys Asn Glu Ser Cys Leu Ala Thr Arg 85 90 95 Glu Thr Ser
Ser Thr Thr Arg Gly Ser Cys Leu Pro Pro Gln Lys Thr 100 105 110 Ser
Leu Met Met Thr Leu Cys Leu Gly Ser Ile Tyr Glu Asp Leu Lys 115 120
125 Met Tyr Gln Thr Glu Phe Gln Ala Ile Asn Ala Ala Leu Gln Asn His
130 135 140 Asn His Gln Gln Ile Ile Leu Asp Lys Gly Met Leu Val Ala
Ile Asp 145 150 155 160 Glu Leu Met Gln Ser Leu Asn His Asn Gly Glu
Thr Leu Arg Gln Lys 165 170 175 Pro Pro Val Gly Glu Ala Asp Pro Tyr
Arg Val Lys Met Lys Leu Cys 180 185 190 Ile Leu Leu His Ala Phe Ser
Thr Arg Val Val Thr Ile Asn Arg Val 195 200 205 Met Gly Tyr Leu Ser
Ser Ala 210 215 6335PRTMus sp.IL-12 p40 6Met Cys Pro Gln Lys Leu
Thr Ile Ser Trp Phe Ala Ile Val Leu Leu 1 5 10 15 Val Ser Pro Leu
Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val 20 25 30 Val Glu
Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu 35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln 50
55 60 Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val
Lys 65 70 75 80 Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly
Gly Glu Thr 85 90 95 Leu Ser His Ser His Leu Leu Leu His Lys Lys
Glu Asn Gly Ile Trp 100 105 110 Ser Thr Glu Ile Leu Lys Asn Phe Lys
Asn Lys Thr Phe Leu Lys Cys 115 120 125 Glu Ala Pro Asn Tyr Ser Gly
Arg Phe Thr Cys Ser Trp Leu Val Gln 130 135 140 Arg Asn Met Asp Leu
Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro 145 150 155 160 Asp Ser
Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys 165 170 175
Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln 180
185 190 Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu
Leu 195 200 205 Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr
Ser Thr Ser 210 215 220 Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro
Pro Lys Asn Leu Gln 225 230 235 240 Met Lys Pro Leu Lys Asn Ser Gln
Val Glu Val Ser Trp Glu Tyr Pro 245 250 255 Asp Ser Trp Ser Thr Pro
His Ser Tyr Phe Ser Leu Lys Phe Phe Val 260 265 270 Arg Ile Gln Arg
Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys 275 280 285 Asn Gln
Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln 290 295 300
Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn 305
310 315 320 Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg
Ser 325 330 335 7219PRTHomo sapiensIL-12 p35 7Met Cys Pro Ala Arg
Ser Leu Leu Leu Val Ala Thr Leu Val Leu Leu 1 5 10 15 Asp His Leu
Ser Leu Ala Arg Asn Leu Pro Val Ala Thr Pro Asp Pro 20 25 30 Gly
Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala Val 35 40
45 Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro Cys
50 55 60 Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys
Thr Ser 65 70 75 80 Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys
Asn Glu Ser Cys 85 90 95 Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr
Asn Gly Ser Cys Leu Ala 100 105 110 Ser Arg Lys Thr Ser Phe Met Met
Ala Leu Cys Leu Ser Ser Ile Tyr 115 120 125 Glu Asp Leu Lys Met Tyr
Gln Val Glu Phe Lys Thr Met Asn Ala Lys 130 135 140 Leu Leu Met Asp
Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met Leu 145 150 155 160 Ala
Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu Thr 165 170
175 Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr Lys
180 185 190 Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala
Val Thr 195 200 205 Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser 210
215 8328PRTHomo sapiensIL-12 p40 8Met Cys His Gln Gln Leu Val Ile
Ser Trp Phe Ser Leu Val Phe Leu 1 5 10 15 Ala Ser Pro Leu Val Ala
Ile Trp Glu Leu Lys Lys Asp Val Tyr Val 20 25 30 Val Glu Leu Asp
Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu 35 40 45 Thr Cys
Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln 50 55 60
Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys 65
70 75 80 Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly
Glu Val 85 90 95 Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu
Asp Gly Ile Trp 100 105 110 Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu
Pro Lys Asn Lys Thr Phe 115 120 125 Leu Arg Cys Glu Ala Lys Asn Tyr
Ser Gly Arg Phe Thr Cys Trp Trp 130 135 140 Leu Thr Thr Ile Ser Thr
Asp Leu Thr Phe Ser Val Lys Ser Ser Arg 145 150 155 160 Gly Ser Ser
Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser 165 170 175 Ala
Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu 180 185
190 Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile
195 200 205 Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn
Tyr Thr 210 215 220 Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp
Pro Pro Lys Asn 225 230 235 240 Leu Gln Leu Lys Pro Leu Lys Asn Ser
Arg Gln Val Glu Val Ser Trp 245 250 255 Glu Tyr Pro Asp Thr Trp Ser
Thr Pro His Ser Tyr Phe Ser Leu Thr 260 265 270 Phe Cys Val Gln Val
Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg 275 280 285 Val Phe Thr
Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala 290 295 300 Ser
Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser 305 310
315 320 Glu Trp Ala Ser Val Pro Cys Ser 325 917DNAArtificial
SequenceSynthetic ecdysone receptor response
elementmisc_feature(9)..(9)n is a,c,t,g, or t 9rrggttcant gacacyy
171013DNAArtificial SequenceSynthetic ecdysone receptor response
elementmisc_feature(7)..(7)n is a, c, g, or t 10aggtcanagg tca
131115DNAArtifical SequenceSynthetic ecdysone receptor response
element 11gggttgaatg aattt 151218DNAArtificial SequenceSynthetic
I-SceI homing endonuclease restriction site 12tagggataac agggtaat
181337323DNAArtificial SequenceSynthetic Ad-RTS-hIL-12
(SP1-RheoIL-12) 13catcatcaat aatatacctt attttggatt gaagccaata
tgataatgag ggggtggagt 60ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg
tagtagtgtg gcggaagtgt 120gatgttgcaa gtgtggcgga acacatgtaa
gcgacggatg tggcaaaagt gacgtttttg 180gtgtgcgccg gtgtacacag
gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 240taaatttggg
cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga
300agtgaaatct gaataatttt gtgttactca tagcgcgtaa tatttgtcta
gggagatccg 360gtaccggcgc gcgcgccgtt tggccgcctc gagtctagag
atccggtgag tattaggcgc 420gcaccaggtg ccgcaataaa atatctttat
tttcattaca tctgtgtgtt ggttttttgt 480gtgaatcgat agtactaaca
tacgctctcc atcaaaacaa aacgaaacaa aacaaactag 540caaaataggc
tgtccccagt gcaagtgcag gtgccagaac atttctctat cgataatgca
600ggtcggagta ctgtcctccg agcggagtac tgtcctccga gcggagtact
gtcctccgag 660cggagtactg tcctccgagc ggagtactgt cctccgagcg
gagtactgtc ctccgagcgg 720agactcttcg aaggaagagg ggcggggtcg
atcgaccccg cccctcttcc ttcgaaggaa 780gaggggcggg gtcgaagacc
tagagggtat ataatgggtg ccttagctgg tgtgtgagct 840catcttcctg
tagatcacgc gtgccaccat gggtcaccag cagttggtca tctcttggtt
900ttccctggtt tttctggcat ctcccctcgt ggccatatgg gaactgaaga
aagatgttta 960tgtcgtagaa ttggattggt atccggatgc ccctggagaa
atggtggtcc tcacctgtga 1020cacccctgaa gaagatggta tcacctggac
cttggaccag agcagtgagg tcttaggctc 1080tggcaaaacc ctgaccatcc
aagtcaaaga gtttggagat gctggccagt acacctgtca 1140caaaggaggc
gaggttctaa gccattcgct cctgctgctt cacaaaaagg aagatggaat
1200ttggtccact gatattttaa aggaccagaa agaacccaaa aataagacct
ttctaagatg 1260cgaggccaag aattattctg gacgtttcac ctgctggtgg
ctgacgacaa tcagtactga 1320tttgacattc agtgtcaaaa gcagcagagg
ctcttctgac ccccaagggg tgacgtgcgg 1380agctgctaca ctctctgcag
agagagtcag aggggacaac aaggagtatg agtactcagt 1440ggagtgccag
gaggacagtg cctgcccagc tgctgaggag agtctgccca ttgaggtcat
1500ggtggatgcc gttcacaagc tcaagtatga aaactacacc agcagcttct
tcatcaggga 1560catcatcaaa cctgacccac ccaagaactt gcagctgaag
ccattaaaga attctcggca 1620ggtggaggtc agctgggagt accctgacac
ctggagtact ccacattcct acttctccct 1680gacattctgc gttcaggtcc
agggcaagag caagagagaa aagaaagata gagtcttcac 1740ggacaagacc
tcagccacgg tcatctgccg caaaaatgcc agcattagcg tgcgggccca
1800ggaccgctac tatagctcat cttggagcga atgggcatct gtgccctgca
gttaggttgg 1860gcgagctcga attcattgat cccccgggct gcaggaattc
gatatcaagc tcgggatccg 1920aattccgccc cccccccccc ccccccccta
acgttactgg ccgaagccgc ttggaataag 1980gccggtgtgc gtttgtctat
atgttatttt ccaccatatt gccgtctttt ggcaatgtga 2040gggcccggaa
acctggccct gtcttcttga cgagcattcc taggggtctt tcccctctcg
2100ccaaaggaat gcaaggtctg ttgaatgtcg tgaaggaagc agttcctctg
gaagcttctt 2160gaagacaaac aacgtctgta gcgacccttt gcaggcagcg
gaacccccca cctggcgaca 2220ggtgcctctg cggccaaaag ccacgtgtat
aagatacacc tgcaaaggcg gcacaacccc 2280agtgccacgt tgtgagttgg
atagttgtgg aaagagtcaa atggctctcc tcaagcgtat 2340tcaacaaggg
gctgaaggat gcccagaagg taccccattg tatgggatct gatctggggc
2400ctcggtgcac atgctttaca tgtgtttagt cgaggttaaa aaaacgtcta
ggccccccga 2460accacgggga cgtggttttc ctttgaaaaa cacgatgata
atatggccac aaccatgggt 2520ccagcgcgca gcctcctcct tgtggctacc
ctggtcctcc tggaccacct cagtttggcc 2580agaaacctcc ccgtggccac
tccagaccca ggaatgttcc catgccttca ccactcccaa 2640aacctgctga
gggccgtcag caacatgctc cagaaggcca gacaaactct agaattttac
2700ccttgcactt ctgaagagat tgatcatgaa gatatcacaa aagataaaac
cagcacagtg 2760gaggcctgtt taccattgga attaaccaag aatgagagtt
gcctaaattc cagagagacc 2820tctttcataa ctaatgggag ttgcctggcc
tccagaaaga cctcttttat gatggccctg 2880tgccttagta gtatttatga
agacttgaag atgtaccagg tggagttcaa gaccatgaat 2940gcaaagcttc
tgatggatcc taagaggcag atctttctag atcaaaacat gctggcagtt
3000attgatgagc tgatgcaggc cctgaatttc aacagtgaga ctgtgccaca
aaaatcctcc 3060cttgaagaac cggattttta taaaactaaa atcaagctct
gcatacttct tcatgctttc 3120agaattcggg cagtgactat tgatagagtg
atgagctatc tgaatgcttc ctaacgtacg 3180tcgacatcga gaacttgttt
attgcagctt ataatggtta caaataaagc aatagcatca 3240caaatttcac
aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
3300tcaatgtatc ttatcatgtc tgggcgcgcc ggcctccgcg ccgggttttg
gcgcctcccg 3360cgggcgcccc cctcctcacg gcgagcgctg ccacgtcaga
cgaagggcgc agcgagcgtc 3420ctgatccttc cgcccggacg ctcaggacag
cggcccgctg ctcataagac tcggccttag 3480aaccccagta tcagcagaag
gacattttag gacgggactt gggtgactct agggcactgg 3540ttttctttcc
agagagcgga acaggcgagg aaaagtagtc ccttctcggc gattctgcgg
3600agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccg
ggtgtggcac 3660agctagttcc gtcgcagccg ggatttgggt cgcggttctt
gtttgtggat cgctgtgatc
3720gtcacttggt gagtagcggg ctgctgggct gggtacgtgc gctcggggtt
ggcgagtgtg 3780ttttgtgaag ttttttaggc accttttgaa atgtaatcat
ttgggtcaat atgtaatttt 3840cagtgttaga ctagtaaatt gtccgctaaa
ttctggccgt ttttggcttt tttgttagac 3900gagctagcgc cgccaccatg
ggccctaaaa agaagcgtaa agtcgccccc ccgaccgatg 3960tcagcctggg
ggacgagctc cacttagacg gcgaggacgt ggcgatggcg catgccgacg
4020cgctagacga tttcgatctg gacatgttgg gggacgggga ttccccgggt
ccgggattta 4080ccccccacga ctccgccccc tacggcgctc tggatatggc
cgacttcgag tttgagcaga 4140tgtttaccga tgcccttgga attgacgagt
acggtgggga attcgagatg cctgtggaca 4200ggatcctgga ggcagagctt
gctgtggaac agaagagtga ccagggcgtt gagggtcctg 4260ggggaaccgg
gggtagcggc agcagcccaa atgaccctgt gactaacatc tgtcaggcag
4320ctgacaaaca gctattcacg cttgttgagt gggcgaagag gatcccacac
ttttcctcct 4380tgcctctgga tgatcaggtc atattgctgc gggcaggctg
gaatgaactc ctcattgcct 4440ccttttcaca ccgatccatt gatgttcgag
atggcatcct ccttgccaca ggtcttcacg 4500tgcaccgcaa ctcagcccat
tcagcaggag taggagccat ctttgatcgg gtgctgacag 4560agctagtgtc
caaaatgcgt gacatgagga tggacaagac agagcttggc tgcctgaggg
4620caatcattct gtttaatcca gaggtgaggg gtttgaaatc cgcccaggaa
gttgaacttc 4680tacgtgaaaa agtatatgcc gctttggaag aatatactag
aacaacacat cccgatgaac 4740caggaagatt tgcaaaactt ttgcttcgtc
tgccttcttt acgttccata ggccttaagt 4800gtttggagca tttgtttttc
tttcgcctta ttggagatgt tccaattgat acgttcctga 4860tggagatgct
tgaatcacct tctgattcat aatctagcct agcccccctc tccctccccc
4920ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
tgtctatatg 4980ttattttcca ccatattgcc gtcttttggc aatgtgaggg
cccggaaacc tggccctgtc 5040ttcttgacga gcattcctag gggtctttcc
cctctcgcca aaggaatgca aggtctgttg 5100aatgtcgtga aggaagcagt
tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg 5160accctttgca
ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
5220cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
gagttggata 5280gttgtggaaa gagtcaaatg gctctcctca agcgtattca
acaaggggct gaaggatgcc 5340cagaaggtac cccattgtat gggatctgat
ctggggcctc ggtgcacatg ctttacatgt 5400gtttagtcga ggttaaaaaa
cgtctaggcc ccccgaacca cggggacgtg gttttccttt 5460gaaaaacacg
atctctaggc gccaccatga agctactgtc ttctatcgaa caagcatgcg
5520atatttgccg acttaaaaag ctcaagtgct ccaaagaaaa accgaagtgc
gccaagtgtc 5580tgaagaacaa ctgggagtgt cgctactctc ccaaaaccaa
aaggtctccg ctgactaggg 5640cacatctgac agaagtggaa tcaaggctag
aaagactgga acagctattt ctactgattt 5700ttcctcgaga agaccttgac
atgattttga aaatggattc tttacaggat ataaaagcat 5760tgttaacagg
attatttgta caagataatg tgaataaaga tgccgtcaca gatagattgg
5820cttcagtgga gactgatatg cctctaacat tgagacagca tagaataagt
gcgacatcat 5880catcggaaga gagtagtaac aaaggtcaaa gacagttgac
tgtatcgccg gaattcccgg 5940ggatccggcc tgagtgcgta gtacccgaga
ctcagtgcgc catgaagcgg aaagagaaga 6000aagcacagaa ggagaaggac
aaactgcctg tcagcacgac gacggtggac gaccacatgc 6060cgcccattat
gcagtgtgaa cctccacctc ctgaagcagc aaggattcac gaagtggtcc
6120caaggtttct ctccgacaag ctgttggtga caaaccggca gaaaaacatc
ccccagttga 6180cagccaacca gcagttcctt atcgccaggc tcatctggta
ccaggacggg tacgagcagc 6240cttctgatga agatttgaag aggattacgc
agacgtggca gcaagcggac gatgaaaacg 6300aagagtcgga cactcccttc
cgccagatca cagagatgac tatcctcacg gtccaactta 6360tcgtggagtt
cgcgaaggga ttgccagggt tcgccaagat ctcgcagcct gatcaaatta
6420cgctgcttaa ggcttgctca agtgaggtaa tgatgctccg agtcgcgcga
cgatacgatg 6480cggcctcaga cagtattctg ttcgcgaaca accaagcgta
cactcgcgac aactaccgca 6540aggctggcat ggccgaggtc atcgaggatc
tactgcactt ctgccggtgc atgtactcta 6600tggcgttgga caacatccat
tacgcgctgc tcacggctgt cgtcatcttt tctgaccggc 6660cagggttgga
gcagccgcaa ctggtggaag agatccagcg gtactacctg aatacgctcc
6720gcatctatat cctgaaccag ctgagcgggt cggcgcgttc gtccgtcata
tacggcaaga 6780tcctctcaat cctctctgag ctacgcacgc tcggcatgca
aaactccaac atgtgcatct 6840ccctcaagct caagaacaga aagctgccgc
ctttcctcga ggagatctgg gatgtggcgg 6900acatgtcgca cacccaaccg
ccgcctatcc tcgagtcccc cacgaatctc taggcggcct 6960ctagagcggc
cgccaccgcg gggagatcca gacatgataa gatacattga tgagtttgga
7020caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
tgatgctatt 7080gctttatttg taaccattat aagctgcaat aaacaagtta
acaacaacaa ttgcattcat 7140tttatgtttc aggttcaggg ggaggtgtgg
gaggtttttt aaagcaagta aaacctctac 7200aaatgtggta tggctgatta
tgatccggct gcctcgcgcg tttcggtgat gacggtgaaa 7260acctctgaca
catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga
7320gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc
gcagccatga 7380ggtcgactct agtccccgcg gtggcagatc tggaaggtgc
tgaggtacga tgagacccgc 7440accaggtgca gaccctgcga gtgtggcggt
aaacatatta ggaaccagcc tgtgatgctg 7500gatgtgaccg aggagctgag
gcccgatcac ttggtgctgg cctgcacccg cgctgagttt 7560ggctctagcg
atgaagatac agattgaggt actgaaatgt gtgggcgtgg cttaagggtg
7620ggaaagaata tataaggtgg gggtcttatg tagttttgta tctgttttgc
agcagccgcc 7680gccgccatga gcaccaactc gtttgatgga agcattgtga
gctcatattt gacaacgcgc 7740atgcccccat gggccggggt gcgtcagaat
gtgatgggct ccagcattga tggtcgcccc 7800gtcctgcccg caaactctac
taccttgacc tacgagaccg tgtctggaac gccgttggag 7860actgcagcct
ccgccgccgc ttcagccgct gcagccaccg cccgcgggat tgtgactgac
7920tttgctttcc tgagcccgct tgcaagcagt gcagcttccc gttcatccgc
ccgcgatgac 7980aagttgacgg ctcttttggc acaattggat tctttgaccc
gggaacttaa tgtcgtttct 8040cagcagctgt tggatctgcg ccagcaggtt
tctgccctga aggcttcctc ccctcccaat 8100gcggtttaaa acataaataa
aaaaccagac tctgtttgga tttggatcaa gcaagtgtct 8160tgctgtcttt
atttaggggt tttgcgcgcg cggtaggccc gggaccagcg gtctcggtcg
8220ttgagggtcc tgtgtatttt ttccaggacg tggtaaaggt gactctggat
gttcagatac 8280atgggcataa gcccgtctct ggggtggagg tagcaccact
gcagagcttc atgctgcggg 8340gtggtgttgt agatgatcca gtcgtagcag
gagcgctggg cgtggtgcct aaaaatgtct 8400ttcagtagca agctgattgc
caggggcagg cccttggtgt aagtgtttac aaagcggtta 8460agctgggatg
ggtgcatacg tggggatatg agatgcatct tggactgtat ttttaggttg
8520gctatgttcc cagccatatc cctccgggga ttcatgttgt gcagaaccac
cagcacagtg 8580tatccggtgc acttgggaaa tttgtcatgt agcttagaag
gaaatgcgtg gaagaacttg 8640gagacgccct tgtgacctcc aagattttcc
atgcattcgt ccataatgat ggcaatgggc 8700ccacgggcgg cggcctgggc
gaagatattt ctgggatcac taacgtcata gttgtgttcc 8760aggatgagat
cgtcataggc catttttaca aagcgcgggc ggagggtgcc agactgcggt
8820ataatggttc catccggccc aggggcgtag ttaccctcac agatttgcat
ttcccacgct 8880ttgagttcag atggggggat catgtctacc tgcggggcga
tgaagaaaac ggtttccggg 8940gtaggggaga tcagctggga agaaagcagg
ttcctgagca gctgcgactt accgcagccg 9000gtgggcccgt aaatcacacc
tattaccggc tgcaactggt agttaagaga gctgcagctg 9060ccgtcatccc
tgagcagggg ggccacttcg ttaagcatgt ccctgactcg catgttttcc
9120ctgaccaaat ccgccagaag gcgctcgccg cccagcgata gcagttcttg
caaggaagca 9180aagtttttca acggtttgag accgtccgcc gtaggcatgc
ttttgagcgt ttgaccaagc 9240agttccaggc ggtcccacag ctcggtcacc
tgctctacgg catctcgatc cagcatatct 9300cctcgtttcg cgggttgggg
cggctttcgc tgtacggcag tagtcggtgc tcgtccagac 9360gggccagggt
catgtctttc cacgggcgca gggtcctcgt cagcgtagtc tgggtcacgg
9420tgaaggggtg cgctccgggc tgcgcgctgg ccagggtgcg cttgaggctg
gtcctgctgg 9480tgctgaagcg ctgccggtct tcgccctgcg cgtcggccag
gtagcatttg accatggtgt 9540catagtccag cccctccgcg gcgtggccct
tggcgcgcag cttgcccttg gaggaggcgc 9600cgcacgaggg gcagtgcaga
cttttgaggg cgtagagctt gggcgcgaga aataccgatt 9660ccggggagta
ggcatccgcg ccgcaggccc cgcagacggt ctcgcattcc acgagccagg
9720tgagctctgg ccgttcgggg tcaaaaacca ggtttccccc atgctttttg
atgcgtttct 9780tacctctggt ttccatgagc cggtgtccac gctcggtgac
gaaaaggctg tccgtgtccc 9840cgtatacaga cttgagaggc ctgtcctcga
gcggtgttcc gcggtcctcc tcgtatagaa 9900actcggacca ctctgagaca
aaggctcgcg tccaggccag cacgaaggag gctaagtggg 9960aggggtagcg
gtcgttgtcc actagggggt ccactcgctc cagggtgtga agacacatgt
10020cgccctcttc ggcatcaagg aaggtgattg gtttgtaggt gtaggccacg
tgaccgggtg 10080ttcctgaagg ggggctataa aagggggtgg gggcgcgttc
gtcctcactc tcttccgcat 10140cgctgtctgc gagggccagc tgttggggtg
agtactccct ctgaaaagcg ggcatgactt 10200ctgcgctaag attgtcagtt
tccaaaaacg aggaggattt gatattcacc tggcccgcgg 10260tgatgccttt
gagggtggcc gcatccatct ggtcagaaaa gacaatcttt ttgttgtcaa
10320gcttggtggc aaacgacccg tagagggcgt tggacagcaa cttggcgatg
gagcgcaggg 10380tttggttttt gtcgcgatcg gcgcgctcct tggccgcgat
gtttagctgc acgtattcgc 10440gcgcaacgca ccgccattcg ggaaagacgg
tggtgcgctc gtcgggcacc aggtgcacgc 10500gccaaccgcg gttgtgcagg
gtgacaaggt caacgctggt ggctacctct ccgcgtaggc 10560gctcgttggt
ccagcagagg cggccgccct tgcgcgagca gaatggcggt agggggtcta
10620gctgcgtctc gtccgggggg tctgcgtcca cggtaaagac cccgggcagc
aggcgcgcgt 10680cgaagtagtc tatcttgcat ccttgcaagt ctagcgcctg
ctgccatgcg cgggcggcaa 10740gcgcgcgctc gtatgggttg agtgggggac
cccatggcat ggggtgggtg agcgcggagg 10800cgtacatgcc gcaaatgtcg
taaacgtaga ggggctctct gagtattcca agatatgtag 10860ggtagcatct
tccaccgcgg atgctggcgc gcacgtaatc gtatagttcg tgcgagggag
10920cgaggaggtc gggaccgagg ttgctacggg cgggctgctc tgctcggaag
actatctgcc 10980tgaagatggc atgtgagttg gatgatatgg ttggacgctg
gaagacgttg aagctggcgt 11040ctgtgagacc taccgcgtca cgcacgaagg
aggcgtagga gtcgcgcagc ttgttgacca 11100gctcggcggt gacctgcacg
tctagggcgc agtagtccag ggtttccttg atgatgtcat 11160acttatcctg
tccctttttt ttccacagct cgcggttgag gacaaactct tcgcggtctt
11220tccagtactc ttggatcgga aacccgtcgg cctccgaacg gtaagagcct
agcatgtaga 11280actggttgac ggcctggtag gcgcagcatc ccttttctac
gggtagcgcg tatgcctgcg 11340cggccttccg gagcgaggtg tgggtgagcg
caaaggtgtc cctgaccatg actttgaggt 11400actggtattt gaagtcagtg
tcgtcgcatc cgccctgctc ccagagcaaa aagtccgtgc 11460gctttttgga
acgcggattt ggcagggcga aggtgacatc gttgaagagt atctttcccg
11520cgcgaggcat aaagttgcgt gtgatgcgga agggtcccgg cacctcggaa
cggttgttaa 11580ttacctgggc ggcgagcacg atctcgtcaa agccgttgat
gttgtggccc acaatgtaaa 11640gttccaagaa gcgcgggatg cccttgatgg
aaggcaattt tttaagttcc tcgtaggtga 11700gctcttcagg ggagctgagc
ccgtgctctg aaagggccca gtctgcaaga tgagggttgg 11760aagcgacgaa
tgagctccac aggtcacggg ccattagcat ttgcaggtgg tcgcgaaagg
11820tcctaaactg gcgacctatg gccatttttt ctggggtgat gcagtagaag
gtaagcgggt 11880cttgttccca gcggtcccat ccaaggttcg cggctaggtc
tcgcgcggca gtcactagag 11940gctcatctcc gccgaacttc atgaccagca
tgaagggcac gagctgcttc ccaaaggccc 12000ccatccaagt ataggtctct
acatcgtagg tgacaaagag acgctcggtg cgaggatgcg 12060agccgatcgg
gaagaactgg atctcccgcc accaattgga ggagtggcta ttgatgtggt
12120gaaagtagaa gtccctgcga cgggccgaac actcgtgctg gcttttgtaa
aaacgtgcgc 12180agtactggca gcggtgcacg ggctgtacat cctgcacgag
gttgacctga cgaccgcgca 12240caaggaagca gagtgggaat ttgagcccct
cgcctggcgg gtttggctgg tggtcttcta 12300cttcggctgc ttgtccttga
ccgtctggct gctcgagggg agttacggtg gatcggacca 12360ccacgccgcg
cgagcccaaa gtccagatgt ccgcgcgcgg cggtcggagc ttgatgacaa
12420catcgcgcag atgggagctg tccatggtct ggagctcccg cggcgtcagg
tcaggcggga 12480gctcctgcag gtttacctcg catagacggg tcagggcgcg
ggctagatcc aggtgatacc 12540taatttccag gggctggttg gtggcggcgt
cgatggcttg caagaggccg catccccgcg 12600gcgcgactac ggtaccgcgc
ggcgggcggt gggccgcggg ggtgtccttg gatgatgcat 12660ctaaaagcgg
tgacgcgggc gagcccccgg aggtaggggg ggctccggac ccgccgggag
12720agggggcagg ggcacgtcgg cgccgcgcgc gggcaggagc tggtgctgcg
cgcgtaggtt 12780gctggcgaac gcgacgacgc ggcggttgat ctcctgaatc
tggcgcctct gcgtgaagac 12840gacgggcccg gtgagcttga acctgaaaga
gagttcgaca gaatcaattt cggtgtcgtt 12900gacggcggcc tggcgcaaaa
tctcctgcac gtctcctgag ttgtcttgat aggcgatctc 12960ggccatgaac
tgctcgatct cttcctcctg gagatctccg cgtccggctc gctccacggt
13020ggcggcgagg tcgttggaaa tgcgggccat gagctgcgag aaggcgttga
ggcctccctc 13080gttccagacg cggctgtaga ccacgccccc ttcggcatcg
cgggcgcgca tgaccacctg 13140cgcgagattg agctccacgt gccgggcgaa
gacggcgtag tttcgcaggc gctgaaagag 13200gtagttgagg gtggtggcgg
tgtgttctgc cacgaagaag tacataaccc agcgtcgcaa 13260cgtggattcg
ttgatatccc ccaaggcctc aaggcgctcc atggcctcgt agaagtccac
13320ggcgaagttg aaaaactggg agttgcgcgc cgacacggtt aactcctcct
ccagaagacg 13380gatgagctcg gcgacagtgt cgcgcacctc gcgctcaaag
gctacagggg cctcttcttc 13440ttcttcaatc tcctcttcca taagggcctc
cccttcttct tcttctggcg gcggtggggg 13500aggggggaca cggcggcgac
gacggcgcac cgggaggcgg tcgacaaagc gctcgatcat 13560ctccccgcgg
cgacggcgca tggtctcggt gacggcgcgg ccgttctcgc gggggcgcag
13620ttggaagacg ccgcccgtca tgtcccggtt atgggttggc ggggggctgc
catgcggcag 13680ggatacggcg ctaacgatgc atctcaacaa ttgttgtgta
ggtactccgc cgccgaggga 13740cctgagcgag tccgcatcga ccggatcgga
aaacctctcg agaaaggcgt ctaaccagtc 13800acagtcgcaa ggtaggctga
gcaccgtggc gggcggcagc gggcggcggt cggggttgtt 13860tctggcggag
gtgctgctga tgatgtaatt aaagtaggcg gtcttgagac ggcggatggt
13920cgacagaagc accatgtcct tgggtccggc ctgctgaatg cgcaggcggt
cggccatgcc 13980ccaggcttcg ttttgacatc ggcgcaggtc tttgtagtag
tcttgcatga gcctttctac 14040cggcacttct tcttctcctt cctcttgtcc
tgcatctctt gcatctatcg ctgcggcggc 14100ggcggagttt ggccgtaggt
ggcgccctct tcctcccatg cgtgtgaccc cgaagcccct 14160catcggctga
agcagggcta ggtcggcgac aacgcgctcg gctaatatgg cctgctgcac
14220ctgcgtgagg gtagactgga agtcatccat gtccacaaag cggtggtatg
cgcccgtgtt 14280gatggtgtaa gtgcagttgg ccataacgga ccagttaacg
gtctggtgac ccggctgcga 14340gagctcggtg tacctgagac gcgagtaagc
cctcgagtca aatacgtagt cgttgcaagt 14400ccgcaccagg tactggtatc
ccaccaaaaa gtgcggcggc ggctggcggt agaggggcca 14460gcgtagggtg
gccggggctc cgggggcgag atcttccaac ataaggcgat gatatccgta
14520gatgtacctg gacatccagg tgatgccggc ggcggtggtg gaggcgcgcg
gaaagtcgcg 14580gacgcggttc cagatgttgc gcagcggcaa aaagtgctcc
atggtcggga cgctctggcc 14640ggtcaggcgc gcgcaatcgt tgacgctcta
gcgtgcaaaa ggagagcctg taagcgggca 14700ctcttccgtg gtctggtgga
taaattcgca agggtatcat ggcggacgac cggggttcga 14760gccccgtatc
cggccgtccg ccgtgatcca tgcggttacc gcccgcgtgt cgaacccagg
14820tgtgcgacgt cagacaacgg gggagtgctc cttttggctt ccttccaggc
gcggcggctg 14880ctgcgctagc ttttttggcc actggccgcg cgcagcgtaa
gcggttaggc tggaaagcga 14940aagcattaag tggctcgctc cctgtagccg
gagggttatt ttccaagggt tgagtcgcgg 15000gacccccggt tcgagtctcg
gaccggccgg actgcggcga acgggggttt gcctccccgt 15060catgcaagac
cccgcttgca aattcctccg gaaacaggga cgagcccctt ttttgctttt
15120cccagatgca tccggtgctg cggcagatgc gcccccctcc tcagcagcgg
caagagcaag 15180agcagcggca gacatgcagg gcaccctccc ctcctcctac
cgcgtcagga ggggcgacat 15240ccgcggttga cgcggcagca gatggtgatt
acgaaccccc gcggcgccgg gcccggcact 15300acctggactt ggaggagggc
gagggcctgg cgcggctagg agcgccctct cctgagcggc 15360acccaagggt
gcagctgaag cgtgatacgc gtgaggcgta cgtgccgcgg cagaacctgt
15420ttcgcgaccg cgagggagag gagcccgagg agatgcggga tcgaaagttc
cacgcagggc 15480gcgagctgcg gcatggcctg aatcgcgagc ggttgctgcg
cgaggaggac tttgagcccg 15540acgcgcgaac cgggattagt cccgcgcgcg
cacacgtggc ggccgccgac ctggtaaccg 15600catacgagca gacggtgaac
caggagatta actttcaaaa aagctttaac aaccacgtgc 15660gtacgcttgt
ggcgcgcgag gaggtggcta taggactgat gcatctgtgg gactttgtaa
15720gcgcgctgga gcaaaaccca aatagcaagc cgctcatggc gcagctgttc
cttatagtgc 15780agcacagcag ggacaacgag gcattcaggg atgcgctgct
aaacatagta gagcccgagg 15840gccgctggct gctcgatttg ataaacatcc
tgcagagcat agtggtgcag gagcgcagct 15900tgagcctggc tgacaaggtg
gccgccatca actattccat gcttagcctg ggcaagtttt 15960acgcccgcaa
gatataccat accccttacg ttcccataga caaggaggta aagatcgagg
16020ggttctacat gcgcatggcg ctgaaggtgc ttaccttgag cgacgacctg
ggcgtttatc 16080gcaacgagcg catccacaag gccgtgagcg tgagccggcg
gcgcgagctc agcgaccgcg 16140agctgatgca cagcctgcaa agggccctgg
ctggcacggg cagcggcgat agagaggccg 16200agtcctactt tgacgcgggc
gctgacctgc gctgggcccc aagccgacgc gccctggagg 16260cagctggggc
cggacctggg ctggcggtgg cacccgcgcg cgctggcaac gtcggcggcg
16320tggaggaata tgacgaggac gatgagtacg agccagagga cggcgagtac
taagcggtga 16380tgtttctgat cagatgatgc aagacgcaac ggacccggcg
gtgcgggcgg cgctgcagag 16440ccagccgtcc ggccttaact ccacggacga
ctggcgccag gtcatggacc gcatcatgtc 16500gctgactgcg cgcaatcctg
acgcgttccg gcagcagccg caggccaacc ggctctccgc 16560aattctggaa
gcggtggtcc cggcgcgcgc aaaccccacg cacgagaagg tgctggcgat
16620cgtaaacgcg ctggccgaaa acagggccat ccggcccgac gaggccggcc
tggtctacga 16680cgcgctgctt cagcgcgtgg ctcgttacaa cagcggcaac
gtgcagacca acctggaccg 16740gctggtgggg gatgtgcgcg aggccgtggc
gcagcgtgag cgcgcgcagc agcagggcaa 16800cctgggctcc atggttgcac
taaacgcctt cctgagtaca cagcccgcca acgtgccgcg 16860gggacaggag
gactacacca actttgtgag cgcactgcgg ctaatggtga ctgagacacc
16920gcaaagtgag gtgtaccagt ctgggccaga ctattttttc cagaccagta
gacaaggcct 16980gcagaccgta aacctgagcc aggctttcaa aaacttgcag
gggctgtggg gggtgcgggc 17040tcccacaggc gaccgcgcga ccgtgtctag
cttgctgacg cccaactcgc gcctgttgct 17100gctgctaata gcgcccttca
cggacagtgg cagcgtgtcc cgggacacat acctaggtca 17160cttgctgaca
ctgtaccgcg aggccatagg tcaggcgcat gtggacgagc atactttcca
17220ggagattaca agtgtcagcc gcgcgctggg gcaggaggac acgggcagcc
tggaggcaac 17280cctaaactac ctgctgacca accggcggca gaagatcccc
tcgttgcaca gtttaaacag 17340cgaggaggag cgcattttgc gctacgtgca
gcagagcgtg agccttaacc tgatgcgcga 17400cggggtaacg cccagcgtgg
cgctggacat gaccgcgcgc aacatggaac cgggcatgta 17460tgcctcaaac
cggccgttta tcaaccgcct aatggactac ttgcatcgcg cggccgccgt
17520gaaccccgag tatttcacca atgccatctt gaacccgcac tggctaccgc
cccctggttt 17580ctacaccggg ggattcgagg tgcccgaggg taacgatgga
ttcctctggg acgacataga 17640cgacagcgtg ttttccccgc aaccgcagac
cctgctagag ttgcaacagc gcgagcaggc 17700agaggcggcg ctgcgaaagg
aaagcttccg caggccaagc agcttgtccg atctaggcgc 17760tgcggccccg
cggtcagatg ctagtagccc atttccaagc ttgatagggt ctcttaccag
17820cactcgcacc acccgcccgc gcctgctggg cgaggaggag tacctaaaca
actcgctgct 17880gcagccgcag cgcgaaaaaa acctgcctcc ggcatttccc
aacaacggga tagagagcct 17940agtggacaag atgagtagat ggaagacgta
cgcgcaggag cacagggacg tgccaggccc 18000gcgcccgccc acccgtcgtc
aaaggcacga ccgtcagcgg ggtctggtgt gggaggacga 18060tgactcggca
gacgacagca gcgtcctgga tttgggaggg agtggcaacc cgtttgcgca
18120ccttcgcccc aggctgggga gaatgtttta aaaaaaaaaa aagcatgatg
caaaataaaa 18180aactcaccaa ggccatggca ccgagcgttg gttttcttgt
attcccctta gtatgcggcg 18240cgcggcgatg tatgaggaag gtcctcctcc
ctcctacgag agtgtggtga gcgcggcgcc 18300agtggcggcg gcgctgggtt
ctcccttcga tgctcccctg gacccgccgt ttgtgcctcc 18360gcggtacctg
cggcctaccg gggggagaaa cagcatccgt tactctgagt tggcacccct
18420attcgacacc acccgtgtgt acctggtgga caacaagtca acggatgtgg
catccctgaa 18480ctaccagaac gaccacagca actttctgac cacggtcatt
caaaacaatg actacagccc 18540gggggaggca agcacacaga ccatcaatct
tgacgaccgg tcgcactggg gcggcgacct 18600gaaaaccatc ctgcatacca
acatgccaaa tgtgaacgag ttcatgttta ccaataagtt 18660taaggcgcgg
gtgatggtgt cgcgcttgcc tactaaggac aatcaggtgg agctgaaata
18720cgagtgggtg gagttcacgc tgcccgaggg caactactcc gagaccatga
ccatagacct
18780tatgaacaac gcgatcgtgg agcactactt gaaagtgggc agacagaacg
gggttctgga 18840aagcgacatc ggggtaaagt ttgacacccg caacttcaga
ctggggtttg accccgtcac 18900tggtcttgtc atgcctgggg tatatacaaa
cgaagccttc catccagaca tcattttgct 18960gccaggatgc ggggtggact
tcacccacag ccgcctgagc aacttgttgg gcatccgcaa 19020gcggcaaccc
ttccaggagg gctttaggat cacctacgat gatctggagg gtggtaacat
19080tcccgcactg ttggatgtgg acgcctacca ggcgagcttg aaagatgaca
ccgaacaggg 19140cgggggtggc gcaggcggca gcaacagcag tggcagcggc
gcggaagaga actccaacgc 19200ggcagccgcg gcaatgcagc cggtggagga
catgaacgat catgccattc gcggcgacac 19260ctttgccaca cgggctgagg
agaagcgcgc tgaggccgaa gcagcggccg aagctgccgc 19320ccccgctgcg
caacccgagg tcgagaagcc tcagaagaaa ccggtgatca aacccctgac
19380agaggacagc aagaaacgca gttacaacct aataagcaat gacagcacct
tcacccagta 19440ccgcagctgg taccttgcat acaactacgg cgaccctcag
accggaatcc gctcatggac 19500cctgctttgc actcctgacg taacctgcgg
ctcggagcag gtctactggt cgttgccaga 19560catgatgcaa gaccccgtga
ccttccgctc cacgcgccag atcagcaact ttccggtggt 19620gggcgccgag
ctgttgcccg tgcactccaa gagcttctac aacgaccagg ccgtctactc
19680ccaactcatc cgccagttta cctctctgac ccacgtgttc aatcgctttc
ccgagaacca 19740gattttggcg cgcccgccag cccccaccat caccaccgtc
agtgaaaacg ttcctgctct 19800cacagatcac gggacgctac cgctgcgcaa
cagcatcgga ggagtccagc gagtgaccat 19860tactgacgcc agacgccgca
cctgccccta cgtttacaag gccctgggca tagtctcgcc 19920gcgcgtccta
tcgagccgca ctttttgagc aagcatgtcc atccttatat cgcccagcaa
19980taacacaggc tggggcctgc gcttcccaag caagatgttt ggcggggcca
agaagcgctc 20040cgaccaacac ccagtgcgcg tgcgcgggca ctaccgcgcg
ccctggggcg cgcacaaacg 20100cggccgcact gggcgcacca ccgtcgatga
cgccatcgac gcggtggtgg aggaggcgcg 20160caactacacg cccacgccgc
caccagtgtc cacagtggac gcggccattc agaccgtggt 20220gcgcggagcc
cggcgctatg ctaaaatgaa gagacggcgg aggcgcgtag cacgtcgcca
20280ccgccgccga cccggcactg ccgcccaacg cgcggcggcg gccctgctta
accgcgcacg 20340tcgcaccggc cgacgggcgg ccatgcgggc cgctcgaagg
ctggccgcgg gtattgtcac 20400tgtgcccccc aggtccaggc gacgagcggc
cgccgcagca gccgcggcca ttagtgctat 20460gactcagggt cgcaggggca
acgtgtattg ggtgcgcgac tcggttagcg gcctgcgcgt 20520gcccgtgcgc
acccgccccc cgcgcaacta gattgcaaga aaaaactact tagactcgta
20580ctgttgtatg tatccagcgg cggcggcgcg caacgaagct atgtccaagc
gcaaaatcaa 20640agaagagatg ctccaggtca tcgcgccgga gatctatggc
cccccgaaga aggaagagca 20700ggattacaag ccccgaaagc taaagcgggt
caaaaagaaa aagaaagatg atgatgatga 20760acttgacgac gaggtggaac
tgctgcacgc taccgcgccc aggcgacggg tacagtggaa 20820aggtcgacgc
gtaaaacgtg ttttgcgacc cggcaccacc gtagtcttta cgcccggtga
20880gcgctccacc cgcacctaca agcgcgtgta tgatgaggtg tacggcgacg
aggacctgct 20940tgagcaggcc aacgagcgcc tcggggagtt tgcctacgga
aagcggcata aggacatgct 21000ggcgttgccg ctggacgagg gcaacccaac
acctagccta aagcccgtaa cactgcagca 21060ggtgctgccc gcgcttgcac
cgtccgaaga aaagcgcggc ctaaagcgcg agtctggtga 21120cttggcaccc
accgtgcagc tgatggtacc caagcgccag cgactggaag atgtcttgga
21180aaaaatgacc gtggaacctg ggctggagcc cgaggtccgc gtgcggccaa
tcaagcaggt 21240ggcgccggga ctgggcgtgc agaccgtgga cgttcagata
cccactacca gtagcaccag 21300tattgccacc gccacagagg gcatggagac
acaaacgtcc ccggttgcct cagcggtggc 21360ggatgccgcg gtgcaggcgg
tcgctgcggc cgcgtccaag acctctacgg aggtgcaaac 21420ggacccgtgg
atgtttcgcg tttcagcccc ccggcgcccg cgccgttcga ggaagtacgg
21480cgccgccagc gcgctactgc ccgaatatgc cctacatcct tccattgcgc
ctacccccgg 21540ctatcgtggc tacacctacc gccccagaag acgagcaact
acccgacgcc gaaccaccac 21600tggaacccgc cgccgccgtc gccgtcgcca
gcccgtgctg gccccgattt ccgtgcgcag 21660ggtggctcgc gaaggaggca
ggaccctggt gctgccaaca gcgcgctacc accccagcat 21720cgtttaaaag
ccggtctttg tggttcttgc agatatggcc ctcacctgcc gcctccgttt
21780cccggtgccg ggattccgag gaagaatgca ccgtaggagg ggcatggccg
gccacggcct 21840gacgggcggc atgcgtcgtg cgcaccaccg gcggcggcgc
gcgtcgcacc gtcgcatgcg 21900cggcggtatc ctgcccctcc ttattccact
gatcgccgcg gcgattggcg ccgtgcccgg 21960aattgcatcc gtggccttgc
aggcgcagag acactgatta aaaacaagtt gcatgtggaa 22020aaatcaaaat
aaaaagtctg gactctcacg ctcgcttggt cctgtaacta ttttgtagaa
22080tggaagacat caactttgcg tctctggccc cgcgacacgg ctcgcgcccg
ttcatgggaa 22140actggcaaga tatcggcacc agcaatatga gcggtggcgc
cttcagctgg ggctcgctgt 22200ggagcggcat taaaaatttc ggttccaccg
ttaagaacta tggcagcaag gcctggaaca 22260gcagcacagg ccagatgctg
agggataagt tgaaagagca aaatttccaa caaaaggtgg 22320tagatggcct
ggcctctggc attagcgggg tggtggacct ggccaaccag gcagtgcaaa
22380ataagattaa cagtaagctt gatccccgcc ctcccgtaga ggagcctcca
ccggccgtgg 22440agacagtgtc tccagagggg cgtggcgaaa agcgtccgcg
ccccgacagg gaagaaactc 22500tggtgacgca aatagacgag cctccctcgt
acgaggaggc actaaagcaa ggcctgccca 22560ccacccgtcc catcgcgccc
atggctaccg gagtgctggg ccagcacaca cccgtaacgc 22620tggacctgcc
tccccccgcc gacacccagc agaaacctgt gctgccaggc ccgaccgccg
22680ttgttgtaac ccgtcctagc cgcgcgtccc tgcgccgcgc cgccagcggt
ccgcgatcgt 22740tgcggcccgt agccagtggc aactggcaaa gcacactgaa
cagcatcgtg ggtctggggg 22800tgcaatccct gaagcgccga cgatgcttct
gatagctaac gtgtcgtatg tgtgtcatgt 22860atgcgtccat gtcgccgcca
gaggagctgc tgagccgccg cgcgcccgct ttccaagatg 22920gctacccctt
cgatgatgcc gcagtggtct tacatgcaca tctcgggcca ggacgcctcg
22980gagtacctga gccccgggct ggtgcagttt gcccgcgcca ccgagacgta
cttcagcctg 23040aataacaagt ttagaaaccc cacggtggcg cctacgcacg
acgtgaccac agaccggtcc 23100cagcgtttga cgctgcggtt catccctgtg
gaccgtgagg atactgcgta ctcgtacaag 23160gcgcggttca ccctagctgt
gggtgataac cgtgtgctgg acatggcttc cacgtacttt 23220gacatccgcg
gcgtgctgga caggggccct acttttaagc cctactctgg cactgcctac
23280aacgccctgg ctcccaaggg tgccccaaat ccttgcgaat gggatgaagc
tgctactgct 23340cttgaaataa acctagaaga agaggacgat gacaacgaag
acgaagtaga cgagcaagct 23400gagcagcaaa aaactcacgt atttgggcag
gcgccttatt ctggtataaa tattacaaag 23460gagggtattc aaataggtgt
cgaaggtcaa acacctaaat atgccgataa aacatttcaa 23520cctgaacctc
aaataggaga atctcagtgg tacgaaacag aaattaatca tgcagctggg
23580agagtcctaa aaaagactac cccaatgaaa ccatgttacg gttcatatgc
aaaacccaca 23640aatgaaaatg gagggcaagg cattcttgta aagcaacaaa
atggaaagct agaaagtcaa 23700gtggaaatgc aatttttctc aactactgag
gcagccgcag gcaatggtga taacttgact 23760cctaaagtgg tattgtacag
tgaagatgta gatatagaaa ccccagacac tcatatttct 23820tacatgccca
ctattaagga aggtaactca cgagaactaa tgggccaaca atctatgccc
23880aacaggccta attacattgc ttttagggac aattttattg gtctaatgta
ttacaacagc 23940acgggtaata tgggtgttct ggcgggccaa gcatcgcagt
tgaatgctgt tgtagatttg 24000caagacagaa acacagagct ttcataccag
cttttgcttg attccattgg tgatagaacc 24060aggtactttt ctatgtggaa
tcaggctgtt gacagctatg atccagatgt tagaattatt 24120gaaaatcatg
gaactgaaga tgaacttcca aattactgct ttccactggg aggtgtgatt
24180aatacagaga ctcttaccaa ggtaaaacct aaaacaggtc aggaaaatgg
atgggaaaaa 24240gatgctacag aattttcaga taaaaatgaa ataagagttg
gaaataattt tgccatggaa 24300atcaatctaa atgccaacct gtggagaaat
ttcctgtact ccaacatagc gctgtatttg 24360cccgacaagc taaagtacag
tccttccaac gtaaaaattt ctgataaccc aaacacctac 24420gactacatga
acaagcgagt ggtggctccc gggctagtgg actgctacat taaccttgga
24480gcacgctggt cccttgacta tatggacaac gtcaacccat ttaaccacca
ccgcaatgct 24540ggcctgcgct accgctcaat gttgctgggc aatggtcgct
atgtgccctt ccacatccag 24600gtgcctcaga agttctttgc cattaaaaac
ctccttctcc tgccgggctc atacacctac 24660gagtggaact tcaggaagga
tgttaacatg gttctgcaga gctccctagg aaatgaccta 24720agggttgacg
gagccagcat taagtttgat agcatttgcc tttacgccac cttcttcccc
24780atggcccaca acaccgcctc cacgcttgag gccatgctta gaaacgacac
caacgaccag 24840tcctttaacg actatctctc cgccgccaac atgctctacc
ctatacccgc caacgctacc 24900aacgtgccca tatccatccc ctcccgcaac
tgggcggctt tccgcggctg ggccttcacg 24960cgccttaaga ctaaggaaac
cccatcactg ggctcgggct acgaccctta ttacacctac 25020tctggctcta
taccctacct agatggaacc ttttacctca accacacctt taagaaggtg
25080gccattacct ttgactcttc tgtcagctgg cctggcaatg accgcctgct
tacccccaac 25140gagtttgaaa ttaagcgctc agttgacggg gagggttaca
acgttgccca gtgtaacatg 25200accaaagact ggttcctggt acaaatgcta
gctaactata acattggcta ccagggcttc 25260tatatcccag agagctacaa
ggaccgcatg tactccttct ttagaaactt ccagcccatg 25320agccgtcagg
tggtggatga tactaaatac aaggactacc aacaggtggg catcctacac
25380caacacaaca actctggatt tgttggctac cttgccccca ccatgcgcga
aggacaggcc 25440taccctgcta acttccccta tccgcttata ggcaagaccg
cagttgacag cattacccag 25500aaaaagtttc tttgcgatcg caccctttgg
cgcatcccat tctccagtaa ctttatgtcc 25560atgggcgcac tcacagacct
gggccaaaac cttctctacg ccaactccgc ccacgcgcta 25620gacatgactt
ttgaggtgga tcccatggac gagcccaccc ttctttatgt tttgtttgaa
25680gtctttgacg tggtccgtgt gcaccagccg caccgcggcg tcatcgaaac
cgtgtacctg 25740cgcacgccct tctcggccgg caacgccaca acataaagaa
gcaagcaaca tcaacaacag 25800ctgccgccat gggctccagt gagcaggaac
tgaaagccat tgtcaaagat cttggttgtg 25860ggccatattt tttgggcacc
tatgacaagc gctttccagg ctttgtttct ccacacaagc 25920tcgcctgcgc
catagtcaat acggccggtc gcgagactgg gggcgtacac tggatggcct
25980ttgcctggaa cccgcactca aaaacatgct acctctttga gccctttggc
ttttctgacc 26040agcgactcaa gcaggtttac cagtttgagt acgagtcact
cctgcgccgt agcgccattg 26100cttcttcccc cgaccgctgt ataacgctgg
aaaagtccac ccaaagcgta caggggccca 26160actcggccgc ctgtggacta
ttctgctgca tgtttctcca cgcctttgcc aactggcccc 26220aaactcccat
ggatcacaac cccaccatga accttattac cggggtaccc aactccatgc
26280tcaacagtcc ccaggtacag cccaccctgc gtcgcaacca ggaacagctc
tacagcttcc 26340tggagcgcca ctcgccctac ttccgcagcc acagtgcgca
gattaggagc gccacttctt 26400tttgtcactt gaaaaacatg taaaaataat
gtactagaga cactttcaat aaaggcaaat 26460gcttttattt gtacactctc
gggtgattat ttacccccac ccttgccgtc tgcgccgttt 26520aaaaatcaaa
ggggttctgc cgcgcatcgc tatgcgccac tggcagggac acgttgcgat
26580actggtgttt agtgctccac ttaaactcag gcacaaccat ccgcggcagc
tcggtgaagt 26640tttcactcca caggctgcgc accatcacca acgcgtttag
caggtcgggc gccgatatct 26700tgaagtcgca gttggggcct ccgccctgcg
cgcgcgagtt gcgatacaca gggttgcagc 26760actggaacac tatcagcgcc
gggtggtgca cgctggccag cacgctcttg tcggagatca 26820gatccgcgtc
caggtcctcc gcgttgctca gggcgaacgg agtcaacttt ggtagctgcc
26880ttcccaaaaa gggcgcgtgc ccaggctttg agttgcactc gcaccgtagt
ggcatcaaaa 26940ggtgaccgtg cccggtctgg gcgttaggat acagcgcctg
cataaaagcc ttgatctgct 27000taaaagccac ctgagccttt gcgccttcag
agaagaacat gccgcaagac ttgccggaaa 27060actgattggc cggacaggcc
gcgtcgtgca cgcagcacct tgcgtcggtg ttggagatct 27120gcaccacatt
tcggccccac cggttcttca cgatcttggc cttgctagac tgctccttca
27180gcgcgcgctg cccgttttcg ctcgtcacat ccatttcaat cacgtgctcc
ttatttatca 27240taatgcttcc gtgtagacac ttaagctcgc cttcgatctc
agcgcagcgg tgcagccaca 27300acgcgcagcc cgtgggctcg tgatgcttgt
aggtcacctc tgcaaacgac tgcaggtacg 27360cctgcaggaa tcgccccatc
atcgtcacaa aggtcttgtt gctggtgaag gtcagctgca 27420acccgcggtg
ctcctcgttc agccaggtct tgcatacggc cgccagagct tccacttggt
27480caggcagtag tttgaagttc gcctttagat cgttatccac gtggtacttg
tccatcagcg 27540cgcgcgcagc ctccatgccc ttctcccacg cagacacgat
cggcacactc agcgggttca 27600tcaccgtaat ttcactttcc gcttcgctgg
gctcttcctc ttcctcttgc gtccgcatac 27660cacgcgccac tgggtcgtct
tcattcagcc gccgcactgt gcgcttacct cctttgccat 27720gcttgattag
caccggtggg ttgctgaaac ccaccatttg tagcgccaca tcttctcttt
27780cttcctcgct gtccacgatt acctctggtg atggcgggcg ctcgggcttg
ggagaagggc 27840gcttcttttt cttcttgggc gcaatggcca aatccgccgc
cgaggtcgat ggccgcgggc 27900tgggtgtgcg cggcaccagc gcgtcttgtg
atgagtcttc ctcgtcctcg gactcgatac 27960gccgcctcat ccgctttttt
gggggcgccc ggggaggcgg cggcgacggg gacggggacg 28020acacgtcctc
catggttggg ggacgtcgcg ccgcaccgcg tccgcgctcg ggggtggttt
28080cgcgctgctc ctcttcccga ctggccattt ccttctccta taggcagaaa
aagatcatgg 28140agtcagtcga gaagaaggac agcctaaccg ccccctctga
gttcgccacc accgcctcca 28200ccgatgccgc caacgcgcct accaccttcc
ccgtcgaggc acccccgctt gaggaggagg 28260aagtgattat cgagcaggac
ccaggttttg taagcgaaga cgacgaggac cgctcagtac 28320caacagagga
taaaaagcaa gaccaggaca acgcagaggc aaacgaggaa caagtcgggc
28380ggggggacga aaggcatggc gactacctag atgtgggaga cgacgtgctg
ttgaagcatc 28440tgcagcgcca gtgcgccatt atctgcgacg cgttgcaaga
gcgcagcgat gtgcccctcg 28500ccatagcgga tgtcagcctt gcctacgaac
gccacctatt ctcaccgcgc gtacccccca 28560aacgccaaga aaacggcaca
tgcgagccca acccgcgcct caacttctac cccgtatttg 28620ccgtgccaga
ggtgcttgcc acctatcaca tctttttcca aaactgcaag atacccctat
28680cctgccgtgc caaccgcagc cgagcggaca agcagctggc cttgcggcag
ggcgctgtca 28740tacctgatat cgcctcgctc aacgaagtgc caaaaatctt
tgagggtctt ggacgcgacg 28800agaagcgcgc ggcaaacgct ctgcaacagg
aaaacagcga aaatgaaagt cactctggag 28860tgttggtgga actcgagggt
gacaacgcgc gcctagccgt actaaaacgc agcatcgagg 28920tcacccactt
tgcctacccg gcacttaacc taccccccaa ggtcatgagc acagtcatga
28980gtgagctgat cgtgcgccgt gcgcagcccc tggagaggga tgcaaatttg
caagaacaaa 29040cagaggaggg cctacccgca gttggcgacg agcagctagc
gcgctggctt caaacgcgcg 29100agcctgccga cttggaggag cgacgcaaac
taatgatggc cgcagtgctc gttaccgtgg 29160agcttgagtg catgcagcgg
ttctttgctg acccggagat gcagcgcaag ctagaggaaa 29220cattgcacta
cacctttcga cagggctacg tacgccaggc ctgcaagatc tccaacgtgg
29280agctctgcaa cctggtctcc taccttggaa ttttgcacga aaaccgcctt
gggcaaaacg 29340tgcttcattc cacgctcaag ggcgaggcgc gccgcgacta
cgtccgcgac tgcgtttact 29400tatttctatg ctacacctgg cagacggcca
tgggcgtttg gcagcagtgc ttggaggagt 29460gcaacctcaa ggagctgcag
aaactgctaa agcaaaactt gaaggaccta tggacggcct 29520tcaacgagcg
ctccgtggcc gcgcacctgg cggacatcat tttccccgaa cgcctgctta
29580aaaccctgca acagggtctg ccagacttca ccagtcaaag catgttgcag
aactttagga 29640actttatcct agagcgctca ggaatcttgc ccgccacctg
ctgtgcactt cctagcgact 29700ttgtgcccat taagtaccgc gaatgccctc
cgccgctttg gggccactgc taccttctgc 29760agctagccaa ctaccttgcc
taccactctg acataatgga agacgtgagc ggtgacggtc 29820tactggagtg
tcactgtcgc tgcaacctat gcaccccgca ccgctccctg gtttgcaatt
29880cgcagctgct taacgaaagt caaattatcg gtacctttga gctgcagggt
ccctcgcctg 29940acgaaaagtc cgcggctccg gggttgaaac tcactccggg
gctgtggacg tcggcttacc 30000ttcgcaaatt tgtacctgag gactaccacg
cccacgagat taggttctac gaagaccaat 30060cccgcccgcc taatgcggag
cttaccgcct gcgtcattac ccagggccac attcttggcc 30120aattgcaagc
catcaacaaa gcccgccaag agtttctgct acgaaaggga cggggggttt
30180acttggaccc ccagtccggc gaggagctca acccaatccc cccgccgccg
cagccctatc 30240agcagcagcc gcgggccctt gcttcccagg atggcaccca
aaaagaagct gcagctgccg 30300ccgccaccca cggacgagga ggaatactgg
gacagtcagg cagaggaggt tttggacgag 30360gaggaggagg acatgatgga
agactgggag agcctagacg aggaagcttc cgaggtcgaa 30420gaggtgtcag
acgaaacacc gtcaccctcg gtcgcattcc cctcgccggc gccccagaaa
30480tcggcaaccg gttccagcat ggctacaacc tccgctcctc aggcgccgcc
ggcactgccc 30540gttcgccgac ccaaccgtag atgggacacc actggaacca
gggccggtaa gtccaagcag 30600ccgccgccgt tagcccaaga gcaacaacag
cgccaaggct accgctcatg gcgcgggcac 30660aagaacgcca tagttgcttg
cttgcaagac tgtgggggca acatctcctt cgcccgccgc 30720tttcttctct
accatcacgg cgtggccttc ccccgtaaca tcctgcatta ctaccgtcat
30780ctctacagcc catactgcac cggcggcagc ggcagcaaca gcagcggcca
cacagaagca 30840aaggcgaccg gatagcaaga ctctgacaaa gcccaagaaa
tccacagcgg cggcagcagc 30900aggaggagga gcgctgcgtc tggcgcccaa
cgaacccgta tcgacccgcg agcttagaaa 30960caggattttt cccactctgt
atgctatatt tcaacagagc aggggccaag aacaagagct 31020gaaaataaaa
aacaggtctc tgcgatccct cacccgcagc tgcctgtatc acaaaagcga
31080agatcagctt cggcgcacgc tggaagacgc ggaggctctc ttcagtaaat
actgcgcgct 31140gactcttaag gactagtttc gcgccctttc tcaaatttaa
gcgcgaaaac tacgtcatct 31200ccagcggcca cacccggcgc cagcacctgt
tgtcagcgcc attatgagca aggaaattcc 31260cacgccctac atgtggagtt
accagccaca aatgggactt gcggctggag ctgcccaaga 31320ctactcaacc
cgaataaact acatgagcgc gggaccccac atgatatccc gggtcaacgg
31380aatacgcgcc caccgaaacc gaattctcct ggaacaggcg gctattacca
ccacacctcg 31440taataacctt aatccccgta gttggcccgc tgccctggtg
taccaggaaa gtcccgctcc 31500caccactgtg gtacttccca gagacgccca
ggccgaagtt cagatgacta actcaggggc 31560gcagcttgcg ggcggctttc
gtcacagggt gcggtcgccc gggcagggta taactcacct 31620gacaatcaga
gggcgaggta ttcagctcaa cgacgagtcg gtgagctcct cgcttggtct
31680ccgtccggac gggacatttc agatcggcgg cgccggccgc tcttcattca
cgcctcgtca 31740ggcaatccta actctgcaga cctcgtcctc tgagccgcgc
tctggaggca ttggaactct 31800gcaatttatt gaggagtttg tgccatcggt
ctactttaac cccttctcgg gacctcccgg 31860ccactatccg gatcaattta
ttcctaactt tgacgcggta aaggactcgg cggacggcta 31920cgactgaatg
ttaagtggag aggcagagca actgcgcctg aaacacctgg tccactgtcg
31980ccgccacaag tgctttgccc gcgactccgg tgagttttgc tactttgaat
tgcccgagga 32040tcatatcgag ggcccggcgc acggcgtccg gcttaccgcc
cagggagagc ttgcccgtag 32100cctgattcgg gagtttaccc agcgccccct
gctagttgag cgggacaggg gaccctgtgt 32160tctcactgtg atttgcaact
gtcctaaccc tggattacat caagatctta ttccctttaa 32220ctaataaaaa
aaaataataa agcatcactt acttaaaatc agttagcaaa tttctgtcca
32280gtttattcag cagcacctcc ttgccctcct cccagctctg gtattgcagc
ttcctcctgg 32340ctgcaaactt tctccacaat ctaaatggaa tgtcagtttc
ctcctgttcc tgtccatccg 32400cacccactat cttcatgttg ttgcagatga
agcgcgcaag accgtctgaa gataccttca 32460accccgtgta tccatatgac
acggaaaccg gtcctccaac tgtgcctttt cttactcctc 32520cctttgtatc
ccccaatggg tttcaagaga gtccccctgg ggtactctct ttgcgcctat
32580ccgaacctct agttacctcc aatggcatgc ttgcgctcaa aatgggcaac
ggcctctctc 32640tggacgaggc cggcaacctt acctcccaaa atgtaaccac
tgtgagccca cctctcaaaa 32700aaaccaagtc aaacataaac ctggaaatat
ctgcacccct cacagttacc tcagaagccc 32760taactgtggc tgccgccgca
cctctaatgg tcgcgggcaa cacactcacc atgcaatcac 32820aggccccgct
aaccgtgcac gactccaaac ttagcattgc cacccaagga cccctcacag
32880tgtcagaagg aaagctagcc ctgcaaacat caggccccct caccaccacc
gatagcagta 32940cccttactat cactgcctca ccccctctaa ctactgccac
tggtagcttg ggcattgact 33000tgaaagagcc catttataca caaaatggaa
aactaggact aaagtacggg gctcctttgc 33060atgtaacaga cgacctaaac
actttgaccg tagcaactgg tccaggtgtg actattaata 33120atacttcctt
gcaaactaaa gttactggag ccttgggttt tgattcacaa ggcaatatgc
33180aacttaatgt agcaggagga ctaaggattg attctcaaaa cagacgcctt
atacttgatg 33240ttagttatcc gtttgatgct caaaaccaac taaatctaag
actaggacag ggccctcttt 33300ttataaactc agcccacaac ttggatatta
actacaacaa aggcctttac ttgtttacag 33360cttcaaacaa ttccaaaaag
cttgaggtta acctaagcac tgccaagggg ttgatgtttg 33420acgctacagc
catagccatt aatgcaggag atgggcttga atttggttca cctaatgcac
33480caaacacaaa tcccctcaaa acaaaaattg gccatggcct agaatttgat
tcaaacaagg 33540ctatggttcc taaactagga actggcctta gttttgacag
cacaggtgcc attacagtag 33600gaaacaaaaa taatgataag ctaactttgt
ggaccacacc agctccatct cctaactgta 33660gactaaatgc agagaaagat
gctaaactca ctttggtctt aacaaaatgt ggcagtcaaa 33720tacttgctac
agtttcagtt ttggctgtta aaggcagttt ggctccaata tctggaacag
33780ttcaaagtgc tcatcttatt ataagatttg acgaaaatgg agtgctacta
aacaattcct
33840tcctggaccc agaatattgg aactttagaa atggagatct tactgaaggc
acagcctata 33900caaacgctgt tggatttatg cctaacctat cagcttatcc
aaaatctcac ggtaaaactg 33960ccaaaagtaa cattgtcagt caagtttact
taaacggaga caaaactaaa cctgtaacac 34020taaccattac actaaacggt
acacaggaaa caggagacac aactccaagt gcatactcta 34080tgtcattttc
atgggactgg tctggccaca actacattaa tgaaatattt gccacatcct
34140cttacacttt ttcatacatt gcccaagaat aaagaatcgt ttgtgttatg
tttcaacgtg 34200tttatttttc aattgcagaa aatttcaagt catttttcat
tcagtagtat agccccacca 34260ccacatagct tatacagatc accgtacctt
aatcaaactc acagaaccct agtattcaac 34320ctgccacctc cctcccaaca
cacagagtac acagtccttt ctccccggct ggccttaaaa 34380agcatcatat
catgggtaac agacatattc ttaggtgtta tattccacac ggtttcctgt
34440cgagccaaac gctcatcagt gatattaata aactccccgg gcagctcact
taagttcatg 34500tcgctgtcca gctgctgagc cacaggctgc tgtccaactt
gcggttgctt aacgggcggc 34560gaaggagaag tccacgccta catgggggta
gagtcataat cgtgcatcag gatagggcgg 34620tggtgctgca gcagcgcgcg
aataaactgc tgccgccgcc gctccgtcct gcaggaatac 34680aacatggcag
tggtctcctc agcgatgatt cgcaccgccc gcagcataag gcgccttgtc
34740ctccgggcac agcagcgcac cctgatctca cttaaatcag cacagtaact
gcagcacagc 34800accacaatat tgttcaaaat cccacagtgc aaggcgctgt
atccaaagct catggcgggg 34860accacagaac ccacgtggcc atcataccac
aagcgcaggt agattaagtg gcgacccctc 34920ataaacacgc tggacataaa
cattacctct tttggcatgt tgtaattcac cacctcccgg 34980taccatataa
acctctgatt aaacatggcg ccatccacca ccatcctaaa ccagctggcc
35040aaaacctgcc cgccggctat acactgcagg gaaccgggac tggaacaatg
acagtggaga 35100gcccaggact cgtaaccatg gatcatcatg ctcgtcatga
tatcaatgtt ggcacaacac 35160aggcacacgt gcatacactt cctcaggatt
acaagctcct cccgcgttag aaccatatcc 35220cagggaacaa cccattcctg
aatcagcgta aatcccacac tgcagggaag acctcgcacg 35280taactcacgt
tgtgcattgt caaagtgtta cattcgggca gcagcggatg atcctccagt
35340atggtagcgc gggtttctgt ctcaaaagga ggtagacgat ccctactgta
cggagtgcgc 35400cgagacaacc gagatcgtgt tggtcgtagt gtcatgccaa
atggaacgcc ggacgtagtc 35460atatttcctg aagcaaaacc aggtgcgggc
gtgacaaaca gatctgcgtc tccggtctcg 35520ccgcttagat cgctctgtgt
agtagttgta gtatatccac tctctcaaag catccaggcg 35580ccccctggct
tcgggttcta tgtaaactcc ttcatgcgcc gctgccctga taacatccac
35640caccgcagaa taagccacac ccagccaacc tacacattcg ttctgcgagt
cacacacggg 35700aggagcggga agagctggaa gaaccatgtt ttttttttta
ttccaaaaga ttatccaaaa 35760cctcaaaatg aagatctatt aagtgaacgc
gctcccctcc ggtggcgtgg tcaaactcta 35820cagccaaaga acagataatg
gcatttgtaa gatgttgcac aatggcttcc aaaaggcaaa 35880cggccctcac
gtccaagtgg acgtaaaggc taaacccttc agggtgaatc tcctctataa
35940acattccagc accttcaacc atgcccaaat aattctcatc tcgccacctt
ctcaatatat 36000ctctaagcaa atcccgaata ttaagtccgg ccattgtaaa
aatctgctcc agagcgccct 36060ccaccttcag cctcaagcag cgaatcatga
ttgcaaaaat tcaggttcct cacagacctg 36120tataagattc aaaagcggaa
cattaacaaa aataccgcga tcccgtaggt cccttcgcag 36180ggccagctga
acataatcgt gcaggtctgc acggaccagc gcggccactt ccccgccagg
36240aaccatgaca aaagaaccca cactgattat gacacgcata ctcggagcta
tgctaaccag 36300cgtagccccg atgtaagctt gttgcatggg cggcgatata
aaatgcaagg tgctgctcaa 36360aaaatcaggc aaagcctcgc gcaaaaaaga
aagcacatcg tagtcatgct catgcagata 36420aaggcaggta agctccggaa
ccaccacaga aaaagacacc atttttctct caaacatgtc 36480tgcgggtttc
tgcataaaca caaaataaaa taacaaaaaa acatttaaac attagaagcc
36540tgtcttacaa caggaaaaac aacccttata agcataagac ggactacggc
catgccggcg 36600tgaccgtaaa aaaactggtc accgtgatta aaaagcacca
ccgacagctc ctcggtcatg 36660tccggagtca taatgtaaga ctcggtaaac
acatcaggtt gattcacatc ggtcagtgct 36720aaaaagcgac cgaaatagcc
cgggggaata catacccgca ggcgtagaga caacattaca 36780gcccccatag
gaggtataac aaaattaata ggagagaaaa acacataaac acctgaaaaa
36840ccctcctgcc taggcaaaat agcaccctcc cgctccagaa caacatacag
cgcttccaca 36900gcggcagcca taacagtcag ccttaccagt aaaaaagaaa
acctattaaa aaaacaccac 36960tcgacacggc accagctcaa tcagtcacag
tgtaaaaaag ggccaagtgc agagcgagta 37020tatataggac taaaaaatga
cgtaacggtt aaagtccaca aaaaacaccc agaaaaccgc 37080acgcgaacct
acgcccagaa acgaaagcca aaaaacccac aacttcctca aatcgtcact
37140tccgttttcc cacgttacgt cacttcccat tttaagaaaa ctacaattcc
caacacatac 37200aagttactcc gccctaaaac ctacgtcacc cgccccgttc
ccacgccccg cgccacgtca 37260caaactccac cccctcatta tcatattggc
ttcaatccaa aataaggtat attattgatg 37320atg 37323
* * * * *