U.S. patent application number 15/092937 was filed with the patent office on 2017-01-05 for compositions and methods for detecting and modulating cell death by a translation regulated gene expression system.
The applicant listed for this patent is Wayne State University. Invention is credited to Leon Carlock, Maria Cypher.
Application Number | 20170002377 15/092937 |
Document ID | / |
Family ID | 40351414 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170002377 |
Kind Code |
A1 |
Carlock; Leon ; et
al. |
January 5, 2017 |
COMPOSITIONS AND METHODS FOR DETECTING AND MODULATING CELL DEATH BY
A TRANSLATION REGULATED GENE EXPRESSION SYSTEM
Abstract
The technology relates to a nucleic acid expression cassette
comprising a TR element encoding an mRNA molecule that is
translated in stressed and/or dying cells, and a nucleotide
sequence operably linked to the TR element, that is a first open
reading frame (ORF) sequence and encodes a polypeptide or a
fragment thereof and is co-translated with the TR element. The
technology further relates to mammalian cells and a transgenic
animal comprising such expression cassette. Further included are
kits comprising the expression cassette, and methods for
determining toxicity, and killing a target cell.
Inventors: |
Carlock; Leon; (Bloomfield,
MI) ; Cypher; Maria; (Houston, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wayne State University |
Detroit |
MI |
US |
|
|
Family ID: |
40351414 |
Appl. No.: |
15/092937 |
Filed: |
April 7, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12672989 |
Apr 19, 2011 |
9322034 |
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PCT/US2008/072465 |
Aug 7, 2008 |
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15092937 |
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60955265 |
Aug 10, 2007 |
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60981044 |
Oct 18, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 15/86 20130101;
C12N 2830/42 20130101; C12N 2710/14043 20130101; A61K 48/0066
20130101; C12N 7/00 20130101; A01K 2217/052 20130101; C12N 2840/203
20130101; C12N 2799/026 20130101; A61K 48/00 20130101; C12N
2750/14143 20130101; C07K 14/4713 20130101; C12N 2840/10 20130101;
C12N 2840/44 20130101; G01N 2510/00 20130101; G01N 33/5014
20130101; C12N 2840/85 20130101; C12N 2799/025 20130101; G01N
2333/90241 20130101; A01K 2267/0393 20130101; C12N 2840/00
20130101; C12N 15/85 20130101; A01K 2227/105 20130101 |
International
Class: |
C12N 15/85 20060101
C12N015/85; G01N 33/50 20060101 G01N033/50; C12N 7/00 20060101
C12N007/00; A61K 48/00 20060101 A61K048/00; C12N 15/86 20060101
C12N015/86 |
Claims
1.-122. (canceled)
123. A nucleic acid expression cassette expressible in mammalian
cells, wherein the expression cassette comprises the following
elements in a 5' to 3' direction: at least one transcriptional
effector sequence, a translational regulatory (TR) element encoding
a mammalian mRNA molecule that is at least 80% homologous to a
human Proteolipid Protein (plp) gene corresponding to nts 1-831 of
a FIG. 15 PLP sequence, and is selectively translated in stressed
and/or dying cells, a nucleotide sequence operably linked to the TR
element, that is an open reading frame (ORF) sequence and encodes a
polypeptide or a fragment thereof and is co-translated with the TR
element, and a polyadenylation sequence, wherein selectively
translated in stressed and/or dying cells means that the mRNA
translation activity is observed in more than 95% of any cell line
transformed with the expression cassette at the peak of translation
activity following treatment with an acute toxic agent that induces
cell stress and/or death, and that translational levels of the ORF
of the expression cassette rise to at least 50% of expression
levels of the same ORF when transcribed and translated from the
same expression cassette lacking an operably linked TR element
following treatment with the acute toxic agent.
124. The expression cassette of claim 123, wherein the TR element
is from a mouse.
125. The expression cassette of claim 123, wherein the
transcriptional effector comprises a constitutive, inducible,
tissue specific, tumor specific, or response gene promoter.
126. The expression cassette of claim 125, wherein the promoter is
a constitutive promoter selected from the group consisting of
retroviral Rous sarcoma virus (RSV) long terminal repeat (LTR)
promoter, cytomegalovirus immediate early gene (CMV) promoter,
simian virus early (SV40) promoter, cytoplasmic beta-actin
promoter, adenovirus major late promoter, and phosphoglycerol
kinase (PGK) promoter; an inducible promoter selected from the
group consisting of human metallothionein II (hMT-IIA) promoter,
dexamethasone (Dex)-inducible promoter, mouse mammary tumor virus
(MMTV) promoter, ecdysone-responsive insect promoter, tetracycline
responsive (Tet-On promoter, Tet-Off) promoter, mifepristone
(RU486)-inducible promoter and rapamycin-responsive promoter; a
tissue specific promoter selected from the group consisting of
transferrin (TF) promoter, tyrosinase (TYR) promoter, albumin (ALB)
promoter, muscle creatine kinase (CKM) promoter, mylin basic
protein (MBP) promoter, glial fibrillary acidic protein (GFAP)
promoter, neuron-specific enolase (NSE) promoter, and synapsin I
(SYN1) promoter; a tumor specific promoter for vascular endothelial
growth factor (VEGF), VEGF receptor (KDR), alpha-fetoprotein (AFP),
carcinoembryonic antigen (CEA), v-erb-b2 erythroblastic leukemia
viral oncogene homolog 2 (erbB2), mucin-1 (muc-1/DF3), ALA, bone
gamma-carboxyglutamate protein (BGLAP), secretory leukoproteinase
inhibitor or antileukoproteinase 1 (SLP1), hypoxia-response element
(HRE), hexokinase II (Grp78/BIP, or HK2); or a response gene
promoter for early growth response gene (EGR1), tissue-type
plasminogen activator (t-PA), multidrug-resistance protein 1
(mdr-1), heat shock 70 kDa (hsp70), v-fos murine osteosarcoma viral
oncogene homolog (c-fos), v-jun sarcoma virus 17 oncogene homolog
(c-jun), E2F transcription factor 1 (E2F-1), heat shock 70 kDa
protein 5 (HSPA5), cyclin A1 (CCNA1) or cell division cycle 25C
(cdc25C).
127. The expression cassette of claim 123, wherein the ORF sequence
is selected from the group consisting of reporter gene, cytotoxic
tumor suppressor, toxin gene, prodrug activating gene and
proapoptotic gene.
128. The expression cassette of claim 127, wherein the ORF sequence
is a reporter gene.
129. The expression cassette of claim 128, wherein the reporter
gene is selected from the group consisting of egfp, gfp, eyfp,
luciferase, lacZ, cat, tk, and tksr39.
130. The expression cassette of claim 129, wherein the reporter
gene is luciferase.
131. The expression cassette of claim 127, wherein the ORF sequence
is a cytotoxic tumor suppressor selected from the group consisting
of p53, APC, BRCA-1, BRCA-2, WT-1, retinoblastoma, NF-1, NF-2 and
VHL.
132. The expression cassette of claim 131, wherein the cytotoxic
tumor suppressor is p53.
133. The expression cassette of claim 127, wherein the ORF sequence
is a toxin gene selected from the group consisting of pseudomonas
exotoxin, ricin toxin, and diptheria toxin.
134. The expression cassette of claim 133, wherein the toxin gene
is diphteria toxin.
135. The expression cassette of claim 127, wherein the ORF sequence
is a prodrug activating gene selected from the group consisting of
TK and TKsr39.
136. The expression cassette of claim 135, wherein the prodrug
activating gene is TKsr39.
137. The expression cassette of claim 127, wherein the ORF sequence
is a proapoptotic gene selected from the group consisting of p53,
ASPP1, ASPP2, ASPP3, Bax, BCL2-L-10, AIF, Fas, Caspase-8,
Caspase-9, and Caspase-3.
138. The expression cassette of claim 137, wherein the proapoptotic
gene is p53.
139. The expression cassette of claim 123, wherein the
polyadenylation sequence is selected from the group consisting of
SV40 early gene, SV40 late gene, HSV-TK, and hGH polyA tails.
140. The expression cassette of claim 123, wherein the nucleotide
sequence further comprises a 5' untranslated region, which is
located 3' to the transcriptional effector sequence and 5' to the
TR element and comprises an mRNA transcription initiation site.
141. The expression cassette of claim 140, wherein the 5'
untranslated region comprises an intron sequence which directs mRNA
splicing.
142. The expression cassette of claim 123, wherein the nucleotide
acid sequence further comprises one or more of the following: a
sequence of between about 15-50 nucleotides located 5' to the
transcriptional effector, that comprises one or more restriction
sites for insertion of the cassette into a plasmid, shuttle vector
or viral vector; a sequence of between about 15-50 nucleotides
located 3' to the TR element and 5' to the ORF sequence, that
comprises one or more restriction sites for insertion and operative
linkage of the TR element and the ORF sequence; a sequence of
between about 15-50 nucleotides located 3' to the ORF sequence and
5' to the polyadenylation sequence, that comprises one or more
restriction sites for insertion and operative linkage of the ORF
sequence and the polyadenylation sequence; or a sequence of between
about 15-50 nucleotides located 3' to the polyadenylation sequence,
that comprises one or more restriction sites for insertion of the
cassette into a plasmid, shuttle vector or viral vector, wherein
the addition of any of the sequences keeps translation in
frame.
143. The expression cassette of claim 123, wherein the expression
cassette is contained in a plasmid, shuttle vector, or viral
vector.
144. The expression cassette of claim 143, wherein expression
cassette is contained in a plasmid selected from the group
consisting of pCMVneo, pCMV-MCS, pBluescript II, pET14, and pUC19;
a shuttle vector selected from the group consisting of pCMV,
pEYFP-N1, pEGFP-N1, and pEGFP-C1; or a viral vector selected from
the group consisting of pAAV-MCS, pBac-1, and pBacPAK8/9.
145. The expression cassette of claim 144, wherein the plasmid is
pCMVneo.
146. The expression cassette of claim 144, wherein the shuttle
vector is pEYFP-N1.
147. The expression cassette of claim 144, wherein the viral vector
is pAAV-MCS.
148. A mammalian cell that is transformed with the expression
cassette of claim 123.
149. The mammalian cell of claim 148, wherein the mammalian cell is
selected from the group consisting of HEK293, HT1080, NTERA-2D,
HeLa, Caco2, HepG2, BALBC/3T3, and Cos-7.
150. The mammalian cell of claim 148, wherein the mammalian cell is
an embryonic stem cell.
151. The mammalian cell of claim 150, wherein the embryonic stem
cell is a murine embryonic stem cell mES-D3 or human embryonic stem
cell hES.
152. The mammalian cell of claim 148, wherein the ORF sequence is
selected from the group consisting of reporter gene, cytotoxic
tumor suppressor, toxin gene, prodrug activating gene and
proapoptotic gene.
153. The mammalian cell of claim 152, wherein the ORF sequence is a
reporter gene.
154. The mammalian cell of claim 153, wherein the reporter gene is
selected from the group consisting of EGFP, GFP, EYFP, luciferase,
LacZ, CAT, TK, and TKsr39.
155. The mammalian cell of claim 154, wherein the reporter gene is
luciferase.
156. A method for determining toxicity of a substance, wherein the
method comprises: (a) contacting the mammalian cells of claim 153
with the substance, wherein the ORF sequence encodes a reporter
polypeptide; and (b) detecting presence or measuring a level of the
reporter polypeptide, wherein the toxicity of the substance
correlates to the presence or the increase in the level of the
reporter polypeptide as compared to control cells that are not
exposed to the substance or are not transformed.
157. The method of claim 156, wherein the protein level of the
reporter polypeptide is measured.
158. The method of claim 156, wherein the presence or levels of the
mRNA of the reporter polypeptide are measured.
159. The method of claim 156, wherein the step of contacting the
mammalian cells is performed ex vivo.
160. The method of claim 156, wherein the reporter polypeptide is
luciferase.
161. A method for determining toxicity of a substance to a mammal,
wherein the method comprises: (a) transfecting a mammalian cell or
a mammalian cell line with the expression cassette of claim 128,
wherein the ORF sequence encodes a reporter polypeptide; (b)
contacting transfected cells from (a) with the substance; and (c)
detecting presence or measuring levels of the reporter polypeptide,
wherein the toxicity of the substance correlates to the presence or
the increase in the level of the reporter polypeptide as compared
to control cells that are not exposed to the substance or are not
transfected.
162. The method of claim 161, wherein the protein level of the
reporter polypeptide is measured.
163. The method of claim 161, wherein the presence or levels of the
mRNA of the reporter polypeptide are measured.
164. The method of claim 161, wherein the reporter polypeptide is
luciferase.
165. A kit useful for toxicity assays comprising (a) the expression
cassette of claim 123; and (b) instructions for use of the kit.
166. A kit useful for toxicity assays comprising (a) the mammalian
cells of claim 148; and (b) instructions for use of the kit.
167. A transgenic non-human animal comprising the expression
cassette of claim 123 stably integrated into the genome of the
animal.
168. A method of inducing apoptosis in a target cell comprising
transforming the cell with the expression cassette of claim 131,
and exposing the transformed cell under toxic conditions.
169. A method of inducing apoptosis in a target cell comprising
transforming the cell with the expression cassette of claim 133,
and exposing the transformed cell under toxic conditions.
170. A method of inducing apoptosis in a target cell comprising
transforming the cell with the expression cassette of claim 135,
and exposing the transformed cell under toxic conditions.
171. A method of inducing apoptosis in a target cell comprising
transforming the cell with the expression cassette of claim 137,
and exposing the transformed cell under toxic conditions.
172. A method for detecting cell stress and/or apoptosis, wherein
the method comprises: (a) obtaining the mammalian cells of claim
153, wherein the ORF sequence encodes a reporter polypeptide, or
transfecting a mammalian cell or a mammalian cell line with the
expression cassette of claim 6, wherein the ORF sequence encodes a
reporter polypeptide; and (b) detecting presence or measuring a
level of the reporter polypeptide, wherein the level of cell stress
and/or apoptosis correlates to the presence or the increase in the
level of the reporter polypeptide as compared to control cells that
are not transfected.
173. The method of claim 172, wherein the method includes prior to
step (b) a step of contacting the mammalian cells with a substance
capable of inducing cell stress and/or apoptosis.
174. The method of claim 172, wherein the protein level of the
reporter polypeptide is measured or the presence or levels of the
mRNA of the reporter polypeptide are measured.
175. The method of claim 172, wherein the reporter polypeptide is
luciferase.
176. A method for inhibiting apoptosis in a mammalian cell
comprising transforming the mammalian cell with the expression
cassette of claim 123 wherein the ORF sequence encodes an
antiapoptotic protein.
177. The method of claim 176, wherein the antiapoptotic protein is
selected from the group consisting of BCL2, BCL2L1, BCL2A1, BAG1,
TRAF1, BIRC3, BIRC5, BAK1, and API5.
178. A pharmaceutical composition comprising the expression
cassette of claim 123 and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/672,989, filed Apr. 19, 2011, which is a
U.S. PCT National of PCT/US2008/072465, filed Aug. 7, 2008, which
claims the benefit of U.S. Provisional Patent Application Ser. No.
60/981,044, filed Aug. 18, 2007, and U.S. Provisional Patent
Application Ser. No. 60/955,265, filed Aug. 10, 2007. The entire
content of these applications is incorporated herein by
reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing as a
text file, which is entitled "WSUM.06796.US_ST25," was created on
Dec. 12, 2013, and is 53,809 bytes in size. This sequence listing
was submitted via EFS-Web in ASCII format on Dec. 16, 2013, and is
hereby incorporated by reference in its entirety. This sequence
listing contain 63 sequences, which were submitted on Mar. 8, 2010
in a Sequence Listing in PCT/US2008/072465, filed on Aug. 7, 2008,
of which the instant application is a 35 U.S.C. .sctn.371 national
stage application.
BACKGROUND
[0003] The present technology relates to Translational Regulatory
(e.g. TR) nucleic acid molecules encoding mRNA molecules that are
selectively translated and detected early in stressed and/or dying
cells. In various embodiments, the technology relates to expression
cassettes comprising such TR elements, mammalian cells and
transgenic animals comprising such expression cassettes, and
methods of use and treatment.
[0004] Normal biological activity in a living organism combines
endogenous expression of genes that constitute an individual's
genome with responses to the outside world. In higher eukaryotes,
gene expression begins in the nucleus with transcription of genomic
DNA into a pre-mRNA or "primary" RNA transcript. While still in the
nucleus, the pre-mRNA is modified to include a 5' cap structure,
forms heteronuclear ribonucleoprotein (hnRNP) complexes, acquires a
3' polyadenylate tail and undergoes splicing to remove intervening
DNA sequences (e.g. introns). The mature mRNA is then exported to
the cytoplasm where protein complexes direct (1) association with
ribosomes via the 5' cap structure, termed Cap-dependent
translation, or (2) interaction with cytosolic RNA binding proteins
that facilitate mRNA storage, processing or degradation. Following
ribosome-driven translation, sequential shortening of the
3'-polyadenylate tail results in transport of the mRNA body to a
complex of ribonucleases (RNAses), termed the exosome, which
degrades the aged mRNA and effectively terminates protein
synthesis.
[0005] As expected, gene expression is a highly regulated process
that must produce a desired gene product (typically a polypeptide)
at a particular time, rate and quantity. In addition to
transcriptional regulation, post-transcriptional processes such as
mRNA decay and translation are key checkpoints in gene expression.
It is not surprising that changes in a cellular expression profile,
produced by genetic mutations or aberrant responses to external
stimuli can cause severe abnormalities that often result in cell
death and the manifestation of a disease phenotype.
[0006] Extensive or prolonged cellular stimulation by environmental
factors, such as altered nutrient levels, cytokines, hormones and
temperature shifts, as well as environmental stresses like hypoxia,
hypocalcemia, viral infection and tissue injury, results in the
rapid attenuation of cap-dependent translation. This process is
adaptive as it curtails the global synthesis of proteins which is
not needed for an immediate stress response and recovery. However,
this translational abatement does not completely eliminate ribosome
activity, since many products of stress response and recovery genes
continue to be synthesized by an alternative process, termed
cap-independent translation (reviewed in Guhaniyogi & Brewer,
2001, Gene 265(1-2):11-23).
[0007] Cap-independent translation occurs by direct recruitment of
ribosomes to specific RNA structures termed Internal Ribosome Entry
Sites (IRESs). Bypassing the requirement for a 5' mRNA cap
structure was initially described as a mechanism for translating
viral RNAs irrespective of a near complete inhibition of cellular
cap-dependent translation in infected cells (Jang et al., 1988, J.
Virol., 62:2636-43). Generally, IRES sequences cannot be identified
by sequence homology and well characterized IRES elements have been
verified using functional assays (Mountford and Smith, 1995, TIG,
11(5): 179-184; Baird et al., 2006, NAR, 12(10):1755-85). Current
evidence shows that the conformation of the IRES RNA and the
binding of accessory proteins to specific mRNA sequences enable
ribosome binding. In eukaryotic cells, IRES-directed translation
has often been associated with 5' untranslated regions (5'UTRs) of
mRNAs that contain unusually long and thermodynamically stable RNA
secondary structures with multiple short open reading frames (ORFs)
that dramatically inhibit the initiation of ribosome-dependent
translation. However, functional verification of IRES activity for
many of these 5'UTR IRES elements has been complicated by the
presence of transcriptional effector sequences cloned from the
overlapping 5' gene promoter. Attempts to employ these 5'UTR
elements in IRES reporter vectors have been complicated by this
residual background transcriptional activity which masks any
translational regulation produced by these sequences.
[0008] IRES elements have been identified in a number of eukaryotic
mRNAs (Bonnal S et al., (2003) Nucleic Acids Res. 31:427-428) and
ensure the efficient expression of proteins or fragments thereof
during nuclear inactivity or acute cellular stress when
"cap-dependent" translation initiation is inhibited (i.e.,
apoptosis, starvation, gamma-irradiation, hypoxia, mitosis, or
terminal differentiation). U.S. Published Patent Application No.
2006/0173168 discloses two low molecular peptides from the
C-terminus of the PLP/DM20 gene, PIRP-M and PIRP-L, which are
produced by internal translation initiation at an IRES.
[0009] The impact of chemical or biopharmaceutical intervention on
the overall health of a specific individual is often uncertain.
While a pharmaceutical molecule may remedy a targeted symptom, the
treatment may be accompanied by serious side effects or unexpected
toxicity that can, in some cases be worse than the initial malady.
Although the side effects and toxicity of a pharmaceutical
preparation are often known and may be limited to a small subset of
individuals, these effects may be so severe in this small subset of
individuals that a drug may not achieve FDA approval which results
in huge pharmaceutical losses.
[0010] A large number of chemicals are manufactured in the United
States annually. Over 2,000 new chemicals are introduced into the
market each year, although very few are comprehensively tested for
acute or chronic toxicity. In order to define the potential
toxicity of a novel drug or chemical, the Food and Drug
Administration (FDA) requires a New Drug Application (NDA) to
include a large battery of toxicity, carcinogenicity, mutagenicity
and reproductive/fertility tests in at least two animal species.
The frequent, invasive testing and postmortem endpoint has raised
considerable criticism from animal rights groups and the general
public about animal suffering.
[0011] This situation underscores the need in the art for
alternative, high-throughput molecular and biological screening
technologies capable of detecting cell stress and toxicity in a
broad spectrum of cell types following acute or chronic exposure to
a chemical. Accordingly, novel methods for efficient and less
expensive toxicity testing that provide a reliable alternative to
animal testing are needed.
SUMMARY
[0012] Among the various aspects of the present technology are
nucleic acid expression cassettes, which are expressible in
mammalian cells and have the following elements in a 5' to 3'
direction: [0013] at least one transcriptional effector sequence,
[0014] a TR element encoding an mRNA molecule which is selectively
translated in stressed and/or dying cells, [0015] a nucleotide
sequence operably linked to the TR element, which is a first open
reading frame (ORF) sequence and encodes a polypeptide or a
fragment thereof and is co-translated with the TR element, and
[0016] a polyadenylation sequence, herein referred to as the TR
expression cassettes. The first ORF sequence can be selected from a
reporter gene, cytotoxic tumor suppressor gene, toxin gene, prodrug
activating gene and proapoptotic gene.
[0017] In one embodiment, the transcriptional effector sequence is
a promoter.
[0018] In another aspect, the TR expression cassettes can contain a
second ORF sequence, which is situated 5' to the TR sequence and is
independently translated. The second ORF sequence can be selected
from the same sequences as the first ORF.
[0019] It is another aspect of the present technology to provide
mammalian cells transformed with a TR expression cassette.
Preferably the mammalian cells are embryonic stem (ES) cells.
[0020] In still another aspect, the present technology provides
methods for determining toxicity of a substance. One such method
comprises (a) contacting the mammalian cells transformed with a TR
expression cassette, wherein the first ORF sequence encodes a
reporter polypeptide; and (b) detecting presence or measuring
levels of the reporter polypeptide, wherein the presence or an
increase in the level of said reporter polypeptide, compared to
control cells that are (i) not exposed to said substance or (ii)
not transfected is indicative of the toxicity of the substance.
Another method for determining toxicity of a substance comprises
(a) transfecting or transducing a mammalian cell or stably
transforming a mammalian cell line with a TR expression cassette
wherein the first ORF sequence encodes a reporter polypeptide; (b)
contacting transfected cells from (a) with the substance; and (c)
detecting presence or measuring levels of the reporter polypeptide,
wherein the presence or an increase in the level of said reporter
polypeptide, compared to control cells that are (i) not exposed to
said substance or (ii) not transfected/transduced is indicative of
the toxicity of the substance.
[0021] It is yet another aspect of the present technology to
provide a kit, which includes either a TR expression cassette or
mammalian cells transformed with such expression cassette, and
instructions for use.
[0022] In another aspect, the present technology provides a
transgenic non-human animal, which has a TR expression cassette
stably integrated into its genome. The preferred transgenic animal
is a mouse.
[0023] In still another aspect, the present technology provides a
method for killing a target cell by transforming said cell with the
TR expression cassette, wherein the first ORF sequence is selected
from a cytotoxic tumor suppressor, toxin gene, prodrug activating
gene or proapoptotic gene.
DRAWINGS
[0024] FIGS. 1A-1C are a schematic drawing showing the parental
plasmids used to produce the pTR-ORF vectors. FIG. 1A shows a
restriction map of the pEYFP-N1 vector. FIG. 1B shows the pPLPeyfp
expression vector used to express the PLP isoform of the
proteolipid protein (PLP) as a fusion protein with the Enhanced
Yellow Fluorescence Protein (EYFP). FIG. 1C shows the pDM20eyfp
expression vector that expresses the DM20 proteolipid protein
isoform as a fusion protein with the EYFP protein. Functional
plasmid elements (restriction enzyme sites, origins of replication,
open reading frames, etc.) are represented with vertical lines,
boxes and arrows as needed.
[0025] FIGS. 2A-2B show schematic examples of the monocistronic
pTR-EYFP expression vectors. FIG. 2A shows a map of the
pTR.sub.plp-EYFP vector. The TR-ORF cassette in this vector is
composed of the CMV IE transcriptional promoter, the TR element
derived from the PLP isoform cDNA sequence, the EYFP open reading
frame and the SV40 polyadenylation signal. FIG. 2B shows a map of
the pTR.sub.dm-EYFP vector. This TR-ORF cassette is similar to FIG.
2A except the TR element is derived from the DM20 isoform cDNA
sequence.
[0026] FIGS. 3A-3D show schematic representations of the
bicistronic pORF-TR-ORF vectors. FIG. 3A shows a map of the
pfLuc-TR.sub.plp-EYFP vector. The bicistronic cassette in this
vector is composed of the CMV IE transcriptional promoter, the
firefly Luciferase (fLuc) gene in the "sense" orientation relative
to the direction of TR transcription, the plp-specific TR element
functionally linked to the EYFP ORF and the the SV40
polyadenylation signal. Relevant restriction sites and plasmid
functional elements are shown. FIG. 3B shows a map of the
pfLuc-TR.sub.dm-EYFP plasmid. FIG. 3C displays a map of the
pcuLf-TR.sub.plp-EYFP vector, in which the fLuc ORF is in an
"antisense" orientation in the TR expression cassette. FIG. 3D
shows a map for the pcuLf-TR.sub.dm-EYFP plasmid with an antisense
fLuc ORF.
[0027] FIG. 4 displays the quantitation of a Western blot analysis
showing that cell pools expressing the bicistronic TR cassettes
induce EYFP translation after treatment with toxic doses of the
calcium ionophore A23187 or the proteasome inhibitor MG132. EYFP
protein levels are determined by densitometry and expressed as % of
the protein level detected in control HEK293 cells (adjusted to
100%). Cap-independent translation is independent of the
orientation of the upstream fLuc ORF (shown as fLuc in the "sense"
and cuLf in the "antisense" orientation relative to the TR
cassette).
[0028] FIGS. 5A-5B show schematic representations of plasmid
shuttle vectors that can be used to produce recombinant virus
capable of transducing mammalian cells. FIG. 5A shows a map of the
pAAV-MCS shuttle vector that can be used to produce recombinant
Adeno-associated virus (rAAV) which can transduce mammalian cells
in vitro and in vivo. FIG. 5B shows the map of the pBAC-1 shuttle
vector that can be used to produce the recombinant Baculovirus
(rBAC) virions that transduces mammalian cells to selectively
translate the TR-ORF cassettes in stressed and dying cells.
[0029] FIGS. 6A-6B display the quantitation of a Western blot
analysis showing that cells expressing the monocistronic
TR.sub.plp/dm-fLuc cassettes induce fLuc translation after
treatment with toxic doses of the calcium ionophore A23187. fLuc
protein levels are determined by densitometry and expressed as % of
control HEK293 cells (adjusted to 100%). FIG. 6A shows the fLuc
protein levels that are produced by four subclones (#3, 17, 13 and
16) expressing the TR.sub.plp-fLuc cassette compared to CMV-fLuc
and HEK293 cells, as well as a HEK293 TR.sub.plp-fLuc pool. FIG. 6B
shows fLuc protein quantitation for five subclones (#12, 43, 45, 2
and 8) expressing the TR.sub.dm-fLuc cassette correlated with
protein levels in CMV-fLuc, HEK293 and HEK293 TR.sub.dm-fLuc
cells.
[0030] FIG. 7 shows a TR-specific increase in fluorescent HEK293
TR.sub.plp/dm-EYFP cells at 6 hours and 10 hours post-treatment
with a toxic dose of the calcium ionophore A23187. The histogram
represents the direct microscopic counts of fluorescent cells. Cell
numbers are expressed as the percent of fluorescent cells relative
to control HEK293 cells (adjusted to 100%).
[0031] FIG. 8 displays a TR-specific increase in HEK293
TR.sub.plp/dm-fLuc cells stained by immunofluorescence labeling
with an anti-fLuc antibody following treatment with a toxic dose of
the calcium ionophore A23187. The histogram represents the direct
microscopic count of stained fluorescent cells. Cell numbers are
expressed as the percent of fluorescent cells relative to control
HEK293 cells (adjusted to 100%).
[0032] FIGS. 9A-9B show the TR-dependent translation that can be
produced by a series of cell lines expressing the
TR.sub.plp/dm-EYFP and TR.sub.plp/dm-fLuc cassettes following
exposure to a toxic dose of the calcium ionophore A23187. The
histograms show arbitrary fluorescence or luciferase units that can
be obtained by a microplate reader expressed as the ratio of
treated to untreated cultures. Ratios in excess of 1.0 are
indicative of cells exhibiting TR-dependent translation. FIG. 9A
shows the results for eight cell lines expressing the
TR.sub.plp-EYFP cassette compared to HEK293, CMV-EYFP and a
TR.sub.plp-EYFP pool. FIG. 9B shows the results for five cell lines
expressing the TR.sub.plp-fLuc cassette, three cell lines
expressing the TR.sub.dm-fLuc cassette compared to a CMV-fLuc and
HEK293 controls.
[0033] FIGS. 10A-10D show a TR-dependent dose response that can be
produced by cell lines expressing the TR.sub.plp/dm-fLuc cassettes
following exposure to a toxic dose of the calcium ionophore A23187.
FIG. 10A shows a chart of arbitrary luminescence units that can be
obtained by a microplate reader for HEK293, HEK293 CMV-fLuc, HEK
TR.sub.plp-fLuc (subclone #3) and HEK293 TR.sub.dm-fLuc (subclone
#45) cells after culture in increasing toxin concentrations. FIG.
10B shows only the HEK293 and TR.sub.plp/dm-fLuc results to
emphasize the dose response curve which peaks at 6 .mu.M. FIG. 10C
shows a chart that expresses arbitrary luciferase readings as the %
of luciferase activity in untreated cells. This shows the change in
cap-dependent translation that can be produced by the CMV-fLuc
cells to the increase in cap-independent luciferase activity
exhibited by the TR-ORF cells; however, the shape of the dose
response curve is unchanged. FIG. 10D shows the arbitrary
luciferase readings as the ratio of the reading to CMV-fLuc cells.
This comparison emphasizes the sharp decline in CMV-fLuc activity
at high doses and reduces the apparent decline in TR-dependent
translation at higher toxin doses.
[0034] FIGS. 11A-11D show a TR-dependent temporal response that can
be produced by cell lines expressing the TR.sub.plp/dm-fLuc
cassettes following exposure to a toxic dose of the calcium
ionophore A23187. FIG. 11A shows a chart of arbitrary luciferase
readings that can be obtained by a microplate reader for HEK293,
HEK293 CMV-fLuc, HEK TR.sub.plp-fLuc (subclone #3) and HEK293
TR.sub.dm-fLuc (subclone #45) cells after culture with a toxic dose
of the calcium ionophore A23187 as a function of increasing time.
FIG. 11B is a chart of HEK293 and TR.sub.plp/dm-fLuc results that
show the increase in luciferase activity observed by 1.5 hr
post-treatment. FIG. 11C shows a chart that expresses arbitrary
luciferase readings as the % of luciferase activity at 0 hr
post-treatment. FIG. 11D shows the arbitrary luciferase readings as
the ratio to the CMV-fLuc cells.
[0035] FIG. 12 is a histogram showing the ability of rBAC
TR.sub.dm-EYFP virions to transduce HT1080 cells and exhibit
TR-dependent translation in stressed and dying cells. Cells
transduced with 10 pfu/cell or 25 pfu/cell rBAC virions are
cultured in a toxic concentration of the calcium ionophore A23187
for 13.5 hours or 23 hours. Fluorescent cells are counted
microscopically and expressed as the % of control HEK293 cells
(infected but not treated with toxin).
[0036] FIG. 13 is a chart showing the ability of the
TR.sub.plp-TKsr39 cell pool to respond to the pro-drug ganciclovir
and induce cell death. HEK293, HEK CMV-EYFP and HEK293
TR.sub.plp-TKsr39 cells are cultured in various concentrations of
ganciclovir for 3 or 4 days. Cell viability is determined by the
Trypan blue exclusion assay and cell numbers expressed as % viable
cells. In contrast to the HEK293 and HEK CMV-EYFP cultures that
show no decrease in cell viability at any pro-drug concentration or
timepoint, the TR.sub.plp-TKsr39 cells display reduced viability
after 3 days in ganciclovir supplemented medium. By 4 days, the
TR.sub.plp-TKsr39 cells show significant cell death and a dose
dependent reduction in cell viability.
[0037] FIG. 14 is a sequence comparison table of C-terminal
sequences of myelin proteolipid proteins from a variety of
vertebrates, taken from the following NCBI Genbank numbers: (1)
P60201 Homo sapiens, (2) Q5R6E6 Pongo pygmaeus (orangutan), (3) XP
001140782 Pan troglodytes (chimpanzee), (4) XP 001088537 Macaca
mulatta (rhesus monkey), (5) Q8HXW7 Macaca fascicularis
(crab-eating macaque), (6) NP 999139 Sus scrofa (pig), (7)
NP_035253 Mus musculus (mouse), (8) NP_112252 Rattus norvegicus
(rat), (9) XP_001374483 Monodelphis domestica (opossum), (10)
P47789 Oryctolagus cuniculus (rabbit), (11) CAA08909 Bos taurus
(cattle), (12) 39025 Canis familiaris (dog), (13) CAA43839 Gallus
gallus (chicken), (14) P47790 Taeniopygia guttata (zebra finch),
(15) AAW79015 Gekko japonicus (gecko lizard), (16) CAA79582 Xenopus
laevis (frog), and (17) BAA84207 Latimeria chalumnae (coelacanth).
Insertion mutations present in some species are shown
double-underlined.
[0038] FIG. 15, i.e. 15A-15C, is a sequence alignment chart of
murine and human PLP/DM20 coding sequences and TR elements hereof.
Key: mDM=murine DM20 cDNA; mP=murine PLP cDNA; TRd=TRdm [SEQ ID
NO:1]; TRp=TRplp [SEQ ID NO:2]; hDM=human DM20; and hP=human PLP.
Because DM20 sequences omit part of the sequence present in
full-length PLP coding sequences, the numbering of DM20 sequences
in FIG. 15 is discontinuous and, after the omitted segment, DM20
numbering is shown continuing below the aligned sequences. In
describing sequences herein with reference to FIG. 15, in some
cases dual numbering for PLP/DM20 nucleotide positions is utilized,
e.g., residue 560/455; this usage refers to PLP and DM20 numbering
in the alternative, with PLP numbering as shown above the aligned
sequences, and DM20 numbering as shown below the aligned sequences.
The last expressed codon shown is `ttc` 829/724 to 831/726.
[0039] It should be noted that the figures set forth herein are
intended to exemplify the general characteristics of materials and
methods among those of the present technology, for the purpose of
the description of such embodiments herein. These figures may not
precisely reflect the characteristics of any given embodiment, and
are not necessarily intended to define or limit specific
embodiments within the scope of this technology.
DETAILED DESCRIPTION
[0040] The following description of technology is merely exemplary
in nature of the subject matter, manufacture, and use of one or
more inventions, and is not intended to limit the scope,
application, or uses of any specific invention claimed in this
application or in such other applications as may be filed claiming
priority to this application, or patents issuing therefrom. The
following definitions and non-limiting guidelines must be
considered in reviewing the description of the technology set forth
herein.
[0041] The headings (such as "Introduction" and "Summary,") and
sub-headings (such as "Expression Cassettes") used herein are
intended only for general organization of topics within the
disclosure of the present technology, and are not intended to limit
the disclosure of the technology or any aspect thereof. In
particular, subject matter disclosed in the "Introduction" may
include aspects of technology within the scope of one or more
inventions, and may not constitute a recitation of prior art.
Subject matter disclosed in the "Summary" is not an exhaustive or
complete disclosure of the entire scope of the technology or any
embodiments thereof.
[0042] The citation of references herein does not constitute an
admission that those references are prior art or have any relevance
to the patentability of the technology disclosed herein. Any
discussion of the content of references cited in the Introduction
is intended merely to provide a general summary of assertions made
by the authors of the references, and does not constitute an
admission as to the accuracy of the content of such references. All
references cited in the Description section of this specification
are hereby incorporated by reference in their entirety.
[0043] The description and specific examples, while indicating
embodiments of the present technology, are intended for purposes of
illustration only and are not intended to limit the scope of the
technology. Moreover, recitation of multiple embodiments having
stated features is not intended to exclude other embodiments having
additional features, or other embodiments incorporating different
combinations of the stated features. Specific Examples are provided
for illustrative purposes of how to make, use and practice the
materials and methods of this technology and, unless explicitly
stated otherwise, are not intended to be a representation that
given embodiments of this technology have, or have not, been made
or tested.
[0044] As used herein, the words "preferred" and "preferably" refer
to embodiments of the technology that afford certain benefits,
under certain circumstances. However, other embodiments may also be
preferred, under the same or other circumstances. Furthermore, the
recitation of one or more preferred embodiments does not imply that
other embodiments are not useful, and is not intended to exclude
other embodiments from the scope of the technology.
[0045] As used herein, the terms "comprising," "including," and
"having," and their variants, are intended to be non-limiting, such
that recitation of items in a list is not to the exclusion of other
like items that may also be useful in the materials, compositions,
and methods of this technology.
[0046] As used herein, the term "about," when applied to the value
for a parameter of a composition or method of this technology,
indicates that the calculation or the measurement of the value
allows some slight imprecision without having a substantial effect
on the chemical or physical attributes of the composition or
method.
[0047] When introducing elements of the present technology or the
preferred embodiment(s) thereof, the articles "a," "an," "the," and
"said" are intended to mean that there are one or more of the
elements.
[0048] The term "cytotoxic gene" refers to a nucleotide sequence
which when expressed in a target cell induces death of the cell by
lysis, apoptosis, necrosis or any other mechanism of cell
killing.
[0049] The term "gene" refers to a nucleic acid (e.g., DNA)
sequence that comprises coding sequences necessary for the
production of a polypeptide or precursor or RNA (e.g., tRNA, siRNA,
rRNA, etc.). The polypeptide can be encoded by a full length coding
sequence or by any portion of the coding sequence so long as the
desired activity or functional properties (e.g., enzymatic
activity, ligand binding, signal transduction, etc.) of the
full-length or fragment are retained. The term also encompasses the
coding region of a structural gene and the sequences located
adjacent to the coding region on both the 5' and 3' ends, such that
the gene corresponds to the length of the full-length mRNA. The
sequences that are located 5' of the coding region and which are
present on the mRNA are referred to as 5' untranslated sequences.
The sequences that are located 3' or downstream of the coding
region and that are present on the mRNA are referred to as 3'
untranslated sequences. The term "gene" encompasses both cDNA and
genomic forms of a gene. A genomic form or clone of a gene contains
the coding region, which may be interrupted with non-coding
sequences termed "introns" or "intervening regions" or "intervening
sequences." Introns are removed or "spliced out" from the nuclear
or primary transcript, and are therefore absent in the messenger
RNA (mRNA) transcript. The mRNA functions during translation to
specify the sequence or order of amino acids in a nascent
polypeptide.
[0050] The term "expression vector" refers to both viral and
non-viral vectors comprising a nucleic acid expression
cassette.
[0051] The term "expression cassette" is used to define a
nucleotide sequence containing regulatory elements operably linked
to a coding sequence that result in the transcription and
translation of the coding sequence in a cell.
[0052] A "mammalian promoter" refers to a transcriptional promoter
that functions in a mammalian cell that is derived from a mammalian
cell, or both.
[0053] A "mammalian minimal promoter" refers to a `core` DNA
sequence required to properly initiate transcription via RNA
polymerase binding, but which exhibits only token transcriptional
activity in the absence of any operably linked transcriptional
effector sequences.
[0054] The phrase "open reading frame" or "coding sequence" refers
to a nucleotide sequence that encodes a polypeptide or protein. The
coding region is bounded in eukaryotes, on the 5' side by the
nucleotide triplet "ATG" that encodes the initiator methionine and
on the 3' side by one of the three triplets which specify stop
codons (i.e., TAA, TAG, and TGA).
[0055] "Operably linked" is defined to mean that the nucleic acids
are placed in a functional relationship with another nucleic acid
sequence. For example, a promoter or enhancer is operably linked to
a coding sequence if it affects the transcription of the sequence;
or a ribosome binding site is operably linked to a coding sequence
if it is positioned so as to facilitate translation. Generally,
"operably linked" means that the DNA sequences being linked are
contiguous. However, enhancers do not have to be contiguous.
Linking is accomplished by ligation at convenient restriction
sites. If such sites do not exist, the synthetic oligonucleotide
adaptors or linkers are used in accord with conventional
practice.
[0056] "Recombinant" refers to the results of methods, reagents,
and laboratory manipulations in which nucleic acids or other
biological molecules are enzymatically, chemically or biologically
cleaved, synthesized, combined, or otherwise manipulated ex vivo to
produce desired products in cells or other biological systems. The
term "recombinant DNA" refers to a DNA molecule that is comprised
of segments of DNA joined together by means of molecular biology
techniques.
[0057] "Transfection" is the term used to describe the introduction
of foreign material such as foreign DNA into eukaryotic cells. It
is used interchangeably with "transformation" and "transduction"
although the latter term, in its narrower scope refers to the
process of introducing DNA into cells by viruses, which act as
carriers. Thus, the cells that undergo transfection are referred to
as "transfected," "transformed" or "transduced" cells.
[0058] The term "plasmid" as used herein, refers to an
independently replicating piece of DNA. It is typically circular
and double-stranded.
[0059] A "reporter gene" refers to any gene the expression of which
can be detected or measured using conventional techniques known to
those skilled in the art.
[0060] The term "regulatory element" or "effector element" refer to
a transcriptional promoter, enhancer, silencer or terminator, as
well as to any translational regulatory elements, polyadenylation
sites, and the like. Regulatory and effector elements may be
arranged so that they allow, enhance or facilitate selective
production of a mature coding sequence that is subject to their
regulation.
[0061] The term "vector" refers to a DNA molecule into which
foreign fragments of DNA may be inserted. Generally, they contain
regulatory and coding sequences of interest. The term vector
includes but is not limited to plasmids, cosmids, phagemids, viral
vectors and shuttle vectors.
[0062] A "shuttle" vector is a plasmid vector that is capable of
prokaryotic replication but contains no eukaryotic replication
sequences. Viral DNA sequences contained within this
replication-deficient shuttle vector direct recombination within a
eukaryotic host cell to produce infective viral particles.
[0063] The term "substance" as used herein refers to a matter of
defined chemical composition. It is used herein interchangeably
with the term "compound."
[0064] The term "viral vector" refers to a virus which contains
foreign genetic material for delivery into cells it infects.
[0065] A "replication-deficient" viral vector is incapable of
replication in a "wild-type" or otherwise unmanipulated mammalian
cell. Production of significant quantities of such viruses requires
that a producer cell line be co-transfected with a helper virus or
otherwise modified to supply or complement the missing
function(s).
[0066] A "replication-competent" viral vectors is one that is
capable of infecting cells and undergoing DNA replication, viral
packaging and release from the infected cell.
[0067] "Conditionally replicating" viral vectors as used herein are
replication-competent vectors that are designed to be selectively
expressed in particular cell types so that undesired broad spectrum
infection is avoided. Conditional replication may be achieved by
including in the vector tissue-specific, tumor-specific or cell
type-specific or other selectively induced regulatory control
sequences that are operably linked to early viral genes.
[0068] The terms "stress" and "toxicity" are used to refer to the
disturbance of the natural biochemical and biophysical homeostasis
of the cell. Whereas stress generally leads to recovery of cellular
homeostasis, a toxic response eventually results in cell death.
[0069] The translation regulated (TR) sequence (also referred to as
the "TR element") employed in the present technology is the IRES
element, which can be distinguished from the 5' UTR IRESs by (a)
its nucleic acid sequence context and (b) the cellular activity
which regulates translation (US Published Patent Application No.
2006/0173168). The combination of these two features forms a basis
for selective translation of downstream coding sequences in
stressed and/or dying mammalian cells that are operably linked to
this IRES sequence. Thus, the present technology contemplates the
use of any mammalian IRES as the TR element, which is selectively
expressed in stressed and/or dying cells.
[0070] In some embodiments, the IRES element of this technology has
cap-independent translational activity which localizes within the
ORF of the mammalian Proteolipid Protein (plp) gene. In its native
context, plp IRES activity resides within a multicistronic RNA
containing several upstream ORFs ("uORFs") which effectively block
ribosome scanning to internal AUG codons in normal cells. However,
exposure of cells to toxic agents results in ribosome binding and
translation from specific internal RNA sequences so that an
internal amino acid sequence is translated from the 3' end of the
plp ORF. Thus, the expression of an appropriate coding sequence,
which is regulated by the TR element, permits the visualization,
monitoring and modulation of cell death, which finds use in
numerous applications. Recombinant DNA molecules provided herein
allow for the selective expression of an RNA transcript containing
one or more nucleic acid sequences encoding one or more
polypeptides in stressed or dying cells.
[0071] In some embodiments, the TR element of the present
technology is derived from exons 1-7 of the plp gene. While not
being bound to a particular theory, it is believed that the exons 1
through 4 are sufficient to encode a functional IRES activity based
on mutational analysis data. Furthermore, it is believed that the
TR regulatory system, which plays a role in stress/death-specific
translation is located within exons 6 and/or 7.
[0072] In contrast to the IRES element disclosed in US
2006/0173168, which is expressed in dying cells, a TR element of
the present technology derived from PLP/DM20 differs in all of the
following features:
[0073] 1) nucleotide 1 (in SEQ ID Nos. 1 and 2) was mutated from A
to T to remove the wild type AUG start codon in the myelin
proteolipid protein PLP and DM20 cDNAs that directs the synthesis
of the full length PLP and DM20 in order to prevent such synthesis
from occurring;
[0074] 2) nucleotide 4 was mutated from G to A in order to create a
stop codon in the second possible reading frame of the PLP and DM20
cDNAs to prevent full length synthesis thereof;
[0075] 3) nucleotides 6, 7 and 8 were mutated from C to T, T to G
and T to A respectively to create a stop codon in the third
possible reading frame of the PLP and DM20 cDNAs to prevent
synthesis of the full length PLP and DM20;
[0076] 4) nucleotides 17 and 18 were mutated from G to A and T to
G, respectively to create the first stop codon in the main (first)
open reading frame of the PLP and DM20 cDNAs to prevent their full
length synthesis;
[0077] 5) nucleotide 21 was mutated from T to A in order to create
the second stop codon in the main (first) open reading frame of the
PLP and DM20 cDNAs to prevent full length synthesis thereof;
[0078] 6) nucleotide 27 was mutated from A to T in order to remove
the AUG codon from the third possible reading frame of the PLP and
DM20 cDNAs to prevent out-of frame translation initiation in the
absence of the wild type AUG codon; and
[0079] 7) the stop codon was deleted from the PLP and DM20 cDNAs to
reduce interference with translation of the downstream open reading
frame.
[0080] As a result, the TR elements of the present technology
derived from PLP/DM20 do not direct cap-dependent translation of
either PIRP-M or PIRP-L. In addition to the above changes, the
following mutations were introduced into the TR elements from the
DM 20 variant of the cDNA:
[0081] 1) nucleotide 511 was mutated from A to T in order to remove
the first in-frame internal AUG start codon in the DM20 variant
that directs the synthesis of PIRP-M protein to prevent such
synthesis from occurring; and
[0082] 2) nucleotide 598 was mutated from A to T to remove the
second in-frame internal AUG start codon in the DM20 variant that
directs the synthesis of PIRP-L protein in order to prevent such
synthesis from occurring.
[0083] Similarly, the following mutations were introduced into the
TR elements from the PLP variant of the cDNA:
[0084] 1) nucleotide 616 was mutated from A to T in order to remove
the first in-frame internal AUG start codon in the PLP variant that
directs the synthesis of PIRP-M protein to prevent such synthesis
from occurring; and
[0085] 2) nucleotide 703 was mutated from A to T to remove the
second in-frame internal AUG start codon in the PLP variant that
directs the synthesis of PIRP-L protein in order to prevent such
synthesis from occurring.
[0086] The TR cassette of the present technology finds many uses in
methods such as
[0087] detecting cell death ex vivo or in vivo; determining the
cytotoxicity of a compound in vivo or ex vivo; in vivo diagnostics;
inducing apoptosis in a cell in vivo or ex vivo; preventing
apoptosis in a cell in vivo or ex vivo; and combining the imaging
of cell stress and/or death with subsequent treatment. In addition,
the present technology details the methods for screening for
additional TR cassettes, i.e., the IRES elements which are
selectively expressed in stressed and/or dying cells.
[0088] Expression Cassettes
[0089] One aspect of the present technology is directed to a
nucleic acid expression cassette expressible in mammalian cells.
The expression cassette contains the following elements in a 5' to
3' direction: at least one transcriptional effector sequence, a TR
element encoding an mRNA molecule that is selectively translated in
stressed and/or dying cells, a nucleotide sequence operably linked
to the TR element, and a polyadenylation sequence. The nucleotide
sequence is a first open reading frame (ORF) sequence and encodes a
polypeptide or a fragment thereof and is co-translated with the TR
element.
[0090] In various embodiments, the TR elements of the present
technology exhibit selective translation in stressed and/or dying
cells. The term "selectively translated" or "selective translation"
in stressed and/or dying cells means that the mRNA translation
activity is observed in more than 95% of any cell line transformed
with the TR expression cassette at the peak of the translation
activity, e.g., within about 9 to about 18 hours following
treatment with an acute toxic agent that induces cell stress and/or
death, and that the translational levels of the first ORF of the
inventive expression cassette rise to at least 50% of the
expression levels of the same ORF when transcribed and translated
from the same expression cassette lacking an operably linked TR
element following treatment with the acute toxic agent. For
example, a TR element within an expression cassette of the
technology exhibits selective translation in stressed and/or dying
cells within about 9 hours following treatment with calcium
ionophore A23187 at a concentration of 5 .mu.M, with mRNA
translation being observed in more than 95% of a HEK293 cell line
transformed with the expression cassette, and translation levels of
the first ORF of the expression cassette being at least 50% of the
translation levels of the same ORF when transcribed and translated
from the same expression cassette lacking an operably linked TR
element following the treatment. In some instances, a TR element
within an expression cassette of the technology exhibits selective
translation in stressed and/or dying cells within about 6 to about
9 hours following treatment with calcium ionophore A23187 at a
concentration of 5 .mu.M, with mRNA translation being observed in
about 96, 97, 98, 99, 99.5 or 99.9% of a HEK293 cell line
transformed with the expression cassette, and translation levels of
the first ORF of the expression cassette being about 55, 60, 65,
70, 75, 80, 85, 90, or 95% of the translation levels of the same
ORF when transcribed and translated from the same expression
cassette lacking an operably linked TR element following the
treatment.
[0091] In some embodiments of the present invention, the TR element
is a plp IRES element, which does not direct translation of PIRP-M
or PIRP-L. In other embodiments, the TR element is not derived from
the plp IRES.
[0092] Thus, in one embodiment, the present technology relates to a
nucleic acid expression cassette expressible in mammalian cells,
wherein the expression cassette has the following elements in a 5'
to 3' direction: at least one transcriptional effector sequence; a
TR element encoding a mRNA molecule which is translated in stressed
and/or dying cells; a 3' sequence flanking the TR element that
contains restriction enzyme sites common in the art; a nucleotide
sequence operably linked to the TR element, which is a first open
reading frame (ORF) sequence and encodes a polypeptide or a
fragment thereof and is co-translated with the TR element; and a
polyadenylation sequence.
[0093] In a preferred embodiment, a TR element is selected from a
human or a mouse TR element. More preferably, the TR element is
selected from murine sequences TR.sub.dm (SEQ ID NO: 1) and
TR.sub.plp (SEQ ID NO: 2).
[0094] TR.sub.dm nucleic sequence (SEQ ID NO: 1) was derived from
the DM20 splice variant cDNA of the mouse proteolipid protein gene
1, but has been modified at nucleotide positions 1, 4, 6, 7, 8, 17,
18, 21, 27, 511, and 598. In addition, the last 3 nucleotides
encoding the stop codon were removed.
[0095] TR.sub.plp nucleic sequence (SEQ ID NO: 2) was derived from
the PLP splice variant cDNA of the mouse proteolipid protein gene
1, and it contains modifications at nucleotide positions 1, 4, 6,
7, 8, 17, 18, 21, 27,616, and 703. TR.sub.plp differs from
TR.sub.dm by the presence of nucleotides 349-453. The last 3
nucleotides encoding the stop codon were removed.
[0096] In addition to the TR element, the expression cassettes of
the present technology comprise an upstream transcriptional
effector sequence which regulates gene expression. In one
embodiment, the transcriptional effector sequence is a mammalian
promoter. In addition, the transcriptional effector can also
include additional promoter sequences and/or transcriptional
regulators, such as enhancer and silencers or combinations thereof.
These transcriptional effector sequences can include portions known
to bind to cellular components which regulate the transcription of
any operably linked coding sequence. For example, an enhancer or
silencer sequence can include sequences that bind known cellular
components, such as transcriptional regulatory proteins. The
transcriptional effector sequence can be selected from any suitable
nucleic acid, such as genomic DNA, plasmid DNA, viral DNA, mRNA or
cDNA, or any suitable organism (e.g., a virus, bacterium, yeast,
fungus, plant, insect or mammal). It is within the skill of the art
to select appropriate transcriptional effector sequences based upon
the transcription and/or translation system being utilized. Any
individual regulatory sequence can be arranged within the
transcriptional effector element in a wild-type arrangement (as
present in the native genomic order), or in an artificial
arrangement. For example, a modified enhancer or promoter sequence
may include repeating units of a regulatory sequence so that
transcriptional activity from the vector is modified by these
changes.
[0097] In one embodiment, the promoters are selected from
constitutive, inducible, tissue specific, tumor specific and
response gene promoters. Constitutive promoters can be selected,
e.g., from Rous sarcoma virus (RSV) long terminal repeat (LTR)
promoter, cytomegalovirus immediate early gene (CMV) promoter,
simian virus 40 early (SV40E) promoter, cytoplasmic beta-actin
promoter, adenovirus major late promoter, and the phosphoglycerol
kinase (PGK) promoter. In a preferred embodiment, a constitutive
promoter is a CMV promoter. In another preferred embodiment, a
constitutive promoter is an SV40E promoter.
[0098] Selection of a promoter that is regulated in response to
specific physiologic or synthetic signals can permit inducible
transcription of the gene product. For example, in the case where
expression of a transgene is toxic to the cells in which the vector
is produced, it may be desirable to prevent or reduce transcription
of the transgene. By way of example, a proapoptotic transgene can
be toxic to the cell in which it is produced. Thus, several
inducible promoter systems are available for production of vectors,
which contain a transgene encoding a toxic protein.
[0099] Non-limiting examples of inducible promoters include the
metal-regulated human metallothionine (hMT-IIA) promoter, the
zinc-inducible human Zinc Transporter 1 (hZnT-1) promoter,
dexamethasone (Dex)-inducible promoter, mouse mammary tumor virus
(MMTV) promoter, ecdysone-responsive insect promoter, tetracycline
responsive Tet-On.RTM. and Tet-Off.RTM. systems, RU486-inducible
promoter and rapamycin-responsive promoter. In one embodiment, the
inducible promoter is a metallothionine hMT-IIA promoter. In
another embodiment, the inducible promoter is a zinc-inducible
hZnT-1 promoter.
[0100] Methods and compositions are provided for the controlled
induction of gene expression in a mammalian host cell. For example,
DNA sequences which comprise the human metallothionein II (hMT-IIA)
and zinc-inducible Zinc Transporter 1 (hZnT-1) transcriptional
regulatory systems are induced by elevated concentrations of heavy
metal ions and glucocorticoids. These inducible promoters are
composed of multiple metal-regulatory elements (e.g., MRE) adjacent
to a basal level transcriptional regulator.
[0101] In other indications, it may be desirable to activate
transcription using promoters responsive to hormones or
antibiotics. The ecdysone system (Invitrogen, Carlsbad, Calif.)
consists of a tightly regulated expression mechanism that prevents
basal level transgene expression, but allows for an over 200-fold
induction of transcription. The system is based on the
heterodimeric ecdysone receptor of Drosophila, and when ecdysone or
an analog thereof (such as muristerone A) binds to the receptor,
the receptor activates a promoter to turn on expression of the
downstream transgene. In this system, both monomers of the
heterodimeric receptor are constitutively expressed from one
vector, whereas the ecdysone-responsive promoter which drives the
expression of transgene is on a second plasmid. Thus,
cotransfection of the two plasmids containing the regulated
transgene and the receptor monomers into a reporter cell allows for
the inducible expression of even toxic transgenes.
[0102] The Tet-Off.RTM. or Tet-On.RTM. system (Clontech, Palo Alto,
Calif.) originally developed by Gossen and Bujard (Gossen and
Bujard, 1992; Gossen et al., 1995) utilize tetracycline or
tetracycline derivatives, such as doxycycline, to regulate
transgene expression. In the Tet-On.RTM. system, gene expression is
induced by tetracycline or doxycycline, whereas in the Tet-Off.RTM.
system, antibiotic exposure eliminates gene expression. These
systems are based on two regulatory elements derived from the
tetracycline resistance operon of E. coli, namely the tetracycline
operator DNA sequence and the tetracycline repressor protein. A
Tet-regulated plasmid contains a minimal promoter with
tetracycline-responsive operator elements. A second plasmid
contains the tetracycline-controlled transactivator protein, which
is a fusion protein comprised of the VP16 transcriptional activator
domain and the wild-type tetracycline repressor protein of the
Tet-Off.RTM. system. In the Tet-On.RTM. system, the tetracycline
repressor protein has been altered so that transcription is
activated by the presence of tetracycline or doxycycline.
[0103] Tissue specific promoters can be selected, e.g., from the
transferrin (TF), tyrosinase (TYR), albumin (ALB), muscle creatine
kinase (CKM), myelin basic protein (MBP), glial fibrillary acidic
protein (GFAP), neuron-specific enolase (NSE), and synapsin I
(SYN1) promoters. In one embodiment, the tissue specific promoter
is a synapsin I (SYN1) promoter. In another embodiment, the tissue
specific promoter is the ALB promoter.
[0104] Tumor specific promoters include but are not limited to
promoters for vascular endothelial growth factor (VEGF), a VEGF
receptor (i.e. KDR, E-selectin, or endoglin), alpha-fetoprotein
(AFP), carcinoembryonic antigen (CEA), erbB2 (v-erb-b2
erythroblastic leukemia viral oncogene homolog 2), osteocalcin
(bone gamma-carboxyglutamate protein, BGLAP), SLP1 (secretory
leukoproteinase inhibitor or antileukoproteinase 1),
hypoxia-response element (HRE), L-plastin (lymphocyte cytosolic
protein 1) and hexokinase II (HK2). In one embodiment, the tumor
specific promoter is an alpha fetoprotein (AFP) promoter. In
another embodiment, the tumor specific promoter is a SLP1
promoter.
[0105] Response gene promoters, which stimulate transcription
preferentially or uniquely under certain cellular states and/or in
response to external chemical or environmental stimuli (i.e., heat
or cold shock), can be selected, e.g., from promoters for early
growth response gene 1 (EGR1/ZIF268), tissue-type plasminogen
activator (t-PA), multidrug-resistance protein 1 (mdr-1),
HSPA5/Grp78/BIP (heat shock 70 kDa protein 5), c-fos (v-fos FBJ
murine osteosarcoma viral oncogene homolog), c-jun (v-jun sarcoma
virus 17 oncogene homolog) or from cell cycle-regulated genes such
as, but not limited to, E2F-1 (E2F transcription factor 1), cyclin
A1 (CCNA1) and CDC25C (cell division cycle 25C). In a preferred
embodiment, the response gene promoter is a promoter for HSPA5. In
another preferred embodiment, the response gene promoter is an EGR1
promoter.
[0106] In some embodiments, a specific transcriptional effector
element is isolated and then operatively linked to a minimal
promoter to produce an expression cassette whose transcriptional
activity is dependent upon a single or limited type of cellular
response (e.g., a heat shock response or metal-regulated
element).
[0107] The expression cassette can include species-specific
transcriptional regulatory sequences. Such DNA regulatory sequences
can be selected on the basis of the cell type into which the
expression cassette will be inserted and can be isolated from
prokaryotic or eukaryotic cells, including but not limited to
bacteria, yeast, plant, insect, mammalian cells or from viruses. In
such example, a mammalian promoter would be selected to express a
nucleic acid of choice in a mammalian cell.
[0108] The TR expression cassettes of the present technology enable
selective gene expression in stressed and dying cells, allowing for
a heterologous ORF to be inserted 3' to the TR sequence and 5' of a
polyadenylation signal. The heterologous gene can be either a full
genomic sequence (e.g., including introns), synthetic nucleic acid
or a cDNA copy of a gene of interest, which encodes a protein or a
polypeptide of interest, wherein the polypeptide includes
biologically active ("bioactive") protein fragments. In a preferred
embodiment, cDNA sequences are used for the purposes of the present
technology due to the reduction in genomic complexity provided by
removal of mRNA splice sites.
[0109] Thus, in one embodiment, a first ORF sequence is selected
from the group of reporter genes, cytotoxic tumor suppressor genes,
toxin genes, prodrug activating genes and proapoptotic genes.
[0110] In various embodiments, the first ORF sequence is a reporter
gene. As the name implies, a reporter gene does not confer any
selective advantage on the cell into which it is introduced.
Rather, a reporter gene encodes a product that confers on the cell
a detectable biochemical or visually observable (e.g., fluorescent)
phenotype. The reporter polypeptide can also include a fused or
hybrid polypeptide in which another polypeptide is fused at the
N-terminus or the C-terminus of the polypeptide or fragment
thereof. A fused polypeptide is produced by cloning a nucleic acid
sequence (or a portion thereof) encoding one polypeptide in-frame
with a nucleic acid sequence (or a portion thereof) encoding
another polypeptide. Techniques for producing fusion polypeptides
are known in the art, and include, ligating the coding sequences
encoding the polypeptides so that they are in-frame and translation
of the fused polypeptide is under the control of the TR cassette.
For example, cloning the plp ORF in-frame with the enhanced green
fluorescent protein (EGFP) ORF produced a fusion protein that was
used to monitor the expression, subcellular localization and
biological effect of the fusion protein in cultured cells (Ghandour
S et al. Glia (2002) 40(3):300-11; Boucher S et al. J Neurosci
(2002) 22(5): 1772-83).
[0111] One commonly used class of reporter genes encodes an enzyme
or other biochemical marker, which, when expressed in a mammalian
cell, cause a visible change in the cell or the cell environment.
Such a change can be observed directly, can involve the addition of
an appropriate substrate that is converted into a detectable
product or the addition and binding of a metabolic tracer. Examples
of these reporter genes are the bacterial lacZ gene which encodes
the .beta.-galactosidase (.beta.-gal) enzyme, the Chloramphenicol
acetyltransferase (CAT) enzyme, Firefly luciferase (Coleoptera
beetle), Renilla luciferase (sea pansy), Herpes Simplex 1 thymidine
kinase (HSV1-TK) and the mutant Herpes Simplex 1 thymidine kinase
(HSV1-sr39tk) genes. In the case of .beta.-gal, incubation of
expressing cells with halogen-derivatized galactose results in a
colored or fluorescent product that can be detected and quantitated
histochemically or fluorimetrically. In the case of CAT, a cell
lysate is incubated with radiolabeled chloramphenicol or another
acetyl donor molecule such as acetyl-CoA, and the acetylated
chloramphenicol product is assayed chromatographically. Other
useful reporter genes encode proteins that are naturally
fluorescent, including the (green fluorescent protein (GFP),
enhanced yellow fluorescent protein (EYFP), or monomeric red
fluorescent protein (mRFP1).
[0112] As can be seen from above, exemplary reporter genes can be
selected from GFP, EYFP, mRFP1, .beta.-Gal, and CAT, but any other
reporter gene known in the art can be used. See, e.g., the http
World Wide Web olympusconfocal.com/applications/fpcolorpalette.html
site. In a preferred embodiment, the reporter gene is Firefly
Luciferase. In another preferred embodiment, the reporter gene is
Renilla Luciferase.
[0113] The first ORF sequence can also encode a cytotoxic tumor
suppressor gene that encodes a polypeptide capable of suppressing
the neoplastic phenotype and/or inducing apoptosis. Examples of
tumor suppressor genes useful in the practice of the present
technology include the p53, adenomatous polyposis coli (APC),
Breast Cancer-1 (BRCA-1), BRCA-2, Wilm's Tumor (WT-1),
retinoblastoma gene (Rb), Neurofibromatosis-1 (NF-1), NF-2 and von
Hippel-Lindau (VHL) genes. In a preferred embodiment, the cytotoxic
tumor suppressor gene is the p53 gene.
[0114] In another embodiment, the first ORF sequence encodes a
"toxin gene" that binds to cellular receptor proteins and after
uptake interferes with protein synthesis by blocking ribosome
assembly or function. Examples of toxin genes include proteins such
as Pseudomonas exotoxin (e.g., Exotoxin A or "ETA"), ricin toxin,
diphtheria toxin, and the like. In a preferred embodiment, the
toxin gene is the diphtheria toxin gene.
[0115] In another embodiment, the first ORF sequence is a prodrug
activating gene (e.g., drug-susceptibility or suicide gene), which
codes for a protein that converts a prodrug, which lacks a
therapeutic effect into a drug which renders a cell expressing said
gene susceptible to death following exposure to said prodrug.
Examples of pro-drug genes include the thymidine kinase of Herpes
Simplex Virus (HSV-tk), cytochrome P450, human deoxycytidine kinase
and the bacterial enzymes cytosine deaminase and guanine
phosphoribosyl transferase (gpt) genes. Cells which express these
genes are rendered sensitive to the prodrugs ganciclovir (HSV-tk),
cyclophosphamide (cytochrome P450), cytosine arabinoside
(deoxycytidine kinase), 5-fluorocytosine (bacterial cytosonine
deaminase) or thioxanthine (gpt). In a preferred embodiment, the
prodrug activating gene is the HSV-tk gene which can also provide
an important therapeutic advantage. During TK catalysis of the
antiviral guanosine analogue ganciclovir, apoptotic molecules are
released that kill surrounding cells by a process termed
"bystander" killing. Although a limited number of target cells may
initially express the HSV-tk gene, this localized cytocidal effect
provides a therapeutic effect to adjacent non-expressing, undesired
bystander cells.
[0116] In embodiments in which the first ORF sequence is a
proapoptotic gene, such a sequence causes programmed cell death or
apoptosis of an expressing cell. Examples of pro-apoptotic genes
include p53, the Apoptosis Stimulating Proteins of p53 (e.g. ASPP1,
ASPP2, and ASPP3), the Bcl-2 homologs Bax and Bcl2-L-10 (Diva), the
Apoptosis-Inducing Factor (AIF), Fas, initiator caspases such as
caspase-8 and caspase-9 or an effector caspase such as caspase-3.
In a preferred embodiment, the proapoptotic gene is the caspase-3
gene.
[0117] In another embodiment, the first ORF sequence encodes a
recombinant intracellular antibody ("intrabody") comprising an Fab
or single chain Fv (scFv) molecule but does not encode an operable
secretory sequence and hence are restricted to intracellular
compartments where they bind, neutralize or modify the activity of
a target antigen. This interaction may result in the direct
inhibition of target antigen function, restoration of a mutant
deficient activity, interference with the intracellular trafficking
of the antigen or restriction of the folding of a pathological
mutant protein. To exert their function, recombinant intrabodies
are directed to a subcellular compartment where the antigen is
located. This can be achieved by incorporating signal sequences
routinely fused to the N- or C-terminus. For example, the KDEL
peptide sequence allows the retention of recombinant antibodies
within the endoplasmic reticulum and hence, can be used to block
the processing of cell surface targeted proteins. Other signal
sequences can be incorporated into the intrabody ORF and produce
nuclear localization, ER or Golgi routing, nucleolar localization,
as well as transport to the endosomal or liposomal
compartments.
[0118] Methods for the production of single chain antibodies are
well known to those with skill in the art. By way of example, the
skilled artisan is referred to U.S. Pat. No. 5,359,046 for such
methods. A single chain antibody ("scFv" or "SCA") is composed of
an antibody variable light-chain amino acid sequence (VL) tethered
to a variable heavy-chain sequence (VH) by a designed peptide that
links the carboxyl terminus of the VL sequence to the amino
terminus of the VH sequence, thereby reconstituting an antigen
binding site on a single molecule. SCAs have the binding
specificity and affinity of monoclonal antibodies and, in their
native form, are about one-fifth to one-sixth of the size of a
monoclonal antibody. In addition to these benefits, fully-human
intrabodies can be isolated directly from human libraries (such as
the human single-fold, single-chain variable fragment (scFv)
libraries) without the need for costly and time consuming
"humanization" procedures.
[0119] Almost any kind of biologic molecule can serve as an
intrabody target antigen, for example, intermediate metabolites,
sugars, lipids, and hormones as well as macromolecules such as
complex carbohydrates, phospholipids, nucleic acids and proteins.
The preferred target molecule is an endogenous protein. Intrabodies
have been developed for a number of target proteins involved in
cancer, infectious disease, transplantation, neurodegenerative
disease and other diseases associated with protein overexpression
or mutagenesis. Specific examples of intrabody target proteins
include erbB-2 (androgen receptor), IL-2 receptor, epidermal growth
factor receptor, vascular endothelial growth factor receptor 2, the
folate receptor, HIV gp120 protein, CCR5, CXCR4, alphaV integrin,
metalloproteinase MMP-2 and MMP-9, the RelA subunit of NF-kappaB,
the prion-like protein PrP, the huntingtin protein and the
beta-amyloid precursor protein. In one embodiment, the intrabody is
directed to the RelA subunit of NF-kappaB.
[0120] In embodiments in which the first ORF encodes a secreted
antibody fusion protein, such proteins can induce apoptosis or an
enhanced immune response in targeted cells. Examples include any
antibody fusion protein that delivers a therapeutic response such
as the human interleukin-2-truncated diphtheria toxin, anti-CD22
dsFv-truncated Pseudomonas exotoxin, anti-CD25 scFv-truncated
Pseudomonas exotoxin and the anti-B4-blocked ricin (anti-CD19)
immunotoxin proteins. In one preferred embodiment, the antibody
fusion protein is the anti-B4-blocked ricin immunotoxin
protein.
Sequence Variants
[0121] In certain instances, sequence elements operably linked to
the TR sequences might disrupt the selective translational activity
displayed by the TR expression cassette or exhibit sub-optimal
translational activity. To alleviate any effect on TR activity by
the linked ORF, the present technology provides for codon-usage
variants of the disclosed nucleotide sequences, that employ
alternate codons which do not alter the polypeptide sequence (and
thereby do not affect the biological activity) of the ORF products.
These variants are based on the degeneracy of the genetic code,
whereby several amino acids are encoded by more than one codon
triplet. An example would be the codons CGT, CGG, CGC, and CGA,
which all encode the amino acid, arginine (R). Thus, a protein can
be encoded by a variant nucleic acid sequence that differs in its
precise sequence, but still encodes a polypeptide with an identical
amino acid sequence. Based on codon utilization/preference, codons
can be selected to optimize the translation efficiency of an ORF
without affecting regulated translation from the TR expression
cassette.
[0122] Site directed mutagenesis is one particularly useful method
for producing sequence variants by altering a nucleotide sequence
at one of more desired positions. Site directed (or site specific)
mutagenesis uses oligonucleotide sequences comprising a DNA
sequence with the desired mutation, as well as a sufficient number
of adjacent nucleotides to provide a sequence of sufficient size
and complexity to form a stable duplex on both sides of the
proposed mutation. Typically, a synthetic primer of about 20 to 25
nucleotides in length is preferred, with about 5 to 10 residues on
both sides of the proposed mutation of the sequence being altered.
Typical vectors useful in site directed mutagenesis include the
disclosed vectors, as well as any commercially or academically
available plasmid vector. In general, nucleotide substitutions are
introduced by annealing the appropriate DNA oligonucleotide
sequence with the target DNA and amplifying the target sequence by
PCR procedures known in the art. The present technology
contemplates the use of every possible codon in a coding sequence
for producing the desired ORF sequence for use in accordance with
this invention.
[0123] Directed evolution techniques can be used to prepare
sequence variants having improved TR function. In a directed
evolution technique, at least one round of nucleic acid mutation or
nucleic acid splicing or homologous recombination can be performed,
starting from a TR-containing polynucleotide. Mutation, splicing,
and homologous recombination can be performed in a directed or
random manner. For example, one or more oligonucleotides can be
designed for site-directed mutagenesis of the TR element, as
described above, or one or more randomly generated oligonucleotides
can be contacted with the initial TR-containing polynucleotide
template. Alternatively, or in addition, PCR amplification of the
initial template can be performed under error-permissive conditions
and/or an error-prone polymerase to permit introduction of
mutations, a technique referred to a "sloppy" PCR.
[0124] Similarly, a set of homologous, TR-element-containing
polynucleotides can be spliced or recombined in a directed or
random manner. For example, one or more restriction endonucleases
can be used to digest the homologous polynucleotide templates,
randomly or in a predetermined manner, and the resulting fragments
can then be ligated together. Alternatively or in addition, the set
of TR-element-containing polynucleotides can be pooled and treated
under conditions favoring homologous recombination among them,
either in vitro or in cyto. A combination of mutation and splicing
or recombination techniques can be employed. One or more than one
rounds of any of these can be performed.
[0125] After one or more rounds of mutation, splicing, and/or
recombination, the resulting polynucleotides are then tested to
screen them for TR activity. Typically, this can be done by placing
a reporter molecule coding sequence under the operative control of
one or more of the TR variants that have been produced. The
resulting construct(s) are then expressed in a cell that is placed
under conditions, such as a condition of stress, for which TR
translation can take place. The testing can be used to detect a
desired improvement in TR element function. For example, any one of
improvement in specificity of TR element translation to a stress
condition, sensitivity of TR element activation to a cellular
stress response (e.g., a biochemical change antecent to cell stress
and/or death), or efficiency (i.e. magnitude) of translation
initiation upon TR element activation can be the focus of the
assay.
[0126] Based on the assay result, one or more improved TR elements
can be selected for use, or for further development; in some
embodiments, the selected improved TR element nucleic acids can be
used as a starting polynucleotide or as a starting set of
polynucleotides for another round, or course of rounds, of directed
evolution.
[0127] In various embodiments herein, a TR element can comprise, or
can be made by mutation of a PLP/DM20 polynucleotide comprising
bases of, or corresponding to, bases from about 27 to about 615/510
of a murine or human PLP/DM20 DNA sequences of FIG. 15; and this
can comprise further bases of, or corresponding to, bases from
about 616/511 to about 702/597, bases from about 703/598 to about
772/666, and/or bases from about 773/667 to about 810/705. For
example, a TR element can comprise, or can be made by mutation of a
PLP/DM20 polynucleotide comprising bases of, or corresponding to,
bases from about 27 to about 810/705, with or without omission of
bases from about 616/511 to about 702/597, numbered with reference
to FIG. 15.
[0128] In PLP/DM20 coding sequences, and TR elements thereof or
constructed therefrom, mutations can be made, without adverse
effect on TR-element function, at one or more positions
corresponding to the following PLP/DM20 positions stated with
reference to FIG. 15, i.e. positions: 01, 02, 03, 04 to 21
(including deletion of all of part of this segment), 25, 26, 314,
332, 560/455, 614/509, 622/518 to 696/591 (including deletion of
all or part of this segment, which removes exon 5), 616/511,
703/598, 806/701, 811/706, 817/712, 818/713, and 827/722. In
various embodiments, other nucleobases than the foregoing can be
conserved in PLP/DM20 coding sequences.
[0129] For example, in various embodiments, a nucleobase sequence
of a PLP/DM20 coding sequence hereof can comprise polypyrimidine
motifs at nucleotide positions corresponding to PLP nucleotide
positions 41-48, 50-56, 75-81, 150-156, 200-205, 227-244, 251-257,
and 563-570. In some embodiments, such a sequence can further
comprise polypyrimidine motifs at one or more of PLP positions
270-274, 299-303, 490-494, 578-582, 597-601; and in some
embodiments, also at one or more of PLP positions 626-632, 642-648,
669-674, 707-712, 755-761, 767-771, and 800-804.
[0130] Similarly, in various embodiments, a nucleobase sequence of
a PLP/DM20 coding sequence hereof can comprise GNRA motifs at
nucleotide positions corresponding to PLP nucleotide positions:
130-133, 142-145, 190-193, 220-223, 305-308; and in some
embodiments further at 635-638; and in other embodiments further at
one or more of positions 329-332, 343-346, and 572-575; and in
some, still further at one or more of positions 650-653 and
683-686.
[0131] However, as mentioned, mutation of the following positions
can be undertaken with no adverse effect, and in some cases with an
enhancing effect: 01, 04, 06, 07, 08, 17, 18, 21, 27. In some
embodiments, these mutations can be one or more of: 01t, 04a, 06t,
07g, 08a, 17a, 18g, 21a, and 27t. Other positions that can be
mutated with no adverse effect on function include mutations at one
or more of PLP positions: 25, 26, 314, 332, 560/455, 616/511,
703/598, 806/701, 811/706, 817/712, 818/713, and 827/722. In some
embodiments, these can be one or more of: 25g, 26c, 314g, 332g,
560/455c, 616/511t, 703/598t, 806/701g, 811/706t, 817/712a,
818/713a, and 827/722g. In addition, insertions, e.g., insertions
of up to or about 5 nucleotides, can be made at PLP position
614/509, with no adverse effect on function. In addition, fusions
to position 831/726, e.g., in-frame fusions thereto of reporter or
other target gene coding sequences, do not exhibit any adverse
effect on TR element function.
[0132] In various embodiments, a PLP or DM20 sequences useful
herein can be a vertebrate sequence; in some embodiments, this can
be a human, primate, rodent, equine, bovine, ovine, porcine,
canine, feline, lapine, marsupial, avian, piscine, amphibian, or
reptilian sequence. In various embodiments, a vertebrate sequence
can be a native sequence, whether wild-type or variant; in some
embodiments, a vertebrate sequence can be a wild-type sequence.
[0133] As used herein in regard to PLP/DM20 sequences, "vertebrate
consensus sequence" refers to the DNA sequence:
TABLE-US-00001 1 atgggyykgy wdgakkgytg yrynmgmtgy mtbrtwgggg
ymccmttygc ytchbtsrtb 61 gccacwgkvy tvtgyttyky tggrgtsgcv
ctvttctgyg gmtgyggrca ygargchytv 121 asygghacmg armagytvat
ygagacmtay ttytccaara aytaccaaga mtaygartay 181 ctcatyvayg
tsatymaygc yttycagtay gtcatctatg gaaywgccwy yttcttctty 241
cthtwyggrr ycctvctkyt ggcygarggm ttctacacca cmrsygchrt cargcavatc
301 ythggsgast wcmrrmccmc mryywkmrrs rrkggsctga kykcwacrgt
racwggrggm 361 cmkaarggga grrghdcsmg rggmmvvcak cvagyycayw
cywtrsagck srtstgtcrb 421 tgyttgggaa artggctmgg acayccygay
aagtttgtsg gyrtyacyta tryyhtsacy 481 rtyktvtggm tmctrrystt
ygcctgctcd gcygtdccyg tvtacatyta yttyaayacc 541 tggrycacyt
gycagtctat ygcckyccch rssaagacyw cwrccagyrt mrgyasbcts 601
tgykcdgayg symgvatgta yggtgtycts ccmtggaayg cbttycchgg saargtktgy
661 ggswccarcc tkctbkccat ctgcaaracm rsygagttcc aratgacntt
ycayctbttt 721 atygckgcvt tygtgggkgc wgcngchacw ctdgtbkcmc
tgctcacytw yatgrthgsy 781 gcmwcwtwca actwygcygt sctbmrastb
aykggccgrr gcwcmaagtt ytga
and to DNA complements thereof, to RNA sequences corresponding to
any of the foregoing, to nucleic acid analogs having a nucleobase
sequence corresponding to any of these, and to amino acid sequences
encoded thereby.
[0134] As used herein in regard to PLP/DM20 amino acid and
nucleotide sequences, "vertebrate specific sequence" refers to the
PLP or DM20 sequences of the species listed in FIG. 14, i.e. Homo
sapiens, Pongo pygmaeus (orangutan), Pan troglodytes (chimpanzee),
Macaca mulatta (rhesus monkey), Macaca fascicularis (crab-eating
macaque), Sus scrofa (pig), Mus musculus (mouse), Rattus norvegicus
(rat), Monodelphis domestica (opossum), Oryctolagus cuniculus
(rabbit), Bos taurus (cattle), Canis familiaris (dog), Gallus
gallus (chicken), Taeniopygia guttata (zebra finch), Gekko
japonicus (gecko lizard), Xenopus laevis (frog), and Latimeria
chalumnae (coelacanth). In some embodiments, the vertebrate
specific sequence can comprise, or encode, any one of the amino
acid sequences having Genbank numbers: P60201 (human), Q5R6E6
(orangutan), XP_001140782 (chimpanzee), XP_001088537 (rhesus
monkey), Q8HXW7 (crab-eating macaque), NP_999139 (pig), NP_035253
(mouse), NP_112252 (rat), XP_001374483 (opossum), P47789 (rabbit),
CAA08909 (cattle), 39025 (dog), CAA43839 (chicken), P47790 (zebra
finch), AAW79015 (gecko lizard), CAA79582 (frog), or BAA84207
(coelacanth).
[0135] DNA sequences encoding these are readily available to one of
ordinary skill in the art by searching NCBI Genbank in the
Nucleotide menu at the http World Wide Web
ncbi.nlm.nih.gov/sites/entrez website. For example, useful DNA
sequences include those listed under Genbank accession numbers:
AJ006976 (human), CR860432 (orangutan), XM_001140782 (chimpanzee),
XM_001088537 (rhesus monkey), AB083324 (crab-eating macaque), NM
213974 (pig), NM_011123 (mouse), NM_030990 (rat), XM_001374446
(opossum), NM_001082328 (rabbit), AJ009913 (cattle), X55317 (dog),
X61661 (chicken), NM_001076703 (residues 113-946, zebra finch),
AY880400 (gecko lizard), Z19522 (frog), and
AB025938(coelacanth).
[0136] In various embodiments, a PLP or DM20 sequence useful herein
can be a mammalian sequence; in some embodiments, this can be a
human, primate, rodent, equine, bovine, ovine, porcine, canine,
feline, lapine, or marsupial sequence.
[0137] As used herein in regard to PLP/DM20 sequences, "mammalian
consensus sequence" refers to the DNA sequence:
TABLE-US-00002 1 atgggcytgt tagagtgytg ygcnagatgy ctsgtagggg
ccccctttgc ttccytggtg 61 gccactggat trtgtttctt tggrgtggca
ctsttctgtg gmtgtggaca tgaagchytm 121 actggyacag aaaagytaat
tgagacmtat ttctccaaaa aytaccaaga ctaygagtat 181 ctcatyaatg
tgatycatgc yttccagtat gtcatctatg gaactgcctc tttcttcttc 241
ctttatgggg ccctcctgct ggcygagggc ttctacacca ccggygcwgt caggcagatc
301 tttggcgact acaagaccac catctgcggs aagggcctga gygcaacggt
aacagggggc 361 cagaagggga ggggttccag aggccaacat caagctcatt
ctttggagcg ggtgtgtcat 421 tgtttgggaa aatggctagg acatcccgac
aagtttgtgg gcatcaccta tgccytgacy 481 gttgtrtggc tcctrgtgtt
tgcctgctck gctgtrcctg tgtacattta yttcaayacc 541 tggaccacyt
gycagtctat tgcckycccy agcaagacyt ctgccagyat aggcastctc 601
tgygctgatg ccagaatgta tggtgttctc ccatggaatg ctttyccwgg caargtktgt
661 ggctccaacc ttctgtccat ctgcaaaaca gctgagttcc aaatgacstt
ccayctgttt 721 attgctgcvt tygtgggkgc tgcrgcyaca ctrgtktccc
tgctcacctt catgattgct 781 gccacttaca acttygccgt cctkaaactc
atgggccgag gcaccaagtt ctga
and to DNA complements thereof, to RNA sequences corresponding to
any of the foregoing, to nucleic acid analogs having a nucleobase
sequence of any of these, and to amino acid sequences encoded
thereby.
[0138] As used herein in regard to PLP/DM20 amino acid and
nucleotide sequences, "mammalian specific sequence" refers to the
PLP or DM20 sequences of the mammalian species listed in FIG. 14,
i.e. Homo sapiens, Pongo pygmaeus (orangutan), Pan troglodytes
(chimpanzee), Macaca mulatta (rhesus monkey), Macaca fascicularis
(crab-eating macaque), Sus scrofa (pig), Mus musculus (mouse),
Rattus norvegicus (rat), Monodelphis domestica (opossum),
Oryctolagus cuniculus (rabbit), Bos taurus (cattle), or Canis
familiaris (dog). In some embodiments, the mammalian specific
sequence can comprise, or encode, any one of the amino acid
sequences having Genbank numbers: P60201 (human), Q5R6E6
(orangutan), XP_001140782 (chimpanzee), XP_001088537 (rhesus
monkey), Q8HXW7 (crab-eating macaque), NP_999139 (pig), NP_035253
(mouse), NP_112252 (rat), XP_001374483 (opossum), P47789 (rabbit),
CAA08909 (cattle), or 39025 (dog). TR elements comprising such TR
polynucleotides are useful herein, as are expression cassettes
comprising those TR elements.
[0139] In various embodiments, a TR element can have a PLP or DM20
nucleotide sequence that is at least 62% identical to a FIG. 15 PLP
sequence or a FIG. 15 DM20 sequence, respectively. The sequence
identicality can be at least or about 65, 70, 75, 80, 85, 90, or
95% thereto. In some embodiments, the sequence can be 97, 98, 99%
or more identical thereto. Such a non-identical sequence will
retain operative features a PLP or DM20 TR element, i.e. the
defined polypyrimidine tracts, GNRA motifs, and 19S rRNA binding
site thereof.
[0140] In various embodiments, TR elements hereof can be used to
identify agents that induce, enhance, or inhibit a cellular stress
response, e.g., a reponse to heat stress, cold stress, oxidation
stress, tonic stress, toxication, or a combination thereof. The
cellular stress response can comprises apoptosis and/or necrosis.
In some embodiments, a process for identifying such agents can
involve identifying the degree to which an agent induces, enhances,
inhibits, or reverses a cellular stress response; this can be
accomplished, e.g., by identifying a degree of cellular stress
response that is proportional to the magnitude of a signal detected
from a reporter molecule expressed under the control of the TR
element.
[0141] In various embodiments, TR elements hereof can be used in
processes for prophylactic, curative, or palliative treatment of a
human subject who has need for protection against a cellular stress
response. In such an embodiment, the TR element being part of a
construct in which it is operatively attached to a polynucleotide
comprising a coding sequence encoding an expression product that
provides protection against a cellular stress response, and the
treatment comprises administering a composition comprising such a
construct. The protection provided thereby can be, e.g., an
activity that sequesters or degrades a toxifying agent, that
stabilizes biomolecules in the cell, that catalyzes the formation
of a protective agent, or that causes expression of a protective
agent from a different coding sequence.
[0142] Bicistronic TR Expression Cassettes
[0143] The expression cassette as described in the above sections
is referred to as a monocistronic cassette due to the presence of
only a single ORF sequence is the TR transcript. In addition to the
monocistronic cassette, the present technology also contemplates
the use of bicistronic TR expression cassettes, which include two
ORF sequences.
[0144] Accordingly, in one embodiment of the present technology,
the bicistronic TR expression cassette includes the second ORF
sequence located 5' to the TR sequence in addition to the same
elements described above for the monocistronic TR expression
cassette, wherein the second ORF sequence is not operatively linked
to the TR element. Thus, one skilled in the art will readily
recognize that while the second ORF sequence is transcribed with
the TR sequence and the first ORF in a single mRNA species, it is
translated independently of the TR element and the first ORF
sequence via a cap-dependent mechanism. The second ORF sequence
finds utility when one would like to observe the differential
effect of any agent or molecule on cap-dependent or cap-independent
translation. For example, toxic agents will induce a transition
from cap-dependent to cap-independent translation. Where the second
ORF sequence is a reporter gene, the loss of reporter gene activity
provides a temporal and quantitative measure of the transition from
cap-dependent to cap-independent translation. Hence, a number of
embodiments include a bicistronic expression cassette in which the
first ORF sequence is a reporter gene, cytotoxic tumor suppressor,
toxin gene, prodrug activating gene, antibody, derivative antibody
or a proapoptotic gene, and the second ORF sequence is a reporter
gene. Alternatively, a bicistronic TR expression cassette can
include a first ORF sequence which is a reporter gene, and a second
ORF sequence which is selected from a cytotoxic tumor suppressor,
toxin gene, prodrug activating gene, single chain antibody or a
proapoptotic gene. In this embodiment, the second ORF provides for
the translation of a putative toxic gene product which stimulates
the transition from cap-dependent to cap-independent translation
which can subsequently be measured by reporter gene activity
produced by TR-regulated translation of the first ORF. One skilled
in the art can readily prepare any of these combinations. Exemplary
monocistronic and bicistronic cassettes are shown in Tables
1-4.
[0145] In addition, SEQ ID NOs: 3-4 and SEQ ID NOs: 5-6 describe
nucleic acid sequences for specific examples of monocistronic and
bicistronic TR expression cassettes, respectively.
[0146] TR.sub.dm monocistronic cassette (pCMV-TR.sub.dm-Luc) (SEQ
ID NO: 3) contains the TR.sub.dm nucleic sequence operatively
linked to firefly luciferase coding sequence. Nucleotides 1 to 589
of the cassette correspond to human cytomegalovirus immediate early
promoter from pEYFP-N1 plasmid (Clontech). Nucleotides 590 to 630
correspond to an artificial linker sequence, which was extensively
modified from the original pEYFP-N1 version. Nucleotides 631 to
1356 correspond to TR.sub.dm. The next eight nucleotides are an
artificial linker. Nucleotides 1369 to 1371 correspond to a Kozak
consensus translation initiation site derived from the pEYFP-N1
plasmid. Nucleotides 1372 to 3024 correspond to the firefly
luciferase open reading frame derived from phCMV-Luc-FSR plasmid
(Genlantis). Nucleotides 3025 to 3171, 3179 to 3200, and 3207 to
3223 correspond to linker DNA derived from pEYFP-N1 plasmid.
Nucleotides 3172 to 3178 and 3201 to 3207 correspond to the simian
virus 40 (SV40) early gene polyadenylation signals derived from
pEYFP-N1 plasmid. The mRNAs transcribed from this cassette start at
nucleotide 583 and end at nucleotides 3211 or 3223.
[0147] TR.sub.plp monocistronic cassette (pCMV-TR.sub.plp-Luc) (SEQ
ID NO: 4) contains the TR.sub.plp nucleic sequence operatively
linked to firefly luciferase coding sequence. Nucleotides 1 to 589
of the cassette correspond to human cytomegalovirus immediate early
promoter from pEYFP-N1 plasmid (Clontech). Nucleotides 590 to 630
correspond to an artificial linker sequence (extensively modified
from the original pEYFP-N1 version). Nucleotides 631 to 1461
correspond to TR.sub.plp. The next eight nucleotides are an
artificial linker. Nucleotides 1470 to 1476 correspond to a Kozak
consensus translation initiation site derived from pEYFP-N1
plasmid. Nucleotides 1477 to 3129 correspond to the firefly
luciferase open reading frame derived from phCMV-Luc-FSR plasmid
(Genlantis). Nucleotides 3230 to 3276, 3284 to 3305, and 3313 to
3328 correspond to a linker DNA derived from pEYFP-N1 plasmid.
Nucleotides 3277 to 3283 and 3306 to 3312 correspond to the
SV40early gene polyadenylation signals derived from pEYFP-N1
plasmid. The mRNAs transcribed from this cassette start at
nucleotide 583 and end at nucleotides 3316 or 3328.
[0148] The TR.sub.dm bicistronic cassette (pCMV-Luc-TR.sub.dm-EYFP)
(SEQ ID NO: 5) contains the TR.sub.dm nucleic acid sequence with
the second ORF encoding the firefly luciferase coding sequence and
the operably linked first ORF encoding the EYFP coding sequence
(the enhanced yellow-green variant of the Aequorea victoria green
fluorescent protein). Nucleotides 1 to 589 of the cassette
correspond to human cytomegalovirus immediate early promoter from
pEYFP-N1 plasmid (Clontech). Nucleotides 590 to 630 correspond to
an artificial linker sequence (extensively modified from the
original pEYFP-N1 version). Nucleotides 631 to 2283 correspond to
the firefly luciferase open reading frame derived from
phCMV-Luc-FSR plasmid (Genlantis). The next six nucleotides are an
artificial linker. Nucleotides 2290 to 3015 correspond to
TR.sub.dm. Next 23 nucleotides (3016 to 3038) correspond to an
artificial linker DNA from the pEYFP-N1 plasmid. Nucleotides 3039
to 3045 correspond to a Kozak consensus translation initiation site
derived from pEYFP-N1 plasmid. Nucleotides 3046 to 3760 correspond
to EYFP open reading frame derived from pEYFP-N1 plasmid.
Nucleotides 3761 to 3912, 3920 to 3941, and 3949 to 3964 correspond
to linker DNA derived from pEYFP-N1 plasmid. Nucleotides 3913 to
3919 and 3942 to 3948 correspond to the SV40early gene
polyadenylation signals derived from pEYFP-N1 plasmid. The mRNAs
transcribed from this cassette start at nucleotide 583 and end at
nucleotides 3952 or 3964.
[0149] The TR.sub.plp bicistronic cassette
(pCMV-Luc-TR.sub.plp-EYFP) (SEQ ID NO: 6) contains the TR.sub.plp
nucleic acid sequence with the second ORF encoding the firefly
luciferase coding sequence and the operably linked first ORF
encoding the EYFP coding sequence (the enhanced yellow-green
variant of the Aequorea victoria green fluorescent protein).
Nucleotides 1 to 589 of the cassette correspond to human
cytomegalovirus immediate early promoter from pEYFP-N1 plasmid
(Clontech). Nucleotides 590 to 630 correspond to an artificial
linker sequence (extensively modified from the original pEYFP-N1
version). Nucleotides 631 to 2283 correspond to the firefly
luciferase open reading frame derived from phCMV-Luc-FSR plasmid
(Genlantis). Next 6 nucleotides are an artificial linker.
Nucleotides 2290 to 3120 correspond to TR.sub.plp. The next 23
nucleotides (3121 to 3143) correspond to an artificial linker DNA
from the pEYFP-N1 plasmid. Nucleotides 3144 to 3150 correspond to a
Kozak consensus translation initiation site derived from pEYFP-N1
plasmid. Nucleotides 3151 to 3865 correspond to EYFP open reading
frame derived from pEYFP-N1 plasmid. Nucleotides 3866 to 4017, 4025
to 4046, and 4054 to 4069 correspond to linker DNA derived from
pEYFP-N1 plasmid. Nucleotides 4018 to 4024 and 4047 to 4053
correspond to the SV40early gene polyadenylation signals derived
from pEYFP-N1 plasmid. The mRNAs transcribed from this cassette
would start at nucleotide 583 and end at nucleotides 4057 or
4069.
[0150] Polyadenylation Sequence
[0151] One skilled in the art will readily recognize that any
polyadenylation (polyA) signal can be incorporated into the 3'
untranslated (3'UTR) of the monocistronic or bicistronic TR
expression cassettes described herein. Examples of polyA sequences
useful for the present technology include the SV40 early and late
gene, the HSV-TK, and human growth hormone (hGH) sequences. In a
preferred embodiment, the polyA sequence is the SV40 early gene
sequence.
[0152] Optional Elements
[0153] In various embodiments, the 3'UTR of the TR cassette
sequence can include one or more elements which regulate TR gene
expression by altering mRNA stability. Typically, mRNA decay is
exemplified by the loss of the mRNA polyA tail, recruitment of the
deadenylated RNA to the exosome, and ribonuclease (RNAse)
degradation. In select mRNAs, this process is accelerated by
specific RNA instability elements that promote the selective
recognition of a mRNA by cellular degradation systems. In this
invention, an unstable TR cassette mRNA contains 3'UTR AU-rich
element ("ARE") sequences derived from mRNA species encoding
cellular response/recovery genes.
[0154] Examples of ARE sequences available to this technology
include 3'UTR sequences from the c-fos, the granulocyte-macrophage
colony stimulating factor (GM-CSF), c-jun, tumor necrosis factor
alpha (TNF-.alpha.), and IL-8 mRNAs. In a preferred embodiment, the
ARE sequences from the c-fos gene are used.
[0155] The monocistronic and bicistronic expression cassettes of
the present technology can also include a 5' untranslated region
(5'UTR), which is located 3' to the promoter and 5' to the TR
element. In some embodiments, such a region comprises a mRNA
transcription initiation site. In other embodiments, the 5'
untranslated region comprises an intron sequence, which directs
mRNA splicing and is required for the efficient processing of some
mRNA species in vivo. A general mechanism for mRNA splicing in
eukaryotic cells is defined and summarized in Sharp (Science 235:
736-771 (1987)). There are four nucleic acid sequences which are
necessary for mRNA splicing: a 5' splice donor, a branch point, a
polypyrimidine tract and a 3' splice acceptor. Consensus 5' and 3'
splice junctions (Mount, Nucl. Acids. Res. 10:459-472 (1992)) and
branch site sequences (Zhuang et al., PNAS 86:2752-2756 (1989)) are
known in the art.
[0156] In some embodiments, the 5' UTR sequences comprise natural
introns which exist in a native gene sequence or an artificial
intron, such as the human beta-globin-immunoglobulin sequence
present in the pAAV-MCS vector (Stratagene).
[0157] Additionally, the expression cassettes of the present
technology can include one or more of the following: [0158] a
sequence of between about 15-50 nucleotides located 5' to the
promoter, that includes one or more restriction sites for insertion
of the TR cassette into a plasmid, shuttle vector or viral vector;
[0159] a sequence of between about 15-50 nucleotides located 3' to
the TR element and 5' to the ORF sequence, that includes one or
more restriction sites for insertion and operative linkage of the
TR element and the ORF sequence; [0160] a sequence of between about
15-50 nucleotides located 3' to the ORF sequence and 5' to the
polyadenylation signal, that includes one or more restriction sites
for insertion and operative linkage of the ORF sequence and the
polyadenylation sequence; and [0161] a sequence of between about
15-50 nucleotides located 3' to the polyadenylation sequence, that
includes one or more restriction sites for insertion of the TR
cassette into a plasmid, shuttle vector or viral vector.
[0162] Vectors
[0163] The TR expression cassettes described herein can be inserted
into plasmid or viral ("shuttle") vectors depending upon the host
cell which is used to replicate the TR cassette. In general, the TR
DNA expression cassette is inserted into the appropriate
restriction endonuclease site(s) in the disclosed vectors using
techniques known in the art. Numerous vectors useful for this
purpose are generally known (Miller, Human Gene Therapy 15-14,
1990; Friedman, Science 244:1275-1281, 1989; Eglitis and Anderson,
BioTechniques 6:608-614, 1988; Tolstoshev and Anderson, Current
Opinion in Biotechnology 1:55-61, 1990; Sharp, The Lancet
337:1277-1278, 1991; Cornetta et al., Nucleic Acid Research and
Molecular Biology 36:311-322, 1987; Anderson, Science 226:401-409,
1984; Moen, Blood Cells 17:407-416, 1991; Miller et al.,
Biotechniques 7:980-990, 1989; Le Gal La Salle et al., Science
259:988-990, 1993; and Johnson, Chest 107:77S-83S, 1995).
[0164] A plasmid vector is selected in part based upon the host
cell that is to be transformed with the plasmid. For example, the
presence of bacterial or mammalian selectable markers present in
the plasmid, the origin of replication, plasmid copy number, an
ability to direct random or site specific recombination with
chromosomal DNA, etc. can influence the choice of an appropriate
vector. In some embodiments, bacterial plasmids such as pBluescript
II, pET14, pUC19, pCMV-MCS and pCMVneo are employed for propagating
a TR cassette of the present technology in bacterial cells. In a
preferred embodiment, a plasmid is the pCMVneo vector. In another
preferred embodiment, the plasmid is the pBluescript II vector.
[0165] In another embodiment, a TR expression cassette is inserted
into a mammalian or viral shuttle vector. Whereas mammalian shuttle
vectors contain mammalian selectable markers and provide for the
isolation of cells containing stable genomic integrants, viral
shuttle vectors provide for the reconstitution of a viral genome
using recombination or genetic complementation. In some
embodiments, a mammalian shuttle vector is selected from the pCMV,
pEYFP-N1, pEGFP-N1, or pEGFP-C1 plasmids. In a preferred
embodiment, the mammalian shuttle vector is pEYFP-N1. In some
embodiments, a viral shuttle vector is selected from the pAAV-MCS
(Adeno-associated Virus serotype 2 or AAV2 genome) or pBac-1,
pBacPAK8/9 (Autographa californica baculovirus genome) plasmids. In
one preferred embodiment, the viral shuttle vector is pAAV-MCS. In
another preferred embodiment, the viral shuttle vector is the
pBac-1 plasmid.
[0166] To insure efficient delivery of the expression cassette to a
particular cell, tissue or organ, it can be incorporated into a
non-viral delivery system, which facilitates cellular targeting.
For example, a mammalian shuttle plasmid that includes a TR
cassette may be encapsulated into liposomes. Liposomes include
emulsions, foams, micelles, insoluble monolayers, liquid crystals,
phospholipid dispersions, lamellar layers and the like. The
delivery of DNA sequences to target cells using liposome carriers
is well known in the art as are methods for preparing such
liposomes.
[0167] The viruses useful in the practice of the present technology
include recombinantly modified enveloped or non-enveloped DNA and
RNA viruses, preferably selected from the baculoviridiae,
parvoviridiae, picornoviridiae, herpesviridiae (e.g., HSV),
poxviridiae, and adenoviridiae viruses. In some embodiments, the
recombinant virus is a baculoviridiae virus. In a preferred
embodiment, the baculovirus is an Autographa californica derivative
virus. In other embodiments, the virus is a parvoviridiae virus,
e.g., an adeno-associated virus ("AAV"). In a preferred embodiment,
the AAV is an AAV serotype 2. In another embodiment, the AAV is an
AAV serotype 1. This list is non-exclusive and can include, e.g.,
retroviridae such as lentivirus. A selected virus can be avirulent,
or can be made avirulent as part of preparation as a viral vector
hereof.
[0168] The viral genomes are preferably modified by recombinant DNA
techniques to include the TR expression cassette and may be
engineered to be replication deficient, conditionally replicating
or replication competent. For example, it may prove useful to use a
conditionally replicating virus to limit viral replication to
specific, regulated cell culture conditions.
[0169] Chimeric viral vectors which exploit advantageous elements
of more than one "parent" virus properties are included herein.
Minimal vector systems in which the viral backbone contains only
the sequences needed for packaging of the viral vector and
optionally includes the expression cassette may also be produced
and used in the present invention. It is generally preferred to
employ a virus from the species to be treated, such as a human
herpes virus when a human cell or a human cell line is transduced
with it. In some instances, viruses which originated from species
other than the one which is to be transduced therewith can be used.
For example, adeno-associated viruses (AAV) of serotypes derived
from non-human sources may be useful for treating humans because
the non-human serotypes should not be immediately recognized by
natural or pre-existing human antibodies. By minimizing immune
responses to the vectors, rapid systemic clearance of the vector is
avoided and the duration of the vector's effectiveness in vivo is
increased.
[0170] Mammalian Cells
[0171] Mammalian cells of this technology containing a TR cassette
can be used to screen for molecules (such as chemicals, drugs,
peptides and nucleic acids) or environmental conditions (such as
culture conditions or manipulations) that affect metabolism or
cellular stasis and induces cell stress or death. These cells also
enable the study of drug absorption, metabolism and safety, the
identification of factors that affect drug metabolism and the
evaluation and validation of pharmacological effects. A
monocistronic or a bicistronic TR expression cassette in any of the
mammalian shuttle vectors described above can be transformed into a
mammalian cell. A shuttle vector can be introduced into the host
cell by any technique available to those of skill in the art. These
include, but are not limited to, chemical transfection (e.g.,
calcium chloride method, calcium phosphate method), lipofection,
electroporation, cell fusion, microinjection, and infection with
virus (Ridgway, A. "Mammalian Expression Vectors" Ch24, pg 470-472,
Rodriguez and Denhardt, Eds., Butterworhs, Boston Mass. 1988).
[0172] A mammalian cell can be a mammalian cell that is isolated
from an animal (i.e., a primary cell) or a mammalian cell line.
Methods for cell isolation from animals are well known in the art.
In some embodiments, a primary cell is isolated from a mouse. In
other embodiments, a primary cell is isolated from a human. In
still other embodiments, a mammalian cell line can be used.
Exemplary cell lines include HEK293 (human embryonic kidney),
HT1080 (human fibrosarcoma), NTera2D (human embryonic teratoma),
HeLa (human cervical adenocarcinoma), Caco2 (human colon
adenocarcinoma), HepG2 (human liver hepatocellular carcinoma),
Cos-7 (monkey kidney), ES-D3 (mouse embryonic stem cell), BALBC/3T3
(mouse fibroblast), and hES H1 (human embryonic stem cell). Host
cell lines are typically available from, for example, the American
Tissue Culture Collection (ATCC), any approved Budapest treaty site
or other biological depository.
[0173] In still other embodiments, a mammalian embryonic stem (ES)
cell can be used, such as a mouse ES cell mES-D3 or a human ES cell
hES H1.
[0174] Cell Selection
[0175] The foregoing method requires the preparation of mammalian
cell cultures. The cells used in the assays may be recombinant
cells tailored to express the TR cassette. Once a mammalian cell or
cell line is transformed with the TR expression cassette described
herein, it is desirable to select cells with high expression of the
first and/or second ORF. Several methods for doing so are known in
the art, and are briefly described below.
[0176] A drug resistance gene, referred to as a "dominant
selectable marker" that is present on the mammalian shuttle vector
is utilized for such selection. The selective marker allows for the
isolation of cells that have stably integrated the exogenous
expression vector into the genomic DNA so that the cells that
functionally incorporate the exogenous DNA develop constitutive
resistance to the corresponding drug. This is most typically
followed by the selective growth of cells in restrictive mediums
and the establishment of a continuous supply of recombinant
expressing cells. Examples of selectable markers include neomycin
phosphotransferase ("NeoR or G418R"), hygromycin phosphotransferase
("HygR"), and puromycin N-acetyltransferase ("PurR").
[0177] Two methods are typically employed in the art to establish,
characterize and store an expressing cell. The first involves the
establishment, collection and storage of the entire population of
transformed and drug resistant cells, in which each cell comprises
at least one integration event of the transgene conferring drug
resistance, termed a "cell pool." The second involves the isolation
of individual cell colonies/clones derived from a single drug
resistant cell, screening for a desired trait and storage as a
cellular stock termed a "cell line."
[0178] In contrast to the first approach, which provides a mixed
population of resistant cells with a wide array of gene expression
levels, the second approach requires the selection of distinct
clones from hundreds of isolated cell colonies to identify a select
group of colonies which express the desired gene product at a
desired level. Once these cells are identified, they are amplified,
and either maintained in cell culture or frozen for future use.
[0179] Embryonic Stem Cells
[0180] Stem cells in particular find application in the methods of
the present invention. Pluripotent, adult, blastocyst-derived,
gonadal, teratoma-derived, totipotent, multipotent, embryonic (ES),
embryonic germ (EG), and embryonic carcinoma (EC) cells are all
examples of stem cells for use in these methods.
[0181] Pluripotent stem cells can be produced from the fetal
material of any animal, such as any mammal. However, in one
embodiment, the mammal is a rodent, such as a mouse, guinea pig or
rat. In a preferred embodiment, the mouse ES cell is mES-D3. The
fetal material can be from livestock, such as cattle, horses, pigs,
sheep, goats, etc. The fetal material can also be from primates,
including humans. Pluripotent stem cell lines have been reported,
for example but not limited to, in chicken (Pain, B. et al., (1996)
Development (Cambridge, U.K.) 122, 2339-2348), mink (Sukoyan, M. A.
et al., (1993) Mol. Reprod. Dev. 36, 148-158), hamster (Doetschman,
T. et al., (1988) Dev. Biol. 127, 224-227), pig (Wheeler, M. B.
(1994) Reprod. Fertil. Dev. 6, 563-568; Shim, H. et al., (1997)
Biol. Reprod. 57, 1089-1095), rhesus monkey (Thomson, J. A. et al.,
(1995) Proc. Natl. Acad. Sci. USA 92, 7844-7848), and common
marmoset (Thomson, J. A. et al., (1996) Biol. Reprod. 55, 254-259).
The derivation of stem cell lines is described in the references
cited in the above paragraph.
[0182] Stem cells exhibit a variety of distinct properties and
categories of properties. For example, in some forms, stem cell
lines are capable of prolonged proliferation ex vivo (>1 year)
in an undifferentiated state. Stem cells can also maintain a normal
karyotype while proliferating and/or differentiating. Stem cells
can also exhibit the ability to form every cell type in an organism
(i.e. totipotent trait). Other stem cells retain the ability to
differentiate into mesoderm, endoderm and ectoderm tissues,
including germ cells, eggs and sperm. Some stem cells can form
embryoid bodies (EBs) under certain growth conditions, such as
culture conditions which do not maintain an undifferentiated state.
Moreover, stem cells can often form chimeras through fusion with
blastocysts, which is required for producing transgenic
animals.
[0183] In addition to being kept in an undifferentiated state, the
ES cells can be manipulated through changing growth conditions to
induce differentiation into a particular cell type (referred to as
"directed differentiation"). For example, pluripotent stem cells
can be directed towards a specific lineage by molecules such as
drugs, prodrugs, peptides and nucleic acids that 1) activate
endogenous transcription programs which regulate differentiation;
2) introduce exogenous nucleic acids that ubiquitously express
differentiation-specific transcription factors; 3) provide cell
cultures with medium containing growth factors/regulatory molecules
that induce differentiation; or 4) allow cocultures of stem cells
and cell types capable of lineage induction. A number of ectodermal
derivatives (ED) directed differentiation methods are described
below, and can be used in the methods of the present invention.
[0184] In various embodiments, the present technology provides cell
based systems for the identification of toxic agents. More
specifically, transgenic ES cells, transformed with the TR
cassette, may be programmed by directed differentiation to
differentiate into specific cell lineages, to provide ex vivo
cell-specific screens of toxic agents. In one embodiment the
TR-regulated ORF is a reporter gene, more specifically, the firefly
luciferase gene. In another embodiment, the TR-regulated ORF is the
EYFP protein.
[0185] Genetic manipulation can be used to alter the properties of
the stem cells. A modified stem cell is a stem cell that has a
genetic background different than the original genotype of the
cell. For example, a modified stem cell can be a stem cell that
expresses protein sequences from an extra-chromosomal or integrated
DNA sequence. Stem cell properties can be modified using selection
for dominant selectable markers. For example,
transformation/transduction with a vector encoding an antibiotic
resistance gene can be used to select for a cell population that
can survive antibiotic application. Cells that express the marker
gene can also integrate cis-linked transgenes such as the TR
cassette so that these transgenes are stably incorporated into the
genome. Various methods exist in the art to prepare cell lines of
genetically modified stem cells. One application of this technology
is a method to employ genetically modified stem cell lines, capable
of expressing the TR cassette, for cell based cytotoxicology
assays. In one embodiment, the TR cassette encodes a reporter gene
such as firefly luciferase, from the CMV promoter, providing a
method for constitutive imaging of cell death. In another
embodiment, the TR cassette encodes a reporter gene such as firefly
luciferase, from the EGR-1 promoter, providing a method for imaging
cell death during early stress responses. It is anticipated that a
skilled artisan could design similar methods of imaging cell death
in transgenic stem cells based upon a particular need or process of
measuring and/or inducing cell death. By way of example, a number
of methods are described in the art for producing the directed
differentiation of stem cells ex vivo. Some of these are summarized
in the subsequent sections. The formation of ectodermal derivative
cells is common in spontaneously differentiating stem cells and is
generally considered a default developmental pathway. The
neuroectoderm cell fate can be selectively promoted to generate
neural progenitors and differentiated neural cell types (e.g.
neurons, astroglia and oligodendroglia) (Carpenter M K, et al.
2001). Oligodendrocytes can be produced from stem cell lines using
FGF (e.g. FGF2) and epidermal growth factor (EGF), followed by
supplementation with retinoic acid (RA). These oligodendrocyte
precursors are able to mature and remyelinate neurons (Nistor G I,
et al. 2005). Alternative multistep methods can produce
dopaminergic neurons (Park S, et al. 2004; Perrier A L, et al.
2004) and motor neurons by culturing stem cells in RA and FGF-2,
then RA and sonic hedgehog (SHH), and finally brain-derived
neurotrophic factor (BDNF), glial-derived neurotrophic factor
(GDNF), insulin-like growth factor-1 (IGF1) and low levels of
SHH.
[0186] In contrast, treating stem cell cultures with bone
morphogenetic protein (BMP), an antagonist of Noggin, generates
stem cells with a flattened epithelial morphology and a gene
expression pattern characteristic of extra-embryonic endoderm, a
cell type commonly associated with the yolk sac and placenta in
developing embryos. Thus, prolonged culture of stem cells in
serum-free medium with BMP4 will produce flat epithelial cells that
express genes (e.g., MSX2), and proteins (e.g., human chorionic
gonadotrophin) associated with trophoblast or placental
development.
[0187] Similarly, coculture of stem cells with the mouse bone
marrow mesenchymal PA6 cell line, which expresses stromal cell
derived inducing activity (SDIA), will produce a mixture of
midbrain neurons that are tyrosine hydrolase positive (TH+) and
express the nurr1 and LMX1b genes (Kawasaki H, et al. 2002;
Mizuseki K, et al. 2003), as well as pigmented retinal epithelium
cells. Further manipulation of culture conditions with BMP4 induces
the formation of neural crest cells and dorsal-most central nervous
system cells. Suppression of SHH promotes the formation of motor
neurons (Trounson A. 2004). Stem cells can also be directed into
midbrain dopamine neurons when grown with mouse bone marrow
mesenchyme cell lines (e.g. MS5 and S2 cells), where there is
sequential expression of the Pax2, PaxS and engrailed-1
transcription factors in response to FGF-8, SHH, ascorbic
acid/vitamin C and BDNF (Perrier A L, et al. 2004).
[0188] Exposure of partially differentiated neuroepithelial
derivatives to FGF-8 and SHH promotes the production of
dopaminergic neurons with a forebrain phenotype; however, early
exposure to FGF-8 during neuroepithelial specification promotes a
midbrain phenotype and a differentiation pathway leading to
midbrain dopaminergic neurons. Hence, the order of administering
FGF-8 and SHH can determine neuronal fate.
[0189] Coculture methodologies have also been used to produce
differentiated cardiomyocytes from stem cells. 15-20% of cultures
of stem cells grown with the mouse visceral endoderm cell type
END-2, form beating heart muscle colonies (e.g. cardiomyocytes)
(Mummery C, et al. 2002; Mummery C, et al. 2003). Beating heart
muscle cells derived from stem cells express cardiomyocyte markers
including alpha-myosin heavy chain, cardiac troponins and atrial
natriuretic factor as well as transcription factors typical of
cardiomyocytes, (e.g. GATA4 and MEF3) (Kehat I, et al. 2001; Xu C,
et al. 2002). These cells respond to pharmacological drugs and
exhibit cardiomyocyte action potentials most commonly observed in
human fetal left ventricular cardiomyocytes, which can be easily
distinguished from mouse cardiomyocytes (Mummery C, et al. 2003; He
J Q, et al. 2003). Atrial- and pacemaker-like cells can also be
formed in the differentiating stem cell cultures. These stem cell
derived cardiomyocytes integrate normally into transplanted rodent
and porcine heart muscle, form normal gap junction connections
between stem cell myocytes and the recipient mouse adult
cardiomyocytes (Xue T, et al. 2005; Kehat I, et al. 2004; Hassink R
J, et al. 2003).
[0190] Type II pneumocytes that express Surfactant Protein C (SPC),
a respiratory specific marker, can be generated by coculture of
stem cells with mouse embryonic foregut mesenchyme (Denham M, et
al. 2002). Stem cells can also be induced to form airway epithelial
tissue when differentiated as embryoid bodies or grown on type 1
collagen, and then the resulting Clara cells grown in an air-fluid
interface to form a pseudostratified surface epithelium (Coraux C,
et al. 2005).
[0191] Keratinocytes can be derived from stem cells by replating
embryoid bodies (Green H, et al. 2003). Cells expressing the
transcription factor p63 in the periphery of the secondary cultures
identify the keratinocyte progenitors that produce more mature cell
types in which cytokeratin 14 and basonuclin are detected. These
cells can form terminally differentiated stratifying epithelium but
are not the same as keratinocyte epithelium isolated from neonatal
or adult skin.
[0192] Embryoid bodies (EBs) can also be used to produce
hematopoietic progenitors using a cocktail of hematopoietic
cytokines and BMP-4 (Kaufman D S, 2001, Chadwick K, et al. 2003).
EBs are formed by withdrawal of leukemia inhibitory factor (LIF)
from the ES cell culture and manifest as cell clusters or spherical
multicellular aggregates. These progenitors are immunologically
similar to hematopoietic progenitors of the dorsal aorta. Growth
factors such as stem cell factor (SCF), interleukins-3 and -6
(IL-3, IL-6), granulocyte colony-stimulating factor (GCSF), Flt-3
ligand, as well as vascular endothelial growth factor-A (VEGF-A)
(Cerdan C, et al. 2004).
[0193] Endodermal cells can be detected in stem cells cultures
following exposure to Activin A (Kubo A, et al. 2004). Insulin
producing cells can be formed by differentiating neuroectodermal
cells using the method of Lumelsky et al. (Lumelsky N, et al.
2001). Similarly, Segev et al. (Segev H, et al. 2004) produced
islet-like clusters by culturing embryoid bodies in medium
containing insulin, transferrin, selenium and fibronectin.
Disaggregated cultures were allowed to form clusters in medium
containing FGF-2 and then exposed to nicotinamide with low glucose
in suspension culture. A high percentage of cell clusters expressed
insulin, glucagon and somatostatin similar to immature pancreatic
cells. Responsiveness to glucose and other antagonists suggested
that these cells were immature, fetal-like pancreatic beta-islet
cells.
[0194] Rambhatla et al. (2003) reported differentiation of stem
cells into cells that expressed markers of hepatocytes (albumin,
alpha-1-antitrypsin, cytokeratin 8 and 18) and accumulate glycogen.
Treating embryoid bodies with sodium butyrate or adherent stem cell
cultures with dimethyl sulfoxide followed by sodium butyrate
resulted in hepatic-like endodermal cells (Lavon N, et al. 2004).
Cellular morphology in differentiated adherent stem cell cultures
can be used to select for endodermal populations that express
markers of fetal liver (Stamp L A, et al. 2003).
[0195] Transgenic Animals
[0196] The present technology further relates to transgenic
animals, which contain a TR expression cassette stably integrated
into its genome. In some embodiments, the targeting nucleic acid
constructs comprising the TR cassette are introduced into a
pluripotent cell (e.g., ES cell, Robertson, E. J., In: Current
Communications in Molecular Biology, Capecchi, M. R. (ed.), Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), pp. 39-44).
Suitable ES cells may be derived or isolated from any species or
from any strain of a particular species. In some embodiments, the
species is a mouse, such as a 129 or C57BL/6 strain. In other
embodiments, the species is a rat. Although not required, the
pluripotent cells are typically derived from the same species as
the intended recipient. ES cells may also be obtained from
commercial sources (e.g., Genome Systems, Inc), from International
Depositories (e.g., the ATCC), from University facilities (e.g.,
the Siteman Cancer Center Murine Embryonic Stem Cell Core,
Washington University, St. Louis, Mo.) or, alternatively, may be
derived as described in Robertson, supra. Examples of
clonally-derived ES cells lines include 129/SVJ, RW-4 and C57BL/6
ES cells (Genome Systems, Inc.) or SCC10, B6/Blu, EDJ22, R1 and
B6/GFP ES cells (Washington University).
[0197] ES cells are cultured under suitable conditions, for
example, as described in Ausubel et al., CURRENT PROTOCOLS IN
MOLECULAR BIOLOGY. Preferably, ES cells are cultured on non-mitotic
"feeder layers" of stomal cells (such as STO cells (especially SNC4
STO cells) and/or primary embryonic fibroblast cells) as described
by E. J. Robertson, supra, pp 71-112. Culture media preferably
includes leukocyte inhibitory factor ("lif") (Gough, N. M. et al.,
Reprod. Fertil. Dev. 1:281-288 (1989); Yamamori, Y. et al., Science
246:1412-1416 (1989)), which prevents ES cell differentiation ex
vivo. Stomal cells transformed with and constitutively expressing
the lif growth factor can also be used as feeder cells.
[0198] The targeting constructs are introduced into the ES cells by
any method which will permit the introduced molecule to undergo
recombination at its regions of homology or by random integration,
for example, but not limited to, micro-injection, calcium phosphate
transformation, lipofection, viral vector or electroporation
(Toneguzzo, F. et al., Nucleic Acids Res. 16:5515-5532 (1988);
Quillet, A. et al., J. Immunol. 141:17-20 (1988); Machy, P. et al.,
Proc. Natl. Acad, Sci. (U.S.A.) 85:8027-8031 (1988)). In some
embodiments, microinjection is used for inserting the constructs
into ES cells with the nucleic acid construct being linearized
prior to introduction into ES cells, e.g., by digestion with
restriction nucleases.
[0199] In one aspect, the technology provides a method of
expressing the TR cassette in a host cell using a promoterless DNA
cassette according to the invention, allowing it to undergo site
directed recombination into the coding sequence of a target gene of
interest. Related aspects provide a method of expressing the TR
cassette in a host cell by engineering a functional expression
construct prior to introducing the construct into the host genome.
One such "genomic transgene" is engineered ex vivo by inserting the
TR cassette into a large genomic sequence (i.e. a cosmid or
artificial eukaryotic chromosome encompassing the ORF of a target
gene of interest), which replaces a target gene ORF and drives
expression of the TR cassette from the transcriptional regulatory
elements of the target gene. Large genomic transgenes then provide
the desired TR cassette expression pattern using the target gene
transcriptional regulatory system following random integration into
the host cell.
[0200] Screening and selection of those cells into which the
targeting construct has been integrated can be achieved using the
positive selection marker and/or the negative selection marker in
the construct. In various embodiments, the construct contains both
positive and negative selection markers. In one aspect, methods
which rely on expression of the selection marker are used, for
example, by adding the appropriate substrate to select only those
cells which express the product of the positive selection marker or
to eliminate those cells expressing the negative selection marker.
For example, where the positive selection marker encodes neomycin
resistance, G418 is added to the transformed ES cell culture media
at increasing dosages. Similarly, where the negative selection
marker is used, a suitable substrate (e.g., gancyclovir if the
negative selection marker encodes HSV-TK) is added to the cell
culture. Either before or after selection using the appropriate
substrate, the presence of the positive and/or negative selection
markers in a recipient cell can also be determined by others
methods, for example, hybridization, detection of radiolabelled
nucleotides, PCR and the like. In various embodiments, cells having
integrated targeting constructs are first selected by adding the
appropriate substrate for the positive and/or negative selection
markers. Cells that survive the selection process are then screened
by other methods, such as PCR or Southern blotting, for the
presence of integrated sequences.
[0201] After suitable ES cells containing the construct in the
proper location have been identified, the cells can be inserted
into an embryo, preferably a blastocyst. The blastocysts are
obtained by perfusing the uterus of pregnant females. Suitable
methods for accomplishing this are known to the skilled artisan,
and are set forth by, e.g., Bradley et al, (1992) Biotechnology,
10:534-539. As an example, naturally cycling or superovulated
female mice mated with males can be used to harvest embryos for the
implantation of ES cells. Embryos of the appropriate age are
recovered approximately 3.5 days after successful mating. Mated
females are sacrificed by CO.sub.2 asphyxiation or cervical
dislocation and embryos are flushed from excised uterine horns and
placed in Dulbecco's modified essential medium plus 10% calf serum
for injection with ES cells. Approximately 10 to 20 ES cells are
injected into blastocysts using a glass microneedle with an
internal diameter of approximately 20 .mu.m. Insertion into the
embryo may be accomplished in a variety of ways known to the
skilled artisan; however, a preferred method is by microinjection.
For microinjection, about 10-30 ES cells are collected into a
micropipet and injected into embryos that are at the proper stage
of development to permit integration of the foreign ES cell
containing the construct into the developing embryo. In one
embodiment, the blastocysts are obtained from, for example, the
FVB/N strain of mice and the ES cells are obtained from, for
example, the C57BL/6 strain of mice. With respect to recipient
female mice, randomly cycling adult females are paired with
vasectomized males. Mouse strains such as Swiss Webster, ICR or
others can be used for this purpose. In one embodiment, recipient
females are mated such that they will be at 2.5 to 3.5 days
post-mating when required for implantation with blastocysts
containing ES cells. At the time of embryo transfer, the recipient
females are anesthetized with an intraperitoneal injection of 0.015
ml of 2.5% avertin per gram of body weight. The ovaries are exposed
by making an incision in the body wall directly over the oviduct
and the ovary and uterus are externalized. A hole is made in the
uterine horn with a 25 gauge needle through which the blastocysts
are transferred. After the transfer, the ovary and uterus are
pushed back into the body and the incision is closed by two
sutures. This procedure is repeated on the opposite side if
additional transfers are to be made.
[0202] While any embryo of the right stage of development is
suitable for use, it is preferred that blastocysts are used. In
addition, preferred blastocysts are male and, furthermore,
preferably have genes encoding a coat color that is different from
that encoded by the genes ES cells. In this way, the offspring can
be screened easily for the presence of the knockout construct by
looking for mosaic coat color (indicating that the ES cell was
incorporated into the developing embryo). Thus, for example, if the
ES cell line carries the genes for black fur, the blastocyst
selected will carry genes for white or brown fur. Southern blots
and/or PCR may also be used to determine the presence of the
sequences of interest. Mosaic (chimeric) offspring are then bred to
each other to generate homozygous animals. Homozygotes and
heterozygotes may be identified by Southern blotting of equivalent
amounts of genomic DNA from mice that are the product of this
cross, as well as mice that are known heterozygotes and wild type
mice. Alternatively, Northern blots can be used to probe the mRNA
to identify the presence or absence of transcripts encoding the TR
cassette, the ORF nucleic sequence, or both. In addition, Western
blots can be used to assess the level of expression of the ORF
coding polypeptide, if a suitable antibody against such polypeptide
exists. By way of example and not of limitation, if the polypeptide
is GFP, an antibody against GFP can be used. Finally, in situ
analysis (such as fixing the cells and labeling with antibody)
and/or FACS (fluorescence activated cell sorting) analysis of
various cells from the offspring can be conducted using suitable
antibodies to look for the presence or absence of the targeting
construct.
[0203] In another embodiment, transgenic animals can be made, which
express the TR cassette and the ORF nucleic sequence only in a
particular organ, tissue, cell or cell condition. The protocol for
making such animals is the same as described above, except that a
targeting construct comprises a promoter which is expressed only in
the desired cells, tissue or organ, as well as cellular condition
such as heavy metal application, thus limiting the expression of
the TR cassette and the ORF coding polypeptide thereto. For
example, if it is desirable that the targeting sequences be
expressed in the liver and small intestine, the fatty acid binding
protein (FABP) promoter can be used. In another example,
transthyretin (TTR) (Ye et al., Mol Cell Biol., 1999 December,
19(12), 8570-80) promoter is also well described and widely used
promoter to achieve liver-specific expression of transgenes. Other
promoters for achieving tissue specific expression of genes are
well known in the art and readily available.
[0204] Methods of Detecting a Reporter Protein or Reporter Nucleic
Acid
[0205] Included in this technology are methods for detecting the
reporter protein expressed in a cell transformed with a TR
expression cassette wherein the first ORF sequence is a reporter
gene. Briefly, the method comprises exposing the cell expressing
such TR cassette to conditions suitable for translation of the
reporter polypeptide, and detecting the presence of the reporter
polypeptide. Any cell such as a primary cell, cell line, a cell
that has been transduced in a subject, or a donor cell implanted in
a subject can be used.
[0206] A variety of ex vivo protein detection methods are known in
the art. For example, the polypeptides may be detected using
methods known in the art that are specific for the polypeptides.
These detection methods may include use of specific antibodies,
formation of an enzyme product or disappearance of an enzyme
substrate. Thus, the detection of reporter proteins can be achieved
using any of the standard methods known in the art, such as
fluorescence microscopy, immunohistochemistry, or ELISA assays. For
example, fluorescence microscopy can be used to detect EYFP and
mRFP1. Similarly, antibodies against Firefly Luciferase or
.beta.-Galactosidase can be used to detect presence thereof
following immunofluorescence staining.
[0207] A number of noninvasive methods are available in the art to
detect protein synthesis in vivo. By way of example, Herpes simplex
virus type 1 thymidine kinase (HSV1-TK) ORF placed downstream of
the TR element can be used to detect in vivo cell death using
metabolic tracers. Unlike mammalian thymidine kinase, this enzyme
can efficiently phosphorylate nucleoside analogues (e.g.,
ganciclovir, penciclovir), as well as various radioactive
derivatives such as
(9-(4-[18F]-fluoro-3-hydroxymethylbutyl)guanine; [18F]FHBG), which
is then retained and accumulates within expressing cells. Thus if a
cell is undergoing stress/death, the HSV1-TK will be translated
from the TR cassette, resulting in accummulation of a radioactive
tracer. The radioactivity can be detected using positron emmission
tomography (PET), which allows for monitoring of the detailed
location, magnitude, and persistence of reporter gene expression.
Methods are also provided for mutant HSV1-TK enzymes (e.g., TKsr39)
that exhibit enhanced enzymatic activity and/or binding constants
that improve the sensitivity of PET imaging by enhancing the
cellular accumulation of a radioactive tracer.
[0208] In a specific embodiment, transgenic cells and animals
expressing TR-regulated light generating proteins, for example the
firefly luciferase enzyme, are described. As demonstrated in
specific examples, TR-regulated luciferase expression serves as a
bioluminescent reporter of cell stress/death. In this embodiment,
luciferase is particularly useful as a reporter for low-light
imaging of bioluminescence in living cells and organisms. Although
resolution is less than with MRI or PET, bioluminescence imaging,
typically with a sensitive charged coupled device (CCD) camera,
allows rapid, high throughput (simple data collection from multiple
animals simultaneously), progressive (repeated analysis of the same
animal), noninvasive, nondestructive data collection in vivo. A
variety of detection devices, image processors and image analysis
systems are available in the art.
[0209] The present technology also provides methods using
transgenic cells and animals containing a TR cassette operably
linked to stress- and response-specific promoters to restrict TR
mRNA synthesis to selective cellular stress and response
conditions. In this method, the TR nucleic acid (mRNA) and
TR-regulated protein translation provide independent measures of
cell stress and death. It is anticipated that this method will
allow the detection of transcriptional activity regulated by cell
stress which does not result in the stress/death-specific
translational changes detected by the TR cassette. Various methods
are available in the art to isolate, purify, and quantitate TR mRNA
levels, such as, e.g., quantitative polymerase chain reaction
(Q-PCR), real time PCR (RT-PCR), reverse transcriptase PCR
(RT-PCR), in situ hybridization or nucleic acid hybridization in
solution or solid support.
[0210] Method of Detecting Cell Stress and Apoptosis
[0211] In one embodiment, methods of detecting a reporter protein
as described above are particularly useful in detecting cell stress
and death. Cellular stress and death detection can be done either
ex vivo or vivo. Thus, the present methods find use in studying
normal biological processes, response of cells to injury or
medication, exposure to a compound or condition thought to induce
cytotoxicity, and the like. For example, as part of studying
biological processes, a skilled artisan may want to determine if,
and to what extent apoptosis plays a role in, e.g., cell
differentiation. It may also be useful to determine the apoptotic
potential of cells after a physical injury such as, e.g., a spinal
cord trauma or after exposure to a cancer treatment drug. In
addition, apoptosis detection is usable in evaluating cytotoxicity,
e.g., of novel drugs.
[0212] In embodiments where detection is done ex vivo, one skilled
in the art can use a primary cell, a cell line, or cells isolated
from an animal, as discussed in above sections. Briefly, the cell
that is to be evaluated for stress or death is initially
transformed with a TR expression cassette, wherein the first ORF
sequence is a reporter gene, such as EYFP. The methods for
transforming cells are discussed above. The cells are cultured for
an appropriate amount of time under toxic conditions, following
which the reporter protein expression is detected using any of the
above-mentioned methods. By way of example, fluorescence microscopy
can be used to evaluate the number of cells that are translating a
TR-EYFP cassette and exhibiting a toxic phenotype. Additionally,
antibodies against a reporter protein, such as EYFP, can be used to
determine the level of reporter protein expression using Western
blot analysis.
[0213] For in vivo use, a TR cassette can be inserted into an ES
cell line by any of the currently used transformation methods, such
as, e.g., liposomes, electroporation, microinjection, etc., and a
transgenic animal produced as described. For tissue specific
expression of the TR cassette, a tissue specific promoter can be
used. In some embodiments, a stress-specific promoter may be
operably linked to the TR cassette to further restrict TR
expression to stressed cells. In one preferred embodiment, a
mammalian shuttle vector is used to deliver the TR cassette to ES
cells. The transgenic animal would be exposed for an appropriate
amount of time to a putative toxic drug or condition, following
which reporter gene transcription and translation can be detected
using the above-mentioned methods. Moreover, various standard
methods for assaying gene expression and protein synthesis using
postmortem animal tissue sections are well known in the art. An
example would be the use of antibodies against a reporter protein,
such as EYFP, to evaluate cell- and tissue-specific protein
synthesis, as well as protein expression levels in stressed and
dying cells in tissue sections. In other embodiments, the TR
cassette provides methods for nondestructive in vivo imaging of
cell stress and death. By way of example, micro-PET scanning can be
used to evaluate the location and number of cells that are
translating a TR-TKsr39 cassette.
[0214] Embryotoxicity and Cytotoxicity Assays
[0215] The technology provides in vitro test procedures for the
detection of chemically-induced embryotoxicity and teratogenic
effects based on differentiated, transgenic pluripotent embryonic
stem cell (ESC) lines from the mouse and rat, as well as transgenic
embryonic germ cells (EGCs) obtained from primordial germ cells of
rodent embryos that have been transformed with the TR expression
cassette. Previous in vitro efforts employing ESC lines to detect
embryotoxic and mutagenic compounds, termed the Embryonic Stem Cell
Test or EST, are known in the art (Laschinski et al., Reproductive
Toxicol. 5, 57-65 (1991); Spielmann et al., Ex vivo Toxicol. 10:
119-27 (1997)). In summary, assaying disturbances of in vitro ES
differentiation can be correlated with embryonic germ-layer
aberrations. Since abnormal mammalian development can also lead to
enhanced cell death and a resulting preimplantative embryo death,
developmental defects, maldevelopment or malformations, early test
procedures measured cytotoxic effects using the MTT test. In this
embodiment, the EST procedure will be modified to examine
cytotoxicity resulting in cell stress/death using TR-specific
translation, which will serve as a measure of embryocidal
properties of teratogenic/embryotoxic substances.
[0216] In some embodiments, the toxicity of a substance to the
transgenic ES cell can be tested. The selection of appropriate ES
cells for a particular substance and selection of factors such as
substance concentration, duration of incubation of the substance
with the ES cells and methods of detection can be easily performed
by a skilled artisan. In general, such assays are performed ex
vivo, utilizing transgenic ES or EG cell cultures transformed with
the TR cassette. Briefly, a substance being tested is contacted
with a population of ESCs for a period of time and assayed for
TR-mediated translational activity, as described above. For
example, a substance can be incubated with several different ES
cell cultures over several different time periods (e.g., 12 hours,
24 hours, 48 hours, 72 hours), wherein the substance is applied to
each culture at 4-20 different concentrations. The TR-based
toxicity assay can then be determined using standard methods for
reporter protein detection, as discussed above. An agent's toxic
potential can be expressed in several ways, for example in terms of
the time needed to achieve cell death or the amount of TR-regulated
reporter protein detectable at a given time point. In one preferred
embodiment, bioluminiscence is used to detect a TR-regulated
reporter protein.
[0217] By way of example and not of limitation, if the substance
that is tested is toxic to the tested cells, the cell will rapidly
undergo stress and death resulting in translation of the TR
cassette and the TR-regulated reporter protein, such as EYFP or
firefly luciferase. The presence and/or amount of the reporter
protein can then be determined by using, for example, fluorescence
or bioluminiscence detection. In another embodiment, a substance
can be administered to animals which have previously incorporated a
TR cassette into at least some of their cells. For example, a mouse
can be exposed to a potentially toxic substance following the
injection of a virus encoding the TR cassette into an appropriate
testing tissue or tissue site, wherein the first ORF sequence is a
reporter gene. This will result in a focal infection and
transduction of susceptible cells at the injection site. Methods
are provided in this technology for measuring the toxicity of the
substance to the animal by detecting the reporter protein in cells
of said animal using any of in vivo or ex vivo methods already
discussed.
[0218] In still another embodiment, the transgenic animals, or
animal cells derived from transgenic animals, are used to screen
compounds or substances for cytotoxicity using standard methodology
and as described above. As an example, in such screening assays,
the substance is administered to the animals, or introduced into
the culture media of cells derived from these animals, over a
period of time and in various dosages, following which the animals
or animal cells are examined for reporter protein expression, as
indicative of the cytotoxicity.
[0219] In addition to the cytotoxicity assays above, an assay to
evaluate the "temporal" transition to a toxic phenotype in the
presence of the toxic cellular stimulus can also be envisioned. In
such an assay, one can compare stress-induced RNA synthesis from
the TR cassette transcribed from inducible or stress-regulated
promoters with death induced TR-dependent protein synthesis.
Another example of a transitional assay employs cell-type specific
or organ-specific transcriptional regulatory elements to allow the
detection of cell type-specific or organ-specific toxins using any
of the previously described detection methods.
[0220] Method of Identifying Additional TR Elements
[0221] The present technology also provides methods for identifying
translationally regulated (TR) genes that are selectively
translated during cell stress/death by stimulating translation of
an unknown target mRNA with a toxic agent that induces stress or
death in cells containing the target mRNA. After the treatment with
the toxic agent, such as MG132 or calcium ionophore A23187,
cellular mRNA is harvested, purified and separated into pools of
actively translated and untranslated mRNA. While not being bound to
a particular theory, it is believed that an mRNA encoding a protein
required for a quick response to a toxic environment is rapidly
translated following exposure to the toxic agent and that the
encoded protein quickly appears. Methods of the present technology
identify mRNAs actively translated during cell death by preparing
mRNA pools containing multiple ribosomes ("polysomal") and
ribosome-free, untranslated mRNAs. Following the induction of
cell/tissue death using any physiological, chemical, or
pathological stress, the mRNAs remaining engaged in translation
(putative TR genes translated via cap-independent translation) can
be separated from those which are untranslated using procedures
such as fractionation on a sucrose density gradient, high
performance gel filtration chromatography, or polyacrylamide gel
matrix separation (Ogishima et al., 1984, Menaker et al., 1974,
Hirama et al., 1986, Mechler, 1987, and Bharucha and Murthy, 1992),
since mRNAs that are being translated contain bound ribosomes and,
therefore, migrate differently than ribosome-free untranslated
mRNAs.
[0222] The mRNA-ribosome fractionation can be enhanced by treating
target cell/tissue with drugs that specifically inhibit or modulate
transcription or translation and prevent mRNA-ribosome
dissociation. Examples of such drugs are actinomycin D and
cyclohexamide, respectively. A further refinement of the polysomal
fraction can be made to discriminate between total polyribosomes or
membrane bound ribosomes by methods known in the art (Mechler,
1987).
[0223] Following polysomal isolation and division into translated
and untranslated pools, the mRNA is isolated utilizing techniques
which are well known to those skilled in the art and are described,
for example, in "Molecular Cloning; A Laboratory Manual" (Cold
Springs Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Other methods for the isolation and extraction of RNA from
cells/tissue can be used and will be known to those of ordinary
skill in the art (Mach et al., 1986, Jefferies et al., 1994). mRNA
is further purified to remove any contaminating ribosomal RNA using
poly A selection, which is well known in the art.
[0224] The relative abundance of the many mRNA species found in
each pool can be compared using any of the differential analysis
technique common in the art including serial analysis of gene
expression ("SAGE"), differential display, oligonucleotide arrays,
representational difference analysis (RDA), cDNA microarrays and
suppressive subtraction hybridization (SSH). Labeled mRNA (in a
cDNA or PCR product form) from translated and untranslated
fractions can be used as probes, to identify cDNA, genomic clones,
or mRNA species that are fixed onto a solid matrix like microarrays
(GEM) or membranes of any kind where clones can be either attached
by electrophoresis, direct loading or capillary action onto the
membrane. The label can be radioactive, fluorescent, or
incorporating a modified base such as digoxigenin or biotin. As a
control, mRNA levels in the translated fraction are compared to the
total unfractionated material to discriminate between differentials
in expression levels produced by transcription modulation from
those that result from modulation of translation per se. A strong
association of a particular mRNA with the translated mRNA pool can
indicate that said gene is selectively expressed during cell
stress/death.
[0225] Following the differential expression analysis, the genes
which have been identified as putatively regulated by
cap-independent translation can be PCR amplified from any of the
available genomic or cDNA gene collections and inserted into the
described TR cassette replacing the existing TR sequence and
producing an "investigational" TR cassette. PCR primers will be
designed to incorporate mutations analogous to those inserted into
the previously described TR element to prevent translation from any
cap-dependent translation initiation site. Any investigational TR
element will be subjected to comparative analysis to determine
whether these clones exhibit selective cell stress and/or
death-specific translation.
[0226] In various embodiments, to exhibit the TR defined activity,
an investigational TR clone displays the following translational
parameters:
[0227] a) Minimal translational activity in normal mitotic cells,
displaying no expression level greater than the normal level of
cell death in these cultures determined by any of the standard
assays present in the art,
[0228] b) A rapid increase in translational activity in cells
treated with an acute dose of a toxic agent, such as MG132 or the
calcium ionophore A23187, which is initiated prior to 6 hours but
no more than 9 hours after treatment,
[0229] c) Translation activity observed in more than 95% of any
cell line transformed with the investigational TR cassette and
treated with an acute toxic agent,
[0230] d) Translational levels that rise to more than 50% of the
expression levels of the ORF transcribed and translated without an
operably linked TR sequence following an acute toxic exposure, and
preferably more than 60%, 70%, 80%, 85%, 90%, 95%, or 100%,
[0231] e) Stess or death-specific translation initiation that
occurs proximal to the initiation site of the operably linked
ORF,
[0232] f) No transcriptional or translational activity in the
absence of any operably linked transcriptional effector
sequence/promoter element in normal or dying cells,
[0233] g) No evidence of TR-specific mRNA splicing in expressing
cells which removes any TR sequence from the investigational TR
cassette in normal or dying cells,
[0234] h) Stress or death-specific translation is detected in
multiple cell types composed of not less than 3 cell lines or
tissues representing 3 distinct tissue types.
[0235] In a preferred embodiment, investigational TR elements will
be derived from mammalian cells, preferably human cells. In another
embodiment, investigational TR elements will be derived from rodent
cells, preferably mouse cells.
[0236] Screening Assays for Substances Affecting Cell Stress or
Toxicity
[0237] Use of the TR expression cassette of this technology in
toxicity testing involves combining a cell line expressing the TR
cassette with the agent to be screened (typically by adding it to
the culture medium). The present technology also provides
high-throughput screening assays of substances or compounds that
induce, ameliorate or prevent cellular stress or toxicity.
[0238] In one embodiment, a compound's cytotoxic ability is
evaluated using any of the cytotoxicity assays described above or
by detecting the reporter protein, which is cotranslated with the
TR element. Briefly, the compound is tested by incubating it with
cells that have previously been transformed with a TR expression
cassette, wherein the first ORF sequence encodes a reporter
protein. Next, the cells are screened for expression of the
reporter protein, wherein the presence of the reporter protein
indicates the cytotoxic potential of the compound. Furthermore, the
higher the concentration of the reporter protein in the cells
and/or the larger the percentage of cells which are expressing the
reporter protein correlate with the increase with cytotoxicity of
the compound.
[0239] In another embodiment, in order to identify a substance that
can alleviate or prevent cytotoxicity, the putative therapeutic
substance is administered to any of the mammalian cells or
transgenic animals described above after exposing the cells or
animals to conditions that promote or induce cellular toxicity. For
example, the cells can be initially exposed to radiation or treated
with drugs which induce cytotoxicity, such as, e.g., methotrexate.
Following the incubation of the putative therapeutic substance with
the cells or transgenic animals, the differential expression of the
reporter protein in cells or animals which were treated compared to
the cells or animals which were untreated indicates the ability of
the substance to alleviate or prevent cytotoxicity. For example, if
the reporter gene is GFP, the reduction of the GFP expression in
treated cells or animals compared to the untreated ones indicates
the ability of a substance to reduce cytotoxicity. It should be
obvious to one of ordinary skill in the art that the greater the
reduction in the reporter protein expression in the treated cells
or animals, the greater the ability of the substance that was used
to treat such cells or animals to prevent or ameliorate cell
death.
[0240] This method may find particular use in identifying
substances with the ability to counter the cytotoxicity of many
drugs that are used in treatment of, e.g., cancers. Such drugs are
generally known as "chemotherapeutic" agents and include DNA
damaging agents such as methotrexate, doxorubicin, daunorubicin,
mitomycin, actinomycin D, bleomycin, plicomycin, taxol,
vincristine, vinblastine, cisplatin, carmustine, melphalan,
cyclophosphamide, chlorambucil, ifosfamide, nitosurea, tamoxifen,
raloxifene, estrogen receptor binding agents, gemcitabien,
navelbine, farnesyl-protein transferase inhibitors, transplatinum,
5-fluorouracil, temazolomide and analogs and derivatives of the
same. Each chemotherapeutic agent is generally associated with
several side effects which include stress and death in the skin,
gastrointestinal tract, bone marrow, liver, and kidney.
[0241] Other drugs that are also associated with toxicity are
exemplified by the generic categories of, sedatives,
anti-inflammatory agents, antibiotics, analgesics, anesthetics,
antiviral drugs, etc. For example, antibiotics exhibit toxic
phenotypes ranging from minor gastrointestinal symptoms to more
severe hepatotoxicity, nephrotoxicity, anemia, myalgias, arthalgias
and cardiotoxicity.
[0242] In addition, substances that reduce the cytotoxicity of
commonly used compounds such as food additives, social drugs, and
alternative medical therapies can be identified using the above
method. One example of a food additive, which is also used in the
production of drugs, cosmetics and certain medical devices (i.e.,
contact lenses) is a color additive. FD&C Yellow No. 5, a
compound used to color beverages, dessert powders, candy, ice
cream, custards and other foods can cause skin trauma (hives) and
general toxicity in a significant fraction of exposed individuals.
Other food additives with potential cytotoxic effects include the
cholesterol substitute olestera, sulfites, and monosodium glutamate
(MSG). In the case of MSG, a small percentage of the population
develops MSG symptom complex, a condition characterized by cell
stress/toxicity with neurological symptoms including a burning
sensation in the neck, forearm and chest numbness, tingling that
radiates to the arms and back, warmth/weakness in the upper torso
and head, chest pain, nausea, difficulty breathing, drowsiness and
general lethargy. Substances that counter the effects of social
drugs such as alcoholic beverages, and caffeinated liquids (e.g.
coffee, colas, teas and the like) can also be identified.
Alternative medical therapies include, e.g., herbs such as ginseng,
ginkgo biloba, St. John's wort, ephedra and kava.
[0243] Any substances or compounds may be used to test for their
ability to induce, ameliorate or prevent cell death. For example,
small molecule libraries, e.g., obtained from a commercial source
can be used. Screening of such libraries, including combinatorial
screening libraries (e.g. peptide, chemical or oligonucleotide
libraries), is a rapid and efficient way to examine a large number
of substances. Combinatorial approaches also lend themselves to the
rapid evolution of potential drugs by the creation of second, third
or fourth generation substances modeled on active, but otherwise
undesirable substances.
[0244] The substances or compounds to be screened can also include
fragments or parts of naturally occurring substances or may be
found as active combinations of known substances, which may be
otherwise inactive. Compounds isolated from natural sources, such
as animals, bacteria, fungi, plant sources, including leaves and
bark, and marine samples in addition to the ones derived or
synthesized from chemical compositions or man-made substances can
also be assayed. Non-limiting examples of such compounds include
proteins, peptides, amino acids, small molecules, nucleic acids,
lipids, nutritional supplements such as vitamins or minerals, drugs
or any other substance that may be designed through rational drug
design starting from known inhibitors or stimulators of toxicity
pathways.
[0245] Method of Inducing Cell Death
[0246] The present technology provides agents for inducing cell
death. Such agents for inducing cell death exert a desired
pharmacological effect on various diseases by inducing cell death
resulting from the activation of cell death or inhibition of
anti-cell death cellular pathways. Such agents include medicaments
which comprise a substance having the aforementioned action as an
active ingredient.
[0247] One application of this technology is in the production of
therapies that improve the efficacy of patient treatment and
simultaneously reduce any deleterious side effects of therapeutic
applications. In many cases, combined therapies are used to address
various clinical problems including those associated with cellular
resistance to therapy for hyperproliferative diseases. In the
context of the present technology, it is contemplated that
supplemental therapy based on TR regulated polypeptides could be
used in conjunction with curative surgery, chemotherapy,
radiotherapy, gene therapy, hormone therapy or immunotherapy
treatments, as well as alternative therapies that employ apoptosis
or cell cycle regulating agents to treat these disorders. A
hyperproliferative disease includes diseases and conditions that
are associated with any sort of abnormal cell growth or abnormal
cell growth regulation.
[0248] In methods of the present technology, the patient is a
mammal, preferably a human. A variety of hyperproliferative and
degenerative diseases can be treated according to the methods of
the present technology. Nonlimiting examples of the
hyperproliferative diseases contemplated for treatment by the
present technology are cancer, psoriasis, rheumatoid arthritis,
inflammatory bowel disease, osteoarthritis, and pre-neoplastic
lesions of the mouth, prostate, breast, skin, etc.
[0249] One preferred embodiment is a method for negatively
affecting cancer in a subject by killing cancer cells, inducing
apoptosis in cancer cells, reducing the incidence or number of
metastases, reducing tumor size, inhibiting tumor growth, reducing
the blood supply to a tumor or cancer cell, promoting an immune
response against cancer cells or a tumor, preventing or inhibiting
the progression of cancer, or increasing the lifespan of a subject
with cancer by combining a TR-based therapeutic with other
treatments that are effective for killing or inhibiting cellular
proliferation. For example, if tumor cells from a patient are not
responding to treatment and not undergoing cell death as determined
by the present methods, a TR cassette transcribed by a
tumor-specific promoter encoding a toxin gene, prodrug activating
gene, tumor suppressor gene or immunotherapeutic could be
administered to stimulate apoptosis.
[0250] By way of example, a TR cassette encoding the thymidine
kinase gene (e.g. HSV1 TKsr39 enzyme derivative) transcribed by a
tumor-specific promoter could be delivered, for example using a
recombinant viral vector, to a specific tumor type where that
tumor-specific promoter is preferentially active. Transcription of
the TR cassette in these cells would allow the selective
translation of this enzyme in tumor cells stressed by modest
chemotherapy or radiotherapy treatments. A particular advantage is
observed by treating HSV-TKsr39 expressing cells with specific
protoxic nucleoside analogues, such as acyclovir and ganciclovir,
since this enzyme produces monophosphate intermediates that are
then phosphorylated by cellular kinases to provide potent DNA
synthesis inhibitors. Cells expressing HSV-TK are rendered
extremely sensitive to the toxic effect of ganciclovir, whereas the
mammalian TK enzyme is relatively insensitive, resulting in a large
therapeutic index. Tumor modeling experiments using gene delivery
of HSV-TK have demonstrated complete regression of established
tumors and long-term animal survival, even though only a portion of
the tumor cells were actually transduced with the HSV-TK gene. This
so-called "bystander" cytocidal effect provides an important
therapeutic advantage, as it avoids the need to infect/transduce
100% of the tumor cells with the HSV-TK gene. It is anticipated
that a skilled artisan could design similar methods of treatment
based on a particular use in which cell death is preferred.
[0251] In the context of the present technology, it is contemplated
that TR regulated proteins could be used simultaneously with other
cell death therapies. Alternatively, TR supplemental therapy may
precede or follow another treatment by intervals ranging from
minutes to weeks. In situations, where the first treatment and the
TR therapeutic are applied separately, one would ensure that a
significant period of time did not expire between the times of each
delivery, such that the first agent and the TR-regulated
therapeutic would still exert an advantageous combinatorial
effect.
[0252] Another embodiment envisions methods for combining TR-based
imaging and cell death systems with standard cell death therapies.
Advantages are provided for imaging cell death as a side effect of
medical treatment, and if so, administering monocistronic and
bicistronic TR expression cassettes encoding reporter and pro-death
ORFs allows for temporal imaging based upon the vector TR cassette.
In one example, two TR expression vectors could be delivered to
tumor cells such that one vector expresses a TR-regulated reporter
ORF and the second vector a TR-regulated cell death ORF. In this
case, cap-independent translation following cell stress by a
standard therapeutic, such as a chemotherapy drug, would direct
TR-dependent cell imaging and supplemental therapy by the TR
pro-death activity to targeted tumor cells. Alternatively, a single
bicistronic TR cassette could be transduced into tumor cells
wherein the upstream ORF encodes a reporter ORF and the
TR-regulated ORF is a cell death regulator protein. In this
example, the upstream ORF would allow cap-dependent translation and
visualization of cell transduction prior to the death, so that the
efficiency of transduction could be visualized prior to
chemotherapy application, which would then be lost when
cap-independent translation was induced by the chemotherapy
application. Therefore, efficacious imaging-treatment paradigms can
be designed by varying TR cassette composition and the timing of
therapeutic applications which are anticipated to allow the
minimization of side effects and off-site activities.
[0253] Furthermore, a bicistronic TR expression cassette can be
used, such that both ORF sequences encode proteins which are
cytotoxic. For example, the first and second ORF sequences can
encode any combination of pro-apoptotic, prodrug activating, single
chain antibodies and toxin genes, as exemplified by the following
non-limiting combinations: p53 and diphtheria toxin, p53 and
sr39TK, sr39TK and ricin toxin, diphtheria toxin and ricin toxin,
and p53 and BRCA-1.
[0254] The TR cassette can be applied to cells as naked plasmid
DNA, or can be transduced into cells using viruses as described
above. In one embodiment, it may be useful for the TR cassette to
be expressed selectively in specific cell or tissue types. For such
purposes, tissue-, cell- and condition-specific viruses which
contain a TR cassette selectively transcribed by a regulated
promoter can be used. For example, if a particular cancer cell type
is to be targeted, a tumor specific promoter can be used as
described above.
[0255] Method of Preventing Cell Death
[0256] One embodiment of the present technology is to provide a
therapeutic that inhibits cell death. The present technology
provides therapeutics for inhibiting cell death that can exert a
desired pharmacological effect on various diseases by inhibiting
cell death resulting from the activation of cell survival or
inhibition of cell death cellular pathways. Such medicament
comprise a substance having the aforementioned action as an active
ingredient.
[0257] One application of this technology is in the production of
therapies that improve the efficacy of patient treatment and
simultaneously reduce deleterious side effects of treatment
applications. In general, anti-cell death therapies are used to
address clinical problems associated with chronic and acute cell
death. In the context of the present technology, it is contemplated
that TR regulated polypeptides could be used in conjunction with
other treatments, such as curative surgery, chemotherapy,
radiotherapy, gene therapy, hormone therapy or immunotherapy
treatments, as well as alternative physical therapies, such as
induced hypothermia, to prevent cell death. Chronic degenerative
diseases would be exemplified by any disease or condition that
produces progressive cell death over a significant fraction of the
lifespan of the patient. An acute degenerative disease would be
exemplified by any intense trauma or condition that produces
immediate cell death.
[0258] In methods of the present technology, preferably the patient
is a mammal, more specifically a human. A variety of degenerative
diseases can be treated according to the methods of the present
technology. Nonlimiting examples of chronic degenerative diseases
would include neurological diseases such as Alzheimer's disease,
Parkinson's disease, as well as diseases of other tissues such as
liver necrosis. Similarly, acute degenerative diseases would be
exemplified by traumatic injuries such as acute spinal cord injury,
acute nerve damage, traumatic brain injury, fractures, stroke,
congestive heart failure and severe burns.
[0259] One preferred embodiment is a method of preventing cell
death in a subject by blocking cell death as a result of in situ
TR-regulated gene expression or in combination with other
treatments that are effective for inhibiting cell death. By way of
example, TR-regulated expression of an antiapoptotic gene can
reduce cell death in apoptotic cells. As used herein, the term
"apotosis" refers to the physiological process known as programmed
cell death. Apoptosis is unlike other forms of cell death that
occur, for example, as a result of ischemia or necrosis, because
apoptosis is an active, ATP-dependent form of cell death that
typically requires new RNA and protein synthesis. A TR cassette
encoding an antiapoptotic gene such as BCL2 (B-cell CLL/Lymphoma
2), BCL2L1 (Bcl-xl; BCL2-like 1), BCL2A1 (Bfl-1/A1; BCL2-related
protein A1), BAG1 (BCL2-associated athanogene), TRAF1 (Tumor
necrosis factor receptor-associated factor 1), BIRC3 (C-IAP2;
Baculoviral inhibitor of apoptosis protein repeat-containing 3),
BIRC5 (survivin; Baculoviral inhibitor of apoptosis protein
repeat-containing 5), BAK1 (BCL2-antagonist/killer 1), or API5
(Apoptosis inhibitor 5) transcribed by a cell-specific promoter
could be delivered, for example, using a recombinant viral vector,
to a spinal cord neuron where that promoter is preferentially
active. Transcription of the TR cassette in these cells would allow
the selective translation of an anti-apoptosis protein in spinal
cord neurons undergoing apoptosis as a result of traumatic spinal
cord injury. It is anticipated that a skilled artisan could design
similar methods of treatment based on a particular use in which the
prevention of cell death is preferred. In one preferred embodiment,
the antiapoptotic gene is BCL2. In another preferred embodiment,
the preferred antiapoptotic gene is TRAF 1.
[0260] In context of the present technology, it is contemplated
that TR regulated proteins could be used simultaneously with other
cell death therapies. TR supplemental therapy may precede or follow
another treatment by intervals ranging from minutes to weeks. In
situations, where the first treatment and the TR therapeutic are
applied separately, one would ensure that a significant period of
time did not expire between the times of each delivery, such that
the first agent and the TR-regulated therapeutic would still exert
an advantageous combinatorial effect.
[0261] Another embodiment envisions methods that combine TR-based
imaging and anti-apoptosis/cell death with standard death
preventative therapies. Advantages are provided for imaging cell
death as a side effect of medical treatment, and if so,
administering monocistronic and bicistronic TR expression cassettes
encoding reporter and anti-death ORFs allows for temporal imaging
based upon the vector TR cassette. For example, healthy cells often
undergo apoptosis in a cancer patient as a side effect of
chemotherapy treatment, often targeting a specific sensitive organ.
In one example, two TR expression vectors could be delivered to the
sensitive organ, for example by injection of viral vectors, such
that one vector expresses a TR-regulated reporter ORF and the
second vector a TR-regulated anti-apoptosis ORF. In this case,
cap-independent translation following cell stress by a
chemotherapeutic would direct TR-dependent cell imaging and
supplemental therapy by the TR anti-apoptotic activity in the
targeted organ. Alternatively, a single bicistronic TR cassette
could be transduced into the sensitive organ wherein the upstream
ORF encodes a reporter ORF and the TR-regulated ORF is an
anti-apoptotic protein. In this example, the upstream ORF would
allow cap-dependent translation and visualization of cell
transduction prior to stress, so that the efficiency of
transduction could be visualized prior to chemotherapy application,
which would be lost when cap-independent translation was induced by
chemotherapy-regulated death. Therefore, efficacious
imaging-treatment paradigms can be designed by varying TR cassette
composition and the timing of therapeutic applications which are
anticipated to minimize side effects and off-site activities.
[0262] To utilize the present method, a TR expression cassette can
be applied to potentially dying cells, i.e., cells undergoing
stress or cell death, either as naked plasmid DNA, or can be
transduced into cells using viruses as described. The routes of
administration vary depending on the type if cellular injury, where
the cell is located in the body, etc. One skilled in the art can
readily determine a preferred route of administration for a
particular situation. For example, for a muscle injury, liposomes
or vectors containing the TR cassette can be used. In one
embodiment, it may be useful for the TR cassette to be selectively
transcribed in specific cells or tissue types. For such purposes,
tissue-, cell- and condition-specific cassettes have been
constructed that are selectively transcribed by a regulated
promoter. For example, if a particular cell type is sensitive to
treatment-induced cell death/apoptosis, a cell-specific promoter
can be used as described above. In another example, if a particular
cell condition induces cell death then a TR cassette regulated by a
responsive promoter might be applied as a therapeutic.
[0263] It is also contemplated that the present method can be used
in growing large scale cell cultures. Generally, when the cells are
expanded, at least some of them will undergo cell stress and/or
apoptosis as a result of mixing, etc. Thus, by transforming the
cells with the TR cassette containing the antiapoptotic gene, fewer
cells will undergo apoptosis. For example, the present method can
be used in bioreactors such as Wave Bioreactor.RTM. in combination
with Cellbag.RTM. disposable bags for cell culture. See, e.g., U.S.
Pat. No. 6,544,788.
[0264] Kits
[0265] The present technology provides methods for providing
services to entities desiring to test the effect of compounds or
agents on cell stress/death. Methods may comprise providing cells,
combinations and reagents of this technology to entities for use in
pharmacological screening. When applied in conjunction with in vivo
therapeutic treatments, the present technology provides diagnostic
kits and methods for determining, imaging and quantifying the
toxicity of medical treatments in terms of the cell stress/death
caused within the cell or tissue.
[0266] Also provided herein are kits comprising one or more of the
components described herein in any number of separate containers,
packets, tubes, vials and the like, or the components may be
combined in any combination in such containers. The kit contains
the TR expression cassette as described above or a mammalian cell
transformed with the TR expression cassette. According to one
embodiment, the plurality of cells in this kit are derived from a
single cell line, wherein each cell contains a specific TR cassette
which undergoes cap-independent translation upon the induction of
cell stress/death. Optionally, a kit of this technology may also
contain one or more reagents useful for detecting transcription of
the TR cassette (such as cassette-specific oligonucleotides useful
for PCR amplification), translation from the TR cassette (such as
an antibody or enzyme substrate), one or more control compounds
known to induce or inhibit promoter activity (and thereby
expression of the TR cassette), one or more control compounds that
produce a defined toxic response (and thereby promotes
stress/death-specific translation of the TR cassette), one or more
molecules or other compounds that inhibit, influence or activate a
drug target or drug metabolizing enzyme expressed from the TR
cassette and/or written information on the use of the vectors,
cells or other components of the kit for drug screening or
validation. The oligonucleotides employed in the above kits and
methods of this technology are chosen based upon their ability to
specifically hybridize under high stringency conditions to the
transcription product synthesized from the TR cassette. Various
methods of selecting the oligonucleotide sequences are known in the
art.
[0267] In cases where the TR expression cassette is provided, it is
preferred that the expression cassette be provided as part of a
vector, such as a plasmid or virus. Any of the TR expression
cassettes described herein can be used in the kits. In some
embodiments, a monocistronic TR expression cassette used in a kit
contains a reporter gene, such as GFP or EGFP as the first ORF
sequence. In other embodiments, a bicistronic TR expression
cassette includes two reporter genes, which are either the same or
different. Mammalian cells that are provided in the kit are
preferably from a mammalian cell line, such as HEK293.
[0268] Pharmaceutical Compositions
[0269] The present technology also provides pharmaceutical
compositions comprising a TR expression cassette and a
pharmaceutically acceptable carrier. The TR expression cassette can
be provided on its own, as part of a vector or as part of a
mammalian cell.
[0270] Pharmaceutical compositions are preferably administered to a
subject in a biologically effective or therapeutically effective
amount, either alone or in combination with one or more other
agents. A therapeutically effective amount is a dosage that, when
given for an effective period of time, achieves the desired
therapeutic or clinical effect.
[0271] A therapeutically active amount of a nucleic acid construct
may vary according to factors such as the disease state, age, sex,
and weight of the individual, and the ability of the composition to
elicit a desired response in the individual. Dosage regimes may be
adjusted to provide the optimum therapeutic response. For example,
several divided doses may be administered daily or the dose may be
proportionally reduced as indicated by the exigencies of the
therapeutic situation. A therapeutically effective amount of the
nucleic acid in cell associated form may be stated in terms of
either the amount of the nucleic acid or in cell equivalents.
[0272] Thus an effective amount is between about 1 ng and about 1
gram per kilogram of body weight of the recipient, more preferably
between about 1 ng and 10 mg/kg, more preferably, between about 1
.mu.g and about 1 mg/kg. Dosage forms suitable for internal
administration preferably contain (for the latter dose range) from
about 0.1 mg to about 100 mg of active ingredient per unit. The
active ingredient may vary from about 0.5 to about 95% by weight
based on the total weight of the composition. Alternatively, an
effective dose of cells expressing the nucleic acid is between
about 10.sup.4 and about 10.sup.9 cells, more preferably between
about 10.sup.6 and about 10.sup.8 cells per subject, preferably in
split doses. Those skilled in the art of cell therapy can readily
adjust these doses without undue experimentation.
[0273] The pharmaceutical composition comprising a TR expression
cassette or a cell transfected with the same can be administered in
any convenient manner, e.g., by injection or infusion. The
preferred routes of administration include intravenous,
intrathecal, intracerebroventricular, subcutaneous, intradermal,
and intramuscular routes. Other possible routes include oral
administration, inhalation, or rectal administration.
[0274] Depending on the route of administration, the pharmaceutical
composition of the present technology may be coated in a material
to protect the composition from the action of enzymes, acids and
other natural conditions which may inactivate the composition. For
example, carriers such as liposomes (including
water-in-oil-in-water emulsions as well as conventional liposomes
(Strejan et al., (1984) J Neuroimmunol 7:27)) can be used. In such
instances, the TR expression cassette either on its own or as part
of a vector can be either dispersed or variously present in
corpuscles consisting of aqueous concentric layers adherent to
lipidic layers. The active protein is preferably present in the
aqueous layer and in the lipidic layer, inside or outside, or, in
any event, in the non-homogeneous system generally known as a
liposomic suspension. The hydrophobic layer, or lipidic layer,
generally comprises phospholipids such as lecithin and
sphingomyelin, steroids such as cholesterol, more or less ionic
surface active substances such as dicetylphosphate, stearylamine or
phosphatidic acid, and/or other materials of a hydrophobic
nature.
[0275] The pharmaceutically acceptable carrier (herein also
referred to as "carrier"), also known in the art as an excipient,
vehicle, auxiliary, adjuvant, or diluent, is typically a substance
which is pharmaceutically inert, confers a suitable consistency or
form to the composition, and does not diminish the efficacy of the
composition. The carrier is generally considered to be
pharmaceutically or pharmacologically acceptable if it does not
produce an unacceptably adverse, allergic or other untoward
reaction when administered to a mammal, especially a human. A
"pharmaceutically acceptable carrier" includes, for example, any
and all solvents, dispersion media, coatings, antibacterial and
antifungal agents, and isotonic and absorption delaying agents. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Formulation of drugs is discussed in, for
example, Hoover, John E., Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa. (1975), and Liberman, H. A. and
Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New
York, N.Y. (1980).
[0276] Injectable preparations, for example, sterile injectable
aqueous or oleaginous suspensions, can be formulated according to
the known art using suitable dispersing or wetting agents and
suspending agents. The sterile injectable preparation may also be a
sterile injectable solution or suspension in a nontoxic acceptable
diluent or solvent. Among the acceptable vehicles and solvents that
may be employed are water, Ringer's solution, and isotonic sodium
chloride solution. In addition, sterile, fixed oils are
conventionally employed as a solvent or suspending medium. In
addition, fatty acids such as oleic acid are useful in the
preparation of injectables. Dimethyl acetamide, surfactants
including ionic and non-ionic detergents, and polyethylene glycols
can be used. Mixtures of solvents and wetting agents such as those
discussed above are also useful. Prolonged absorption of the
injectable compositions can be brought about by including in the
composition an agent which delays absorption, for example, aluminum
monostearate and gelatin.
[0277] Suppositories for rectal administration of the compounds
discussed herein can be prepared by mixing the active agent with a
suitable non-irritating excipient such as cocoa butter, synthetic
mono-, di-, or triglycerides, fatty acids, or polyethylene glycols
which are solid at ordinary temperatures but liquid at the rectal
temperature, and which will therefore melt in the rectum and
release the composition.
[0278] Solid dosage forms for oral administration may include
capsules, tablets, pills, powders, and granules. In such solid
dosage forms, the compounds are ordinarily combined with one or
more adjuvants appropriate to the indicated route of
administration. If administered per os, the compounds can be
admixed with lactose, sucrose, starch powder, cellulose esters of
alkanoic acids, cellulose alkyl esters, talc, stearic acid,
magnesium stearate, magnesium oxide, sodium and calcium salts of
phosphoric and sulfuric acids, gelatin, acacia gum, sodium
alginate, polyvinylpyrrolidone, and/or polyvinyl alcohol, and then
tableted or encapsulated for convenient administration. Such
capsules or tablets can contain a controlled-release formulation as
can be provided in a dispersion of active compound in
hydroxypropylmethyl cellulose. In the case of capsules, tablets,
and pills, the dosage forms can also comprise buffering agents such
as sodium citrate, or magnesium or calcium carbonate or
bicarbonate. Tablets and pills can additionally be prepared with
enteric coatings.
[0279] For therapeutic purposes, formulations for parenteral
administration can be in the form of aqueous or non-aqueous
isotonic sterile injection solutions or suspensions. These
solutions and suspensions can be prepared from sterile powders or
granules having one or more of the carriers or diluents mentioned
for use in the formulations for oral administration. The compounds
can be dissolved in water, polyethylene glycol, propylene glycol,
ethanol, corn oil, cottonseed oil, peanut oil, sesame oil, benzyl
alcohol, sodium chloride, and/or various buffers. Other adjuvants
and modes of administration are well and widely known in the
pharmaceutical art.
[0280] Liquid dosage forms for oral administration can include
pharmaceutically acceptable emulsions, solutions, suspensions,
syrups, and elixirs containing inert diluents commonly used in the
art, such as water. Such compositions can also comprise adjuvants,
such as wetting agents, emulsifying and suspending agents, and
sweetening, flavoring, and perfuming agents.
[0281] In sprayable aerosol preparations, the pharmaceutical
composition of the present technology can be provided in
combination with a solid or liquid inert carrier material. Such
preparation can be packaged in a squeeze bottle or in admixture
with a pressurized volatile, normally gaseous propellant. The
aerosol preparations can contain solvents, buffers, surfactants,
and antioxidants in addition to the composition of the
technology.
[0282] Prevention of the action of microorganisms can be achieved
by various antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the
like.
[0283] Exemplary Expression Cassettes
[0284] Tables 1 and 2 below describe numerous monocistronic and
bicistronic TR cassettes, respectively. As discussed previously,
monocistronic TR cassettes comprise one of the TR elements
operatively linked to a 3' ORF sequence whereas the bicistronic
cassettes comprise a TR element operatively linked to upstream and
downstream ORF sequences. The exemplary monocistronic cassettes are
represented by SEQ ID NOs: 3-4 whereas the bicistronic sequences
are exemplified by SEQ ID NOs: 5-6. For purposes of both Tables,
specific promoters, TR elements and ORF sequences that can be used
are described in greater detail above.
TABLE-US-00003 TABLE 1 Monocistronic TR Expression Cassettes TR
Promoter Element ORF Sequence Constitutive TR.sub.dm Reporter Gene
Constitutive TR.sub.plp Reporter Gene Inducible TR.sub.dm Reporter
Gene Inducible TR.sub.plp Reporter Gene Tissue Specific TR.sub.dm
Reporter Gene Tissue Specific TR.sub.plp Reporter Gene Tumor
Specific TR.sub.dm Reporter Gene Tumor Specific TR.sub.plp Reporter
Gene Response Gene TR.sub.dm Reporter Gene Response Gene TR.sub.plp
Reporter Gene Constitutive TR.sub.dm Cytotoxic Tumor Suppressor
Constitutive TR.sub.plp Cytotoxic Tumor Suppressor Inducible
TR.sub.dm Cytotoxic Tumor Suppressor Inducible TR.sub.plp Cytotoxic
Tumor Suppressor Tissue Specific TR.sub.dm Cytotoxic Tumor
Suppressor Tissue Specific TR.sub.plp Cytotoxic Tumor Suppressor
Tumor Specific TR.sub.dm Cytotoxic Tumor Suppressor Tumor Specific
TR.sub.plp Cytotoxic Tumor Suppressor Response Gene TR.sub.dm
Cytotoxic Tumor Suppressor Response Gene TR.sub.plp Cytotoxic Tumor
Suppressor Constitutive TR.sub.dm Toxin Gene Constitutive
TR.sub.plp Toxin Gene Inducible TR.sub.dm Toxin Gene Inducible
TR.sub.plp Toxin Gene Tissue Specific TR.sub.dm Toxin Gene Tissue
Specific TR.sub.plp Toxin Gene Tumor Specific TR.sub.dm Toxin Gene
Tumor Specific TR.sub.plp Toxin Gene Response Gene TR.sub.dm Toxin
Gene Response Gene TR.sub.plp Toxin Gene Constitutive TR.sub.dm
Prodrug Activating Gene Constitutive TR.sub.plp Prodrug Activating
Gene Inducible TR.sub.dm Prodrug Activating Gene Inducible
TR.sub.plp Prodrug Activating Gene Tissue Specific TR.sub.dm
Prodrug Activating Gene Tissue Specific TR.sub.plp Prodrug
Activating Gene Tumor Specific TR.sub.dm Prodrug Activating Gene
Tumor Specific TR.sub.plp Prodrug Activating Gene Response Gene
TR.sub.dm Prodrug Activating Gene Response Gene TR.sub.plp Prodrug
Activating Gene Constitutive TR.sub.dm Proapoptotic Gene
Constitutive TR.sub.plp Proapoptotic Gene Inducible TR.sub.dm
Proapoptotic Gene Inducible TR.sub.plp Proapoptotic Gene Tissue
Specific TR.sub.dm Proapoptotic Gene Tissue Specific TR.sub.plp
Proapoptotic Gene Tumor Specific TR.sub.dm Proapoptotic Gene Tumor
Specific TR.sub.plp Proapoptotic Gene Response Gene TR.sub.dm
Proapoptotic Gene Response Gene TR.sub.plp Proapoptotic Gene
TABLE-US-00004 TABLE 2 Bicistronic TR Expression Cassettes First
ORF Promoter Sequence TR Element Second ORF Sequence Constitutive
Reporter Gene TR.sub.dm Reporter Gene Constitutive Reporter Gene
TR.sub.plp Reporter Gene Inducible Reporter Gene TR.sub.dm Reporter
Gene Inducible Reporter Gene TR.sub.plp Reporter Gene Tissue
Specific Reporter Gene TR.sub.dm Reporter Gene Tissue Specific
Reporter Gene TR.sub.plp Reporter Gene Tumor Specific Reporter Gene
TR.sub.dm Reporter Gene Tumor Specific Reporter Gene TR.sub.plp
Reporter Gene Response Gene Reporter Gene TR.sub.dm Reporter Gene
Response Gene Reporter Gene TR.sub.plp Reporter Gene Constitutive
Reporter Gene TR.sub.dm Cytotoxic Tumor Suppressor Constitutive
Reporter Gene TR.sub.plp Cytotoxic Tumor Suppressor Inducible
Reporter Gene TR.sub.dm Cytotoxic Tumor Suppressor Inducible
Reporter Gene TR.sub.plp Cytotoxic Tumor Suppressor Tissue Specific
Reporter Gene TR.sub.dm Cytotoxic Tumor Suppressor Tissue Specific
Reporter Gene TR.sub.plp Cytotoxic Tumor Suppressor Tumor Specific
Reporter Gene TR.sub.dm Cytotoxic Tumor Suppressor Tumor Specific
Reporter Gene TR.sub.plp Cytotoxic Tumor Suppressor Response Gene
Reporter Gene TR.sub.dm Cytotoxic Tumor Suppressor Response Gene
Reporter Gene TR.sub.plp Cytotoxic Tumor Suppressor Constitutive
Reporter Gene TR.sub.dm Toxin Gene Constitutive Reporter Gene
TR.sub.plp Toxin Gene Inducible Reporter Gene TR.sub.dm Toxin Gene
Inducible Reporter Gene TR.sub.plp Toxin Gene Tissue Specific
Reporter Gene TR.sub.dm Toxin Gene Tissue Specific Reporter Gene
TR.sub.plp Toxin Gene Tumor Specific Reporter Gene TR.sub.dm Toxin
Gene Tumor Specific Reporter Gene TR.sub.plp Toxin Gene Response
Gene Reporter Gene TR.sub.dm Toxin Gene Response Gene Reporter Gene
TR.sub.plp Toxin Gene Constitutive Reporter Gene TR.sub.dm Prodrug
Activating Gene Constitutive Reporter Gene TR.sub.plp Prodrug
Activating Gene Inducible Reporter Gene TR.sub.dm Prodrug
Activating Gene Inducible Reporter Gene TR.sub.plp Prodrug
Activating Gene Tissue Specific Reporter Gene TR.sub.dm Prodrug
Activating Gene Tissue Specific Reporter Gene TR.sub.plp Prodrug
Activating Gene Tumor Specific Reporter Gene TR.sub.dm Prodrug
Activating Gene Tumor Specific Reporter Gene TR.sub.plp Prodrug
Activating Gene Response Gene Reporter Gene TR.sub.dm Prodrug
Activating Gene Response Gene Reporter Gene TR.sub.plp Prodrug
Activating Gene Constitutive Reporter Gene TR.sub.dm Proapoptotic
Gene Constitutive Reporter Gene TR.sub.plp Proapoptotic Gene
Inducible Reporter Gene TR.sub.dm Proapoptotic Gene Inducible
Reporter Gene TR.sub.plp Proapoptotic Gene Tissue Specific Reporter
Gene TR.sub.dm Proapoptotic Gene Tissue Specific Reporter Gene
TR.sub.plp Proapoptotic Gene Tumor Specific Reporter Gene TR.sub.dm
Proapoptotic Gene Tumor Specific Reporter Gene TR.sub.plp
Proapoptotic Gene Response Gene Reporter Gene TR.sub.dm
Proapoptotic Gene Response Gene Reporter Gene TR.sub.plp
Proapoptotic Gene Constitutive Cytotoxic Tumor Suppressor TR.sub.dm
Reporter Gene Constitutive Cytotoxic Tumor Suppressor TR.sub.plp
Reporter Gene Inducible Cytotoxic Tumor Suppressor TR.sub.dm
Reporter Gene Inducible Cytotoxic Tumor Suppressor TR.sub.plp
Reporter Gene Tissue Specific Cytotoxic Tumor Suppressor TR.sub.dm
Reporter Gene Tissue Specific Cytotoxic Tumor Suppressor TR.sub.plp
Reporter Gene Tumor Specific Cytotoxic Tumor Suppressor TR.sub.dm
Reporter Gene Tumor Specific Cytotoxic Tumor Suppressor TR.sub.plp
Reporter Gene Response Gene Cytotoxic Tumor Suppressor TR.sub.dm
Reporter Gene Response Gene Cytotoxic Tumor Suppressor TR.sub.plp
Reporter Gene Constitutive Toxin Gene TR.sub.dm Reporter Gene
Constitutive Toxin Gene TR.sub.plp Reporter Gene Inducible Toxin
Gene TR.sub.dm Reporter Gene Inducible Toxin Gene TR.sub.plp
Reporter Gene Tissue Specific Toxin Gene TR.sub.dm Reporter Gene
Tissue Specific Toxin Gene TR.sub.plp Reporter Gene Tumor Specific
Toxin Gene TR.sub.dm Reporter Gene Tumor Specific Toxin Gene
TR.sub.plp Reporter Gene Response Gene Toxin Gene TR.sub.dm
Reporter Gene Response Gene Toxin Gene TR.sub.plp Reporter Gene
Constitutive Prodrug Activating Gene TR.sub.dm Reporter Gene
Constitutive Prodrug Activating Gene TR.sub.plp Reporter Gene
Inducible Prodrug Activating Gene TR.sub.dm Reporter Gene Inducible
Prodrug Activating Gene TR.sub.plp Reporter Gene Tissue Specific
Prodrug Activating Gene TR.sub.dm Reporter Gene Tissue Specific
Prodrug Activating Gene TR.sub.plp Reporter Gene Tumor Specific
Prodrug Activating Gene TR.sub.dm Reporter Gene Tumor Specific
Prodrug Activating Gene TR.sub.plp Reporter Gene Response Gene
Prodrug Activating Gene TR.sub.dm Reporter Gene Response Gene
Prodrug Activating Gene TR.sub.plp Reporter Gene Constitutive
Proapoptotic Gene TR.sub.dm Reporter Gene Constitutive Proapoptotic
Gene TR.sub.plp Reporter Gene Inducible Proapoptotic Gene TR.sub.dm
Reporter Gene Inducible Proapoptotic Gene TR.sub.plp Reporter Gene
Tissue Specific Proapoptotic Gene TR.sub.dm Reporter Gene Tissue
Specific Proapoptotic Gene TR.sub.plp Reporter Gene Tumor Specific
Proapoptotic Gene TR.sub.dm Reporter Gene Tumor Specific
Proapoptotic Gene TR.sub.plp Reporter Gene Response Gene
Proapoptotic Gene TR.sub.dm Reporter Gene Response Gene
Proapoptotic Gene TR.sub.plp Reporter Gene
[0285] Further to expression cassettes shown in Tables 1 and 2,
Tables 3 and 4 below describe specific examples of monocistronic
and bicistronic TR expression cassettes.
[0286] One skilled in the art can readily recognize that the
combinations described in Tables 3 and 4 are shown by way of
example and not of limitation.
TABLE-US-00005 TABLE 3 Specific Examples of Monocistronic TR
Expression Cassettes Promoter TR Element ORF Sequence
Cytomegalovirus immediate TR.sub.dm Firefly Luciferase early (CMV)
CMV TR.sub.plp Firefly Luciferase Metallothionein-I TR.sub.dm
Firefly Luciferase Metallothionein-I TR.sub.plp Firefly Luciferase
Synapsin I TR.sub.dm Firefly Luciferase Synapsin I TR.sub.plp
Firefly Luciferase Alpha Fetoprotein TR.sub.dm Firefly Luciferase
Alpha Fetoprotein TR.sub.plp Firefly Luciferase Heat Shock Protein
70 TR.sub.dm Firefly Luciferase Heat Shock Protein 70 TR.sub.plp
Firefly Luciferase CMV TR.sub.dm p53 CMV TR.sub.plp p53
Metallothionein-I TR.sub.dm p53 Metallothionein-I TR.sub.plp p53
Synapsin I TR.sub.dm p53 Synapsin I TR.sub.plp p53 Alpha
Fetoprotein TR.sub.dm p53 Alpha Fetoprotein TR.sub.plp p53 Heat
Shock Protein 70 TR.sub.dm p53 Heat Shock Protein 70 TR.sub.plp p53
CMV TR.sub.dm Diphtheria toxin CMV TR.sub.plp Diphtheria toxin
Metallothionein-I TR.sub.dm Diphtheria toxin Metallothionein-I
TR.sub.plp Diphtheria toxin Synapsin I TR.sub.dm Diphtheria toxin
Synapsin I TR.sub.plp Diphtheria toxin Alpha Fetoprotein TR.sub.dm
Diphtheria toxin Alpha Fetoprotein TR.sub.plp Diphtheria toxin Heat
Shock Protein 70 TR.sub.dm Diphtheria toxin Heat Shock Protein 70
TR.sub.plp Diphtheria toxin CMV TR.sub.dm Thymidine Kinase sr39 CMV
TR.sub.plp Thymidine Kinase sr39 Metallothionein-I TR.sub.dm
Thymidine Kinase sr39 Metallothionein-I TR.sub.plp Thymidine Kinase
sr39 Synapsin I TR.sub.dm Thymidine Kinase sr39 Synapsin I
TR.sub.plp Thymidine Kinase sr39 Alpha Fetoprotein TR.sub.dm
Thymidine Kinase sr39 Alpha Fetoprotein TR.sub.plp Thymidine Kinase
sr39 Heat Shock Protein 70 TR.sub.dm Thymidine Kinase sr39 Heat
Shock Protein 70 TR.sub.plp Thymidine Kinase sr39 CMV TR.sub.dm
Caspase 3 CMV TR.sub.plp Caspase 3 Metallothionein-I TR.sub.dm
Caspase 3 Metallothionein-I TR.sub.plp Caspase 3 Synapsin I
TR.sub.dm Caspase 3 Synapsin I TR.sub.plp Caspase 3 Alpha
Fetoprotein TR.sub.dm Caspase 3 Alpha Fetoprotein TR.sub.plp
Caspase 3 Heat Shock Protein 70 TR.sub.dm Caspase 3 Heat Shock
Protein 70 TR.sub.plp Caspase 3
TABLE-US-00006 TABLE 4 Specific Examples of Bicistronic TR
Expression Cassettes First ORF Promoter Sequence TR Element Second
ORF Sequence CMV Renilla Luciferase TR.sub.dm Firefly Luciferase
CMV Renilla Luciferase TR.sub.plp Firefly Luciferase
Metallothionein-I Renilla Luciferase TR.sub.dm Firefly Luciferase
Metallothionein-I Renilla Luciferase TR.sub.plp Firefly Luciferase
Synapsin I Renilla Luciferase TR.sub.dm Firefly Luciferase Synapsin
I Renilla Luciferase TR.sub.plp Firefly Luciferase Alpha
Fetoprotein Renilla Luciferase TR.sub.dm Firefly Luciferase Alpha
Fetoprotein Renilla Luciferase TR.sub.plp Firefly Luciferase Heat
Shock Protein 70 Renilla Luciferase TR.sub.dm Firefly Luciferase
Heat Shock Protein 70 Renilla Luciferase TR.sub.plp Firefly
Luciferase CMV p53 TR.sub.dm Firefly Luciferase CMV p53 TR.sub.plp
Firefly Luciferase Metallothionein-I p53 TR.sub.dm Firefly
Luciferase Metallothionein-I p53 TR.sub.plp Firefly Luciferase
Synapsin I p53 TR.sub.dm Firefly Luciferase Synapsin I p53
TR.sub.plp Firefly Luciferase Alpha Fetoprotein p53 TR.sub.dm
Firefly Luciferase Alpha Fetoprotein p53 TR.sub.plp Firefly
Luciferase Heat Shock Protein 70 p53 TR.sub.dm Firefly Luciferase
Heat Shock Protein 70 p53 TR.sub.plp Firefly Luciferase CMV
Diphtheria toxin TR.sub.dm Firefly Luciferase CMV Diphtheria toxin
TR.sub.plp Firefly Luciferase Metallothionein-I Diphtheria toxin
TR.sub.dm Firefly Luciferase Metallothionein-I Diphtheria toxin
TR.sub.plp Firefly Luciferase Synapsin I Diphtheria toxin TR.sub.dm
Firefly Luciferase Synapsin I Diphtheria toxin TR.sub.plp Firefly
Luciferase Alpha Fetoprotein Diphtheria toxin TR.sub.dm Firefly
Luciferase Alpha Fetoprotein Diphtheria toxin TR.sub.plp Firefly
Luciferase Heat Shock Protein 70 Diphtheria toxin TR.sub.dm Firefly
Luciferase Heat Shock Protein 70 Diphtheria toxin TR.sub.plp
Firefly Luciferase CMV Thymidine Kinase sr39 TR.sub.dm Firefly
Luciferase CMV Thymidine Kinase sr39 TR.sub.plp Firefly Luciferase
Metallothionein-I Thymidine Kinase sr39 TR.sub.dm Firefly
Luciferase Metallothionein-I Thymidine Kinase sr39 TR.sub.plp
Firefly Luciferase Synapsin I Thymidine Kinase sr39 TR.sub.dm
Firefly Luciferase Synapsin I Thymidine Kinase sr39 TR.sub.plp
Firefly Luciferase Alpha Fetoprotein Thymidine Kinase sr39
TR.sub.dm Firefly Luciferase Alpha Fetoprotein Thymidine Kinase
sr39 TR.sub.plp Firefly Luciferase Heat Shock Protein 70 Thymidine
Kinase sr39 TR.sub.dm Firefly Luciferase Heat Shock Protein 70
Thymidine Kinase sr39 TR.sub.plp Firefly Luciferase CMV Caspase 3
TR.sub.dm Firefly Luciferase CMV Caspase 3 TR.sub.plp Firefly
Luciferase Metallothionein-I Caspase 3 TR.sub.dm Firefly Luciferase
Metallothionein-I Caspase 3 TR.sub.plp Firefly Luciferase Synapsin
I Caspase 3 TR.sub.dm Firefly Luciferase Synapsin I Caspase 3
TR.sub.plp Firefly Luciferase Alpha Fetoprotein Caspase 3 TR.sub.dm
Firefly Luciferase Alpha Fetoprotein Caspase 3 TR.sub.plp Firefly
Luciferase Heat Shock Protein 70 Caspase 3 TR.sub.dm Firefly
Luciferase Heat Shock Protein 70 Caspase 3 TR.sub.plp Firefly
Luciferase CMV Renilla Luciferase TR.sub.dm p53 CMV Renilla
Luciferase TR.sub.plp p53 Metallothionein-I Renilla Luciferase
TR.sub.dm p53 Metallothionein-I Renilla Luciferase TR.sub.plp p53
Synapsin I Renilla Luciferase TR.sub.dm p53 Synapsin I Renilla
Luciferase TR.sub.plp p53 Alpha Fetoprotein Renilla Luciferase
TR.sub.dm p53 Alpha Fetoprotein Renilla Luciferase TR.sub.plp p53
Heat Shock Protein 70 Renilla Luciferase TR.sub.dm p53 Heat Shock
Protein 70 Renilla Luciferase TR.sub.plp p53 CMV Renilla Luciferase
TR.sub.dm Diphtheria toxin CMV Renilla Luciferase TR.sub.plp
Diphtheria toxin Metallothionein-I Renilla Luciferase TR.sub.dm
Diphtheria toxin Metallothionein-I Renilla Luciferase TR.sub.plp
Diphtheria toxin Synapsin I Renilla Luciferase TR.sub.dm Diphtheria
toxin Synapsin I Renilla Luciferase TR.sub.plp Diphtheria toxin
Alpha Fetoprotein Renilla Luciferase TR.sub.dm Diphtheria toxin
Alpha Fetoprotein Renilla Luciferase TR.sub.plp Diphtheria toxin
Heat Shock Protein 70 Renilla Luciferase TR.sub.dm Diphtheria toxin
Heat Shock Protein 70 Renilla Luciferase TR.sub.plp Diphtheria
toxin CMV Renilla Luciferase TR.sub.dm Thymidine Kinase sr39 CMV
Renilla Luciferase TR.sub.plp Thymidine Kinase sr39
Metallothionein-I Renilla Luciferase TR.sub.dm Thymidine Kinase
sr39 Metallothionein-I Renilla Luciferase TR.sub.plp Thymidine
Kinase sr39 Synapsin I Renilla Luciferase TR.sub.dm Thymidine
Kinase sr39 Synapsin I Renilla Luciferase TR.sub.plp Thymidine
Kinase sr39 Alpha Fetoprotein Renilla Luciferase TR.sub.dm
Thymidine Kinase sr39 Alpha Fetoprotein Renilla Luciferase
TR.sub.plp Thymidine Kinase sr39 Heat Shock Protein 70 Renilla
Luciferase TR.sub.dm Thymidine Kinase sr39 Heat Shock Protein 70
Renilla Luciferase TR.sub.plp Thymidine Kinase sr39 CMV Renilla
Luciferase TR.sub.dm Caspase 3 CMV Renilla Luciferase TR.sub.plp
Caspase 3 Metallothionein-I Renilla Luciferase TR.sub.dm Caspase 3
Metallothionein-I Renilla Luciferase TR.sub.plp Caspase 3 Synapsin
I Renilla Luciferase TR.sub.dm Caspase 3 Synapsin I Renilla
Luciferase TR.sub.plp Caspase 3 Alpha Fetoprotein Renilla
Luciferase TR.sub.dm Caspase 3 Alpha Fetoprotein Renilla Luciferase
TR.sub.plp Caspase 3 Heat Shock Protein 70 Renilla Luciferase
TR.sub.dm Caspase 3 Heat Shock Protein 70 Renilla Luciferase
TR.sub.plp Caspase 3
[0287] General Methods
[0288] Molecular biological techniques, biochemical techniques, and
microorganism techniques as used herein are well known in the art
and commonly used, and are described in, for example, Sambrook J.
et al. (1989), Molecular Cloning: A Laboratory Manual, Cold Spring
Harbor and its 3rd Ed. (2001); Ausubel, F. M. (1987), Current
Protocols in Molecular Biology, Greene Pub. Associates and
Wiley-interscience; Ausubel, F. M. (1989), Short Protocols in
Molecular Biology: A Compendium of Methods from Current Protocols
in Molecular Biology, Greene Pub. Associates and
Wiley-interscience; Innis, M. A. (1990), PCR Protocols: A Guide to
Methods and Applications, Academic Press; Ausubel, F. M. (1992),
Short Protocols in Molecular Biology: A Compendium of Methods from
Current Protocols in Molecular Biology, Greene Pub. Associates;
Ausubel, F. M. (1995), Short Protocols in Molecular Biology: A
Compendium of Methods from Current Protocols in Molecular Biology,
Greene Pub. Associates; Innis, M. A. et al. (1995), PCR Strategies,
Academic Press; Ausubel, F. M. (1999), Short Protocols in Molecular
Biology: A Compendium of Methods from Current Protocols in
Molecular Biology, Wiley, and annual updates; Sninsky, J. J. et al.
(1999), PCR Applications: Protocols for Functional Genomics,
Academic Press; Special issue, Jikken Igaku [Experimental Medicine]
"Idenshi Donyu & Hatsugenkaiseki Jikkenho [Experimental Method
for Gene introduction & Expression Analysis]", Yodo-sha, 1997;
and the like. Relevant portions (or possibly the entirety) of each
of these publications are herein incorporated by reference.
[0289] Any technique may be used herein for introduction of a
nucleic acid molecule into cells, including, for example,
transformation, transduction, transfection, and the like. Such a
nucleic acid molecule introduction technique is well known in the
art and commonly used, and is described in, for example, Ausubel F.
A. et al., editors, (1988), Current Protocols in Molecular Biology,
Wiley, New York, N.Y.; Sambrook J. et al. (1987) Molecular Cloning:
A Laboratory Manual, 2nd Ed. and its 3rd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; Special issue, Jikken
Igaku [Experimental Medicine] Experimental Method for Gene
introduction & Expression Analysis", Yodo-sha, 1997; and the
like. Gene introduction can be confirmed by method as described
herein, such as Northern blotting analysis and Western blotting
analysis, or other well-known, common techniques.
[0290] Amino acid deletion, substitution or addition of the
polypeptide of the present technology can be carried out by a
site-specific mutagenesis method which is a well known technique.
One or several amino acid deletions, substitutions or additions can
be carried out in accordance with methods described in Molecular
Cloning, A Laboratory Manual, Second Edition, Cold Spring Harbor
Laboratory Press (1989); Current Protocols in Molecular Biology,
Supplement 1 to 38, John Wiley & Sons (1987-1997); Nucleic
Acids Research, 10, 6487 (1982); Proc. Natl. Acad. Sci., USA, 79,
6409 (1982); Gene, 34, 315 (1985); Nucleic Acids Research, 13, 4431
(1985); Proc. Natl. Acad. Sci. USA, 82, 488 (1985); Proc. Natl.
Acad. Sci., USA, 81, 5662 (1984); Science, 224, 1431 (1984); PCT
WO85/00817(1985); Nature, 316, 601 (1985); and the like.
Examples
[0291] The following non-limiting examples are provided to further
illustrate the present technology. It should be appreciated by
those of skill in the art that the techniques disclosed in the
examples that follow represent approaches the inventors have found
function well in the practice of the technology, and thus can be
considered to constitute examples of modes for its practice.
However, those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments that are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
technology.
Example I
Construction of the Monocistronic TR Cassette
[0292] This example describes the preparation of mammalian
expression vectors containing a "monocistronic" cassette (FIG. 2)
which allows selective translation of open reading frames (ORFs)
under the control of the Translational Regulator (TR) sequence
during cell stress and death. The pTR-ORF plasmid construct
described in this example contains a mammalian promoter, a TR
element operably linked to a single protein coding sequence, as
well as a mRNA polyadenylation signal (i.e., the TR expression
cassette).
A. Preparation of the TR Regulated Expression Cassette
[0293] In order to demonstrate the feasibility and efficacy of TR
regulated translation, an initial series of mammalian expression
vectors are prepared containing a TR expression cassette. The
pTR-EYFP expression vectors are constructed essentially as follows.
DNA fragments corresponding to sequences--16 to +858 of the PLP and
DM20 cDNAs are cloned into the pEYFP-N1 vector generating the
pPLPeyfp and pDM20eyfp expression vectors (FIGS. 1A-1C). The
mammalian expression sequences of the pEYFP-N1 plasmid contain the
CMV early promoter/enhancer, the EYFP ORF and the SV40 early
polyadenylation signal. Other mammalian-specific elements in the
pEYFP-N1 backbone include an SV40 origin of replication, a cassette
consisting of the SV40 early (Large T) gene promoter fused to the
neomycin phosphotransferase gene and polyadenylation signals from
the herpes simplex thymidine kinase gene. The neomycin resistance
provided by this expression cassette can be used as a selectable
marker for preparing stably transformed mammalian cells. For
selection and growth in E. coli, a bacterial promoter upstream of
the SV40 promoter provides kanamycin resistance, whereas, a pUC19
origin of replication allows plasmid propagation in E. coli.
[0294] The initial round of oligonucleotide site directed
mutagenesis, of the pPLPeyfp and pDM20eyfp expression vectors, use
internal oligonucleotide primer sets [L205.5w (SEQ ID NO: 7) and
L205.3w (SEQ ID NO: 8); L235.5 (SEQ ID NO: 9) and L235.3 (SEQ ID
NO: 10)] to remove the translation initation codons at nucleotides
511 and 598 in the TR.sub.dm sequence and nucleotides 616 and 703
in the TR.sub.plp sequence, respectively. The mutagenesis procedure
used in this effort is as described for the QuikChange mutagenesis
of the TK ORF, see subsequent section. An ensuing mutation, using a
5'-specific primer set [RI-stop_s (SEQ ID NO: 11) and RI-stop_a
(SEQ ID NO: 12)] alters both TR sequences so that cap-dependent
translation was eliminated by the insertion of stop codons and the
removal of 5' proximal AUG initiation codons. Subsequently, a
3'-specific primer set [H-Xh-Xb_s (SEQ ID NO: 13) and H-Xh-Xb_a
(SEQ ID NO: 14) removes the PLP/DM20 translation termination codon
and introduces HindIII, XhoI, and XbaI sites into the intervening
sequences between the TR sequence and EYFP AUG codon. Procedures
for site-directed mutagenesis are well known in the art (for
example, QuikChange Mutagenesis Kit, Stratagene) and are discussed
in a subsequent section of this example. These constructs are
designated pTR.sub.plp-EYFP and pTR.sub.dm-EYFP (FIG. 2). Thus the
TR expression cassette in the pTR.sub.plp-EYFP plasmid is composed
of the CMV immediate early promoter/enhancer, the PLP TR sequence,
the EYFP ORF and the SV40 polyadenylation signal. Similarly, the TR
expression cassette in the pTR.sub.dm-EYFP plasmid contains the CMV
promoter/enhancer, the DM20 TR sequence, the EFYP ORF and the SV40
polyadenylation signal.
[0295] The sequence identity of these constructs and subsequent
derivatives are verified using any or all of the following
sequencing primers; SK15 (SEQ ID NO: 15), SK16 (SEQ ID NO: 16),
SK17 (SEQ ID NO: 17), EYFP(-)1 (SEQ ID NO: 18), EYFP(-)2 (SEQ ID
NO: 19), BAC-1 (SEQ ID NO: 20), BAC-2 (SEQ ID NO: 21) or BAC-3 (SEQ
ID NO: 22).
B. Selection and Cloning of ORFs Into the TR Cassette
[0296] To demonstrate TR regulation of a variety of ORFs, a number
of different plasmids are created by varying the TR regulated ORF.
Derivatives of the monocistronic pTR.sub.plp/dm-EYFP plasmids are
used to construct other monocistronic TR-ORF vectors by modifying
some or all of these functional elements, including (a) exchanging
the pCMV 1E promoter, (b) removing the EYFP ORF, and/or (c)
addition or subtraction of restriction sites as needed. To clone
the non-EYFP ORFs into the same position as the EYFP ORF, two
nucleotides are inserted upstream of the H-Xh-Xb sequence using
site directed mutagenesis with the AUGback_s (SEQ ID NO: 23) and
AUGback_a (SEQ ID NO: 24) primer set. This allows subsequent
ORF-specific PCR primer sets to include 1) a common 5' sequence
which replaced the native translational initiation codon with an
identical Kozak consensus sequence and 2) 5' HindIll and 3' XhoI
restriction enzyme sites which allowed directional cloning.
Following digestion with HindIII and XhoI, these ORF-specific PCR
fragments are cloned into the pTR.sub.plp-ORF and pTR.sub.dm-ORF
vectors. The EYFP, fLuc, TK, CAT and LacZ ORFs are PCR amplified
from the pEYFP-N1 (Clontech), phCMV-LUC-FSR (Genlantis), pHSV106
(GenBank sequence V00470), pCAT-Enhancer (Promega), and pAAV-LacZ
(Stratagene) plasmid vectors, respectively. The following exemplary
plasmids are made:
[0297] pTR.sub.plp-fLuc, which includes the plp TR element and the
firefly Luciferase (fLUC) ORF that was PCR amplified using the
Luc-1 (SEQ ID NO: 25) and Luc-2 (SEQ ID NO: 26) primer set;
[0298] pTR.sub.dm-fLuc, which includes the dm20 TR element and the
fLUC ORF that was PCR amplified using the Luc-1 and Luc-2 primer
set;
[0299] pTR.sub.plp-TK, which includes the plp TR element and the
HSV thymidine kinase (TK) ORF that was PCR amplified using the TK-1
(SEQ ID NO: 27) and TK-2 (SEQ ID NO: 28) primer set;
[0300] pTR.sub.dm-TK, which includes the dm20 TR element and the TK
ORF that was PCR amplified using the TK-1 and TK-2 primer set;
[0301] pTR.sub.plp-CAT, which includes the plp TR element and the
bacterial choramphenicol acetyltransferase (CAT) ORF that was PCR
amplified using the CAT-1 (SEQ ID NO: 29) and the CAT-2 (SEQ ID NO:
30) primer set;
[0302] pTR.sub.dm-CAT, which includes the dm20 TR sequence and the
CAT ORF that was PCR amplified using the CAT-1 and the CAT-2 primer
set;
[0303] pTR.sub.plp-LacZ, which includes the plp TR sequence and the
bacterial LacZ ORF encoding the beta-galactosidase protein that was
PCR amplified using the LacZ-1 (SEQ ID NO: 31) and LacZ-2 (SEQ ID
NO: 32) primer set; and pTR.sub.dm-LacZ, which includes the dm20 TR
element and the LacZ ORF that was PCR amplified using the LacZ-1
and LacZ-2 primer set.
C. Site Directed Mutagenesis of the TR-Regulated ORFs
[0304] This example shows that the TR-regulated ORF can be altered
to improve a functional characteristic of a protein translated from
the TR cassette or eliminate any RNA sequence/structure that might
interfere with TR regulation. In this example, the five TK sr39
amino acid mutations (Gambhir et al; Proc Natl Acad Sci USA; 97:
2785-2790) are inserted into the TK ORF using the Sr39-1 (SEQ ID
NO: 33) and Sr39-2 primer set (SEQ ID NO: 34).
[0305] Compared to the wildtype TK protein, the TKsr39 protein
displays beneficial kinetic properties, such as enhanced prodrug
binding (83-fold higher than the wildtype protein for GCV),
increased pro-drug mediated cell killing at lower pro-drug
concentrations, and superior binding of metabolic tracers (for
example 18F-labeled penciclovir). In the latter case, this enhanced
binding efficiency improves noninvasive imaging techniques such as
positron emission tomography.
[0306] To validate the process of site directed mutagenesis in the
TR cassette and produce an improved TK marker protein,
oligonucleotide directed mutagenesis is used to insert the TKsr39
DNA mutations into the wild type TR-ORF sequence. Procedures for
site-directed mutagenesis are well known in the art (for example,
QuikChange Site-Directed Mutagenesis Kits, Stratagene). Basically,
oligonucleotide primers (e.g. Sr39-1 and Sr39-2) were constructed
that contained the desired mutations. PCR amplification of the
pTR.sub.plp-TK and pTR.sub.dm-TK templates with the Sr39 primer set
(1 cycle of 95.degree. C. for 30 sec, 12 cycles of 95.degree. C.
for 30 sec, 55.degree. C. for 1 min, 68.degree. C. for 12 min)
incorporated the mutations into the exponentially amplified DNA
strand. Following amplification, the DNA is digested with the DpnI
restriction enzyme which specifically recognizes methylated and
hemimethylated DNA. Since the pTR.sub.plp-TK and pTR.sub.dm-TK
templates were grown in a methylation-positive bacterial strain,
DpnI digestion removed the parental template prior to bacterial
transformation. The transformed bacteria are plated on selective
media and isolated colonies used to prepare DNA minipreps which are
analyzed by restriction mapping and DNA sequencing.
[0307] The resultant plasmids are termed pTR.sub.plp-TKsr39 and
pTR.sub.dm-TKsr39. The pTR.sub.plp-TKsr39 vector includes the plp
TR element and the HSV-1 TKsr39 mutations in the pHSV106 TK ORF
(SEQ ID NO; 39). Similarly, the pTR.sub.dm-TKsr39 plasmid contains
the dm20 TR element and the TKsr39 mutations in the pHSV106 TK ORF
(SEQ ID NO: 40).
Example II
Construction of a Bicistronic TR Cassette
[0308] This example describes the preparation of mammalian
expression vectors containing a bicistronic TR cassette (FIG. 3)
which allows cap-dependent translation of an
[0309] ORF upstream of the TR cassette and cap-independent
translation of an ORF under the control of the Translational
Regulator (TR) element during cell stress and death. These vectors
allow the examination of cap-dependent and cap-independent
translational processes from a single mRNA.
A. Inserting the Firefly Luciferase ORF Upstream of the TR-EYFP
Cassette
[0310] Bicistronic vectors are constructed by inserting the Firefly
Luciferase (fLuc) ORF (termed ORF2) upstream of the TR cassette
containing the EYFP gene (ORF1). The phCMV-LUC-FSR vector
(Genlantis) are digested with EcoRI, the restriction fragment
containing the fLuc sequence purified and cloned into the EcoRI
site of the pTR.sub.plp-EYFP and pTR.sub.dm-EYFP vectors (FIG. 3).
The orientation of the fLuc ORF is verified by restriction mapping
and forward (sense) and reverse (antisense) inserts are recovered.
The sense vectors are termed the pfLuc-TR.sub.plp-EYFP and
pfLuc-TR.sub.dm-EYFP plasmids and the antisense vectors are termed
the pcuLf-TR.sub.plp-EYFP and pcuLf-TR.sub.dm-EYFP plasmids.
[0311] The sense pORF-TR-ORF vectors encode a single mRNA species
that allows constitutive steady-state cap-dependent translation of
the upstream ORF and selective cap-independent translation of the
ORF operatively linked to the TR element during cell stress and
death. The antisense bicistronic vectors serve as a control for TR
activity by providing a large segment of upstream mRNA sequences
which structurally block cap-dependent translation of the
TR-regulated ORF in normal cells. Protein synthesis from the
antisense vectors in stressed and dying cells is TR-regulated
cap-independent translation.
[0312] For example, the pfLuc-TRplp-EYFP vector encodes a single
mRNA species that constitutively exhibits cap-dependent translation
of the fLuc ORF and selective cap-independent translation of the
EYFP ORF from the plp TR element during cell stress and death.
Similarly, a single mRNA species is transcribed from the
pfLuc-TR.sub.dm-EYFP cassette that provides cap-dependent
translation of the fLUC ORF and cap-independent translation of the
EYFP ORF from the dm20 TR element during cell stress and death
(FIG. 4).
Example III
Construction and Production of Recombinant Viral Vectors for
Expression of TR Cassettes in Mammalian Cells
[0313] Recombinant adenovirus-associated virus (rAAV) and
baculovirus (rBAC) vectors are designed to produce infectious
virions that can transduce mammalian cells with the TR expression
cassette. For illustration, a series of rAAV and rBAC viruses are
prepared that direct constitutive expression of TR expression
cassettes in mammalian cells from the CMV-IE promoter/enhancer.
A. Inserting the TR Cassettes into Recombinant AAV (rAAV)
Virions
[0314] The pAAV-TR.sub.plp and pAAV-TR.sub.dm transfer (or shuttle)
vectors are derived from the pAAV-MCS vector (Stratagene). To allow
bacterial propagation, the pAAV-MCS backbone provides (1) the
bacterial beta-lactamase gene, (2) the pUC19 origin of replication,
and (3) the fl replication origin for single-stranded DNA
synthesis. For AAV viral production and mammalian gene expression,
the pAAV-MCS plasmid contains the Left and Right adeno-associated
virus-2 (AAV2) inverted terminal repeats (ITRs) flanking the CMV IE
promoter/enhancer, the CMV IE transcriptional start site, the
beta-globin intron, multiple unique restriction sites for cloning
(multiple cloning site or MCS), and the human growth hormone
polyadenylation signal (FIG. 5).
[0315] The TR-ORF cassettes are produced by digesting the
appropriate TR-ORF expression plasmid with EcoRI/XhoI and cloning
into the EcoRI/XhoI sites of the pAAV-MCS vector. For example, the
pTR.sub.plp-fLUC and pTR.sub.dm-fLUC plasmids can be digested with
EcoRI/XhoI and cloned into the EcoRI/XhoI sites of the pAAV-MCS
vector to create the pAAV-TR-LUC shuttle vectors listed below.
[0316] Following restriction mapping and/or DNA sequencing, the
following examples of pAAV-TR-ORF shuttle vectors can be
produced:
[0317] pAAV-TR.sub.plp-EYFP, which contains the plp TR sequence and
the EYFP ORF;
[0318] pAAV-TR.sub.dm-EYFP, which contains the dm20 TR element and
the EYFP ORF;
[0319] pAAV-TR.sub.plp-fLuc, which contains the plp TR element and
the firefly Luciferase ORF;
[0320] pAAV-TR.sub.dm-fLuc, which contains the dm20 TR sequence and
the fLUC ORF;
[0321] pAAV-TR.sub.plp-TK, which contains the plp TR element and
the HSV-1 TK ORF;
[0322] pAAV-TR.sub.dm-TK, which contains the dm20 TR element and
the HSV-1 TK ORF;
[0323] pAAV-TR.sub.plp-TKsr39, which contains the plp TR sequence
and the sr39 derivative of the HSV-1 TK ORF;
[0324] pAAV-TR.sub.dm-TKsr39, which contains the dm20 TR element
and the sr39 derivative of the HSV-1 TK ORF;
[0325] pAAV-TR.sub.plp-CAT, which contains the plp TR element and
the bacterial chloramphenicol acetyltransferase (CAT) ORF;
[0326] pAAV-TR.sub.dm-CAT, which contains the dm20 TR sequence and
the CAT ORF;
[0327] pAAV-TR.sub.plp-LacZ, which contains the plp TR sequence and
the bacterial LacZ ORF; and
[0328] pAAV-TR.sub.dm-LacZ, which contains the dm20 TR element and
the LacZ ORF.
[0329] To recover recombinant AAV2 viral particles, HEK293 cells
are transfected using the triple plasmid transfection procedure
that requires a pAAV shuttle vector, the pAAV-RC (replication
competent helper plasmid) and the pHelper (adenovirus helper
plasmid). The rAAV 3-plasmid procedure is well known in the art and
is briefly described below. The pAAV shuttle vector is
cotransfected into a packaging cell line along with the 7.3-kb
pAAV-RC vector and the 11.6-kb pHelper vector. The pAAV-RC vector
encodes the rep (DNA replication protein) and cap (the AAV2 capsid
protein) genes, which are required for infectious virions. The
pHelper vector contains a deleted Adenovirus genome that expresses
various adenovirus genes required for AAV production. Genetic
complementation between proteins expressed from the three plasmids
and the adenovirus E1A and E1B proteins (provided by the HEK293
cells) allows the generation of packaged virions following
recombination at the left and right inverted terminal repeats
(L-ITR and R-ITR) in the pAAV shuttle vector.
[0330] For this example, HEK293 cells are transfected with 15
micrograms of a pAAV-TR-ORF shuttle vector or 15 micrograms of the
pAAV-PLP/DM20eyfp plasmids, 10 micrograms of the pAAV-RC plasmid,
and 10 micrograms of the pHelper vector using a standard calcium
phosphate transfection protocol. Following transfection, the HEK293
cells are incubated for 72 hrs total. At this time, the cells are
collected, the medium removed and a lysate produced using three
freeze-thaw cycles. Clarified lysates are prepared by
centrifugation at 2500 rpm for 10 min.
[0331] Specific examples of rAAV-TR-ORF viruses that can be
generated in this manner include: rAAV-TR.sub.plp-EYFP, which
contains the plp TR element operatively linked to the EYFP ORF;
[0332] rAAV-TR.sub.dm-EYFP, which contains the dm20 TR sequence
linked to the EYFP ORF;
[0333] rAAV-TR.sub.plp-fLuc, which includes the plp TR element and
the firefly Luciferase ORF;
[0334] rAAV-TR.sub.dm-fLuc, which contains the dm20 TR sequence and
the fLUC ORF;
[0335] rAAV-TR.sub.plp-TK, which contains the plp TR element and
the HSV-1 TK ORF;
[0336] rAAV-TR.sub.dm-TK, which contains the dm20 TR element and
the HSV-1 TK ORF;
[0337] rAAV-TR.sub.plp-TKsr39, which contains the plp TR sequence
and the sr39 derivative of the HSV-1 TK ORF;
[0338] rAAV-TR.sub.dm-TKsr39, which contains the dm20 TR element
and the sr39 derivative of the HSV-1 TK ORF;
[0339] rAAV-TR.sub.plp-CAT, which contains the plp TR element and
the CAT ORF;
[0340] rAAV-TR.sub.dm-CAT, which contains the dm20 TR sequence and
the CAT ORF;
[0341] rAAV-TR.sub.plp-LacZ, which contains the plp TR sequence and
the bacterial LacZ ORF; and
[0342] rAAV-TR.sub.dm-LacZ, which contains the dm20 TR element and
the LacZ ORF.
B. Preparation of Recombinant Baculovirus (rBAC) Virions
Transducing the TR Expression Cassette
[0343] A set of mammalian expression vectors are prepared using the
pBAC-1 shuttle transfer backbone (Novagen). The commercial
pBAC.TM.1 vector is a baculovirus transfer plasmid designed for
cloning and expressing recombinant proteins in insect cells using
the polh promoter. The commercial pBAC-1 plasmid contains (1) the
bacterial beta-lactamase gene, as well as (2) the pUC19 origin of
replication and (3) the fl origin of replication (FIG. 5). The
vector also contains the polh promoter and a series of unique
restriction sites commonly used to clone ORFs for expression in
insect cells. Flanking the insect expression elements are
baculovirus sequences needed for DNA recombination, the late
expression factor 2 (lef-2) and the open reading frame 1629
(orf1629) genes. Transfecting insect cells with a pBAC shuttle
vector and replication deficient baculoviral DNA allows
recombination between the lef-2/orf1629 shuttle vector sequences
with homologous sequences in the viral DNA and the generation of a
replication competent recombinant virus.
[0344] To provide mammalian expression, the viral polh promoter and
polh transcription initiation site are removed by ligating the
pBAC-1 vector digested with BglII and BamHI, thus producing the
pBAC polh-plasmid. For this example, the CMV IE promoter/enhancer
is PCR amplified using the oligonucleotide primer set [CMV-1 (SEQ
ID NO: 35) and CMV-2 (SEQ ID NO: 36)] which introduces a 5' StuI
site and a 3' EcoRI site into the PCR fragment. The amplified DNA
is cut with StuI and EcoRI and directionally cloned into the
StuI/EcoRI sites of the pBAC polh-vector, resulting in the pBAC-CMV
plasmid. To introduce a mRNA polyadenylation signal, two
complementary oligonucleotides [PolyA-1 (SEQ ID NO: 37) and PolyA-2
(SEQ ID NO: 38)] containing the SV40 early polyadenylation signal
flanked by AvrII and SphI restriction sites are annealed and cloned
into the AvrII/SphI sites of the pBAC-CMV vector to create the
pBAC-CMV-PolyA plasmid.
[0345] The TR-ORF cassettes are removed from the pTR.sub.plp-EYFP
and pTR.sub.dm-EYFP plasmids with EcoRI/NotI and cloned into the
EcoRI/NotI sites of the pBAC-CMV-PolyA vector to create the pBAC
shuttle vectors listed below. In a related procedure, the other
pBAC shuttle vectors listed below are produced by digesting the
appropriate TR-ORF expression plasmid with EcoRI/XhoI and cloning
into the EcoRI/XhoI sites of the pBAC-CMV-PolyA vector.
[0346] Following restriction mapping and/or DNA sequencing, the
following specific examples of pBAC-TR-ORF shuttle vectors are
produced:
[0347] pBAC-TR.sub.plp-EYFP, which contains the plp TR sequence and
the EYFP ORF;
[0348] pBAC-TR.sub.dm-EYFP, which includes the dm20 TR element and
the EYFP ORF;
[0349] pBAC-TR.sub.plp-fLuc, which includes the plp TR element and
the firefly Luciferase ORF;
[0350] pBAC-TR.sub.dm-fLuc, which contains the dm20 TR sequence and
the fLUC ORF;
[0351] pBAC-TR.sub.plp-TK, which contains the plp TR element and
the HSV-1 TK ORF;
[0352] pBAC-TR.sub.dm-TK, which contains the dm20 TR element and
the HSV-1 TK ORF;
[0353] pBAC-TR.sub.plp-TKsr39, which contains the plp TR sequence
and the sr39 derivative of the HSV-1 TK ORF;
[0354] pBAC-TR.sub.dm-TKsr39, which contains the dm20 TR element
and the sr39 derivative of the HSV-1 TK ORF;
[0355] pBAC-TR.sub.plp-CAT, which contains the plp TR element and
the bacterial chloramphenicol acetyltransferase (CAT) ORF;
[0356] pBAC-TR.sub.dm-CAT, which contains the dm20 TR sequence and
the CAT ORF;
[0357] pBAC-TR.sub.plp-LacZ, which includes the plp TR sequence and
the bacterial LacZ ORF; and
[0358] pBAC-TR.sub.dm-LacZ, which includes the dm20 TR element and
the LacZ ORF.
[0359] The methods for producing infectious rBAC virions are well
known in the art. Briefly, the process can be described as outlined
below. Recovery of recombinant baculovirus particles was
accomplished by transfection of Spodoptera frugiperda (Sf9) cells
with the pBAC-TR shuttle vector and BacVector-2000 (or
BacVector-3000) Triple Cut Virus DNA (Novagen). Genetic
recombination results in insertion of the CMV-TR cassette into the
baculovirus genome and packaging into BAC virions. In this example,
500 ng of shuttle vector was mixed with 100 ng of BacVector Triple
Cut Virus DNA and transfected into 500,000 Sf-9 cells for 1 hour
using the Insect GeneJuice Transfection Reagent (Novagen). Cells
were washed in serum free BacVector Insect Cell Medium and overlaid
with complete medium (containing 5% fetal bovine serum) for 4-5
days. At this time, the cells were collected in the media overlay
and pelleted by centrifugation at 2000 rpm for 10 min. This primary
viral media stock was transferred to sterile tubes and stored at 4
C. High titer rBAC viral stocks (termed secondary or tertiary
stocks) were prepared by low titer infections of Sf-9 cells
(infectivity of 0.1 pfu/cell) using the media overlay procedure
described above. rBAC titers were determined by standard plaque
overlay techniques. Restriction mapping of rBAC viral DNA
preparations was used to verify viral integrity and
composition.
[0360] Specific examples of rBAC-TR-ORF viruses that can be
generated in this manner include:
[0361] rBAC-TR.sub.plp-EYFP, which includes the plp TR element
operatively linked to the EYFP ORF;
[0362] rBAC-TR.sub.dm-EYFP, which includes the dm20 TR sequence
linked to the EYFP ORF;
[0363] rBAC-TR.sub.plp-fLuc, which includes the plp TR element and
the firefly Luciferase ORF;
[0364] rBAC-TR.sub.dm-fLuc, which contains the dm20 TR sequence and
the fLUC ORF;
[0365] rBAC-TR.sub.plp-TK, which contains the plp TR element and
the HSV-1 TK ORF;
[0366] rBAC-TR.sub.dm-TK, which contains the dm20 TR element and
the HSV-1 TK ORF;
[0367] rBAC-TR.sub.plp-TKsr39, which contains the plp TR sequence
and the sr39 derivative of the HSV-1 TK ORF;
[0368] rBAC-TR.sub.dm-TKsr39, which contains the dm20 TR element
and the sr39 derivative of the HSV-1 TK ORF;
[0369] rBAC-TR.sub.plp-CAT, which contains the plp TR element and
the CAT ORF;
[0370] rBAC-TR.sub.dm-CAT, which contains the dm20 TR sequence and
the CAT ORF;
[0371] rBAC-TR.sub.plp-LacZ, which includes the plp TR sequence and
the bacterial LacZ ORF;
[0372] and rBAC-TR.sub.dm-LacZ, which includes the dm20 TR element
and the LacZ ORF.
Example IV
Preparation of Mammalian Cells Stably Expressing the TR-ORF
Cassettes
A. Cell Culture
[0373] All mammalian cells are maintained at 37.degree. C., 5%
CO.sub.2 in complete medium which is Dulbecco's Modified Eagle
Medium (DMEM) (Invitrogen Life Technologies), supplemented with 10%
fetal bovine serum (Hyclone), 3.7 g/L sodium bicarbonate, and 30-50
mg/L gentamicin sulfate (Invitrogen Life Technologies).
B. Transfection and Isolation of Stably Transformed Mammalian
Cells
[0374] Mammalian transfections are performed using the Calcium
Phosphate transfection procedure (using reagents such as the
Profection.RTM. Mammalian Transfection System, Promega) or the
nonlipidic Transfectol Transfection Reagent (Continental Lab
Products) as described by the vendor. In this example, HEK293 cells
are transfected with various monocistronic pTR-ORF vectors, which
include the pTR.sub.plp-EYFP, pTR.sub.dm-EYFP, pTR.sub.plp-fLuc,
pTR.sub.dm-fLuc, pTR.sub.plp-TK, pTR.sub.dm-TK, pTR.sub.plp-CAT,
pTR.sub.dm-CAT, pTR.sub.plp-LacZ, and pTR.sub.dm-LacZ vectors. In
related efforts, the bicistronic vectors pfLuc-TR.sub.plp-EYFP,
pfLuc-TR.sub.dm-EYFP, pcuLf-TR.sub.plp-EYFP and
pcuLf-TR.sub.dm-EYFP are introduced into HEK293 cells.
[0375] Prior to transfection, mammalian cells are grown to 50-70%
confluence and fed 1-3 hrs prior to addition of the
DNA/transfection reagent mixture. A standard transfection assay
contains 15 .mu.g of plasmid DNA. Each DNA/transfection reagent
mixture is incubated with cells overnight. At this time, the
culture medium is replaced, incubated for another 24 hr, and G418
selective DMEM medium (500 .mu.g/mL) applied about 48 hrs post
transfection. The selection medium is changed every second day for
2-3 weeks, during which the majority of cells detach and G418
resistant "primary" colonies emerge.
[0376] Depending upon the number and density of colonies, surviving
cells are grown for 3-5 days in G418-free medium prior to pool
isolation or colony subcloning. Once colonies reach an appropriate
size, each plate is examined by phase contrast or fluorescence
microscopy and colonies marked for subcloning. Flame sterilized
cloning rings are placed around the colonies with a light coating
of grease and the cells removed by treatment with trypsin-EDTA
(Invitrogen). After passage into 24-well trays, subclones are fed
24-48 hrs after plating and grown until 80% confluent.
Alternatively, all of the "primary" colonies on a selection plate
are collected together in one sample, transferred to 100 mm dish or
a T-75 flask, fed 24 hrs after plating, and grown until 80%
confluent. This collection of colonies is termed a stable
cellular"pool".
[0377] In some situations, stable colonies are prepared from pooled
samples. For this effort, cells are diluted and replated prior to
colony subcloning. Cell pools are diluted at ratios ranging from
1:2500 to 1:5000, passaged onto 100 mm dishes, and allowed to grow
into colonies (about 1 week). The resultant colonies are then
processed as described for the original selection plates.
[0378] Each cellular isolate is assayed using one or more of the
cytotoxicity assays described in Examples V-VIII to verify
expression and selective translation of the TR-ORF mRNA in stressed
or dying cells. Candidate cellular resources, whether subclones or
pools, are frozen for storage. For freezing, cells are grown to 90%
confluence, treated with trypsin-EDTA (1 min, RT), collected in 2
mL freezing medium (90% fetal bovine serum, 10% DMSO) per 100 mm
dish, and transferred to cryotubes (1 ml cells per tube). Cryotubes
are placed in a -70/-80 C freezer in a slow freeze container for
16-24 hr, then submerged in liquid nitrogen for long-term
storage.
[0379] For this example, stable HEK293 cell lines are prepared from
colonies isolated after transfection of the pTR.sub.plp-EYFP,
pTR.sub.dm-EYFP, pTR.sub.plp-fLuc, and pTR.sub.dm-fLuc plasmids.
Alternatively, HEK293 pools are isolated after transfection with
the pTR.sub.plp-TK, pTR.sub.dm-TK, pTR.sub.plp-CAT, pTR.sub.dm-CAT,
pTR.sub.plp-LacZ, and pTR.sub.dm-LacZ expression vectors, as well
as the bicistronic pfLuc-TR.sub.plp-EYFP, pfLuc-TR.sub.dm-EYFP,
pcuLf-TR.sub.plp-EYFP and pcuLf-TR.sub.dm-EYFP plasmids. Each
cellular isolate constitutively expresses a unique monocistronic
TR-ORF or bicistronic ORF2-TR-ORF1 mRNA from the CMV IE
promoter/enhancer and selectively translated the ORF operably
linked to the TR element in stressed or dying cells as determined
by one of the cytotoxicity assays described below.
Example V
Procedure for Cytotoxicity Assay Using Western Blot Analysis
A. Western Blot Analysis of Mammalian Cells Expressing a
Monocistronic TR Expression Cassette
[0380] For this example, expression and regulation of the
TR.sub.plp-EYFP, TR.sub.dm-EYFP, TR.sub.plp-fLUC, or TR.sub.dm-fLUC
expression cassettes in stably transformed HEK293 cells are
validated using Western blot analysis. Cell lines or pools are
grown in six-well trays or 60 mm dishes to about 60% confluence and
treated with complete medium supplemented with a toxic chemical.
For this example, the toxic agents were the proteasomal inhibitor
MG132 (50 micromolar) or the Calcium Ionophore A23187 (5-6.7
micromolar), which are shown to produce complete cell death in
HEK293 cells within 24 hr as determined by Trypan blue staining.
Control samples are treated with fresh medium. Treated and control
cells were removed by pipette, collected in media, pelleted for 5
min at 800 rpm (room temperature), and either stored at
-70/-80.degree. C. or immediately processed for total proteins.
[0381] For protein extraction, cells are resuspended in equal
volumes of Suspension Buffer (100 mM NaCl, 10 mM Tris-HCl [pH 7.6],
1 mM EDTA, 1 mg/mL aprotinin, 100 .mu.g/mL PMSF) and 2.times.SDS
Buffer (100 mM Tris-HCl [pH 6.8], 4% SDS, 20% glycerol, 200 mM
DTT). Frozen cells are thawed on ice prior to resuspension. The
extracts are homogenized by several passages through a syringe
fitted with a 26 G needle. Samples are subsequently incubated at RT
for 1-2 hr. Samples are transferred to -70/-80.degree. C. for
storage or applied to an SDS PAGE gel and examined by Western blot
analysis.
[0382] To insure equal protein loading levels on Western blots, 10
.mu.L of each protein extract are initially resolved by SDS PAGE,
fixed overnight in Preblot gel fixer (25% isopropanol, 10% acetic
acid), and stained in 0.05% Coomassie blue (0.05% brilliant blue R,
50% methanol, 10% acetic acid) for 20 min, RT. Gels are destained
in 10% acetic acid and dried under vacuum. Using the dried gels,
any necessary volume adjustment was made to samples prior to
Western blotting.
[0383] Western blot analysis is a well established technique in the
art and is summarized in the following. Following SDS PAGE gel
resolution and protein transfer to a solid membrane support by
electrophoresis, the membranes are dried overnight. Subsequent
Western analysis requires membrane rehydration, washing with a
protein solution (5% powdered milk or 3% BSA) to block stray
protein binding sites on the filter, incubation with an antibody
that recognizes the TR regulated ORF, and chemiluminescent
detection using a labeled secondary antibody. For the EYFP protein,
the primary antibody is an anti-GFP antibody (Molecular Probes;
1:500 to 1:1500 dilution). Similarly, fLUC protein is detected
using an anti-fLUC antibody (Sigma; 1:1000 dilution). After
incubation with a primary antibody and extensive washing
(1.times.PBS-T), proteins binding the primary antibody are detected
by incubation with a horseradish peroxidase (HRP) conjugated
anti-rabbit or anti-mouse antibody (Amersham; 1:5000 to 1:10000
dilution). Following incubation with the secondary antibody and
extensive washing, reactive proteins are detected with the ECL
reagent system (Amersham) as described by the vendor.
[0384] For this example, the results for HEK293 cell lines
expressing the TR.sub.plp-fLUC, TR.sub.dm-fLUC or fLUC mRNAs are
shown in FIG. 6. Following treatment with toxic levels of the
calcium ionophore A23187, highly significant increases in fLuc
protein levels are observed in the stressed and dying cells that
were not evident in untreated cultures. Individual protein bands
are quantitated by densitometry on a Beckman DU7400
spectrophotometer. Cell pools and lines treated with the calcium
ionophore A23187 exhibit increases in fLUC protein levels that
range from 160-800% of the protein levels detected in untreated
TR.sub.plp-fLUC/TR.sub.dm-fLUC cells. The average increase in fLuc
protein levels observed in stressed and dying HEK293
TR.sub.plp-fLuc expressing cells is 402.8% (n=5), which is similar
to the 524.3% (n=6) increase observed in calcium ionophore treated
HEK293 TR.sub.dm-fLuc cells. Furthermore, cells expressing the
TR-fLUC cassette exhibit fLUC protein levels in stressed and dying
cells that are as high as 110% of the protein levels produced by
cells expressing the cap-dependent CMV-fLuc sequence (FIG. 6).
B. Cytotoxicity Assays Using Cells that Stably Express a
Bicistronic TR Cassette
[0385] In this example, HEK293 cell pools expressing the
fLUC-TR.sub.plp-EYFP, fLUC-TR.sub.dm-EYFP, pcuLf-TR.sub.plp-EYFP or
pcuLf-TR.sub.dm-EYFP expression cassettes are treated with the
calcium ionophore A23187 and MG132 as above. Briefly, the
translation of both reporter proteins is assayed using the Western
blot cytotoxicity procedure as described above. Translation of the
upstream reporter protein (fLUC) in the sense vectors reflect
cap-dependent translation, while the level of the downstream
reporter protein (EYFP) correlate with cap-independent (i.e. TR
regulated) translation. Although cap-dependent translation may not
be measured in the antisense constructs, cap-independent
translation can be detected from the ORF operatively linked to the
TR element. The primary and secondary antibodies used in these
assays and the assay procedure are as above.
[0386] Following the induction of cell stress and death,
TR-mediated translation as measured by increases in EYFP protein
levels is detected in all cell pools expressing the TR-expression
cassette. EYFP protein levels increase to 130-750% of untreated
cellular levels. Although the anti-EYFP and anti-fLuc antibodies
may not be applied to a single blot, cap-independent translation is
more efficient from the monocistronic TR cassette than the
bicistronic orientation (FIG. 4). It is also evident that
cap-independent TR-mediated translation is not inhibited by
insertion of the antisense fLuc ORF upstream of the TR expression
cassette.
Example VI
Procedure for Cytotoxicity Assay Using Fluorescent Microscopy
[0387] Fluorescence of single cells, tissues or cell suspensions
can be detected and quantitated by several means such as visual
inspection under a microscope, automated or semiautomated
fluorescence imaging, flow cytometry, fluorescence spectroscopy in
a fluorometer or in a microplate reader, using an appropriate
filter set. For this example, visual inspection under a microscope
(Nikon TE2000-S) was used to validate TR translational regulation
in stressed and dying HEK293 cells expressing the TR.sub.plp-EYFP,
TR.sub.dm-EYFP, TR.sub.plp-fLUC and TR.sub.dm-fLUC mRNAs.
A. Direct Visualization of TR-Mediated EYFP Translation During a
Cytotoxic Event
[0388] In this study, HEK293 cells transformed with the CMV-EYFP,
TR.sub.plp-EYFP or TR.sub.dm-EYFP expression cassettes are directly
visualized following translation of the spontaneously fluorescent
EYFP protein. One day prior to toxin addition, 200,000 cells
(HEK293, HEK293 CMV-EYFP, HEK293 TR.sub.plp-EYFP or HEK293
TR.sub.dm-EYFP) are plated into a six-well tray containing a flame
sterilized glass slide. Cells are cultured for 24 hr in complete
DMEM medium. Fresh medium supplemented with 5 micromolar calcium
ionophore A23187 is applied and slides were collected at 0 hr, 2
hr, 4 hr, 6 hr, 9 hr, 10 hr, 11 hr and 25 hr. Slides were fixed for
10 min in 4% paraformaldehyde, washed extensively with 1.times.PBS
and mounted for fluorescence microscopy.
[0389] Fluorescent cell counts are performed on the 6 hr and 10 hr
timepoints using an EYFP-selective filter set (Nikon) and are shown
in FIG. 7. In untreated cell cultures, the frequency of
EYFP-positive cells range from 1.5-2.5%, which is consistent with
the frequency of nonviable cells in these cultures, as determined
by Trypan blue staining. Following toxin treatment, EYFP
translation from the TR-EYFP cassette, defined as the number of
fluorescent cells in at least 5 random sections containing no fewer
than 500 cells total, increased as a function of time. In contrast,
no significant change in the number of fluorescent cells is
detected in the HEK293 or HEK CMV-EYFP cells. The frequency of
fluorescent cells in the TR.sub.plp-EYFP expressing cells rises by
1000-2300% of untreated control cultures. Similar increases are
observed in TR.sub.dm-EYFP expressing cells with cell numbers
rising 600-1465% of control cultures (FIG. 7).
B. Direct Visualization of TR-Mediated fLUC Translation During a
Cytotoxic Event
[0390] To examine TR-mediated translation from the TR-fLUC
cassettes, the translated fLUC protein requires immunolabeling with
fluorescent antibodies. In this study, HEK293 cells transformed
with the CMV-fLUC, TR.sub.plp-fLUC or TR.sub.dm-fLUC expression
cassettes are visualized following immunodetection of the fLUC
protein using a primary anti-fLUC antibody (Sigma) and a
species-specific rhodamine-labeled secondary antibody (Kirkegaard
and Perry Laboratories, Inc.). DAPI labeling of nuclear DNA is a
well known procedure in the art and was used to label nuclei.
[0391] Forty hours prior to toxin exposure, 300,000 cells (HEK293,
HEK293 CMV-fLUC, HEK293 TR.sub.plp-fLUC subclone #3, HEK293
TR.sub.plp-fLUC subclone #17, HEK293 TR.sub.dm-fLUC subclone #12,
or HEK293 TR.sub.dm-fLUC subclone #45) were plated on flame
sterilized glass coverslips in a 12-well tray. Cells are cultured
in complete DMEM medium. Fresh medium supplemented with 6.7
micromolar calcium ionophore A23187 is applied and slides collected
at 12 hr. Coverslips are fixed in 4% paraformaldehyde (10 min, RT),
washed extensively with 1.times.PBS, permeabilized in 100% methanol
(2 min, RT), washed with 1.times.PBS, blocked in 3% BSA (5 min, RT)
and incubated with a primary anti-fLUC antibody (Sigma; 1:500
dilution) for 1 hr at RT. Following the primary antibody staining,
the coverslips are washed extensively with 1.times.PBS and
incubated with a rhodamine-labeled anti-rabbit secondary antibody
(Kirkegaard and Perry Laboratories; 1:100 to 1:200 dilution) for 1
hr at RT. After this, the coverslips are washed in 1.times.PBS,
labeled with DAPI (30 sec, RT), washed in 1.times.PBS, and mounted
for fluorescence microscopy.
[0392] Fluorescent cell counts are performed using
rhodamine-selective and DAPI filter sets (Nikon) and are shown in
FIG. 8. In untreated cell cultures, the frequency of fLUC-positive
cells ranged from 2.4-7.2%, which is 1-3 times higher than the
normal frequency of nonviable cells, as determined by Trypan blue
staining. Subsequent visual inspection of DAPI stained nuclei
establishes that the anti-fLUC antibody selectively cross-reacts
with telophase cells, which results in elevated estimates of cell
stress in untreated cultures. Following toxin treatment, the
frequency of fLUC positive cells in the HEK293 TR.sub.plp-fLUC
subclone #3 and #17 cell lines increased by 1150% and 2245%
compared to the number of positive cells in untreated samples,
respectively. Similarly, the number of fLUC positive cells in the
HEK293 TR.sub.dm-fLUC subclone #12 and #45 cell lines increased by
3640% and 1440%, respectively.
Example VII
Procedure for Cytotoxicity Assay Using a Microplate Reader
[0393] Microplate readers are designed to scan, analyze and obtain
numerical results using absorbance, fluorescence and luminescence
on high density sample arrays. In this example, a microplate reader
was used to measure fluorescent and luminescent marker proteins
translated from the TR-ORF cassette.
A. Assay Procedures for the Microplate Reader
[0394] For this example, HEK293 cells expressing the
TR.sub.plp-EYFP, TR.sub.dm-EYFP, TR.sub.plp-fLUC and TR.sub.dm-fLUC
expression cassettes are evaluated using a microplate reader. To
quantitate the TR translational response, 24,000 cells are plated
in a 96-well microtiter plates and allowed to grow for about 40 hr
to achieve the proper cell density prior to incubation with a toxic
agent. Each well is cultured with complete DMEM medium containing
either no toxin or a defined concentration of toxin and incubated
at 37 C for specific time periods. At that time, fluorescence or
chemiluminescence on a FLUOstar Optima (BMG Labtech) microplate
reader is used measure TR-ORF response.
[0395] Direct detection of the spontaneously fluorescent EYFP
protein fluorophore is achieved by excitation of samples in black
optical bottom 96-well trays with a fluorescein filter (545 nm) or
a YFP filter (YFPex) and emission measured at 590 nm or 544 nm
using gains of 1000-2000. To reduce the background fluorescence
associated with media components, the trays are centrifuged at 1200
rpm, RT for 3 min to collect any floating cells, and the media
replaced with 200 microliters of 1.times.PBS prior to detection.
For the fLUC protein, protein activity is quantified by
luminescence produced during live cell or lysed cell luciferase
assays. For a live cell assay, cells are cultured as above, but in
white optical bottom 96-well trays. After toxin incubation, the
96-well plates are centrifuged at 1200 rpm for 3 min to pellet
detached cells. Luminescence is developed by injection of 20
microliters of D-luciferin solution dissolved in sodium
citrate/DMSO assay buffer (50% 100 mM sodium citrate, pH5.2, 50%
DMSO, 6.7 mM ATP and 3.35 mM D-luciferin) and a 45 sec incubation
to allow for cell penetration. Luminescence was detected using lens
filter at gains of 2000-3000.
[0396] For a lysed cell assay, cells are cultured as above, but in
regular flat bottom 96-well trays. After toxin incubation, the
96-well plates are centrifuged as above. The media is removed,
replaced with 50 microliters of Cell Lysis Buffer (25 mM
Tris-phosphate (pH7.8), 10% glycerol, 1% TritonX-100, 1 mg/ml BSA,
2 mM EGTA and 2 mM DTT) and incubated for 10 min at RT. Cell lysis
is verified using a phase contrast microscope, and the lysates
transferred to a white bottom 96-well tray. Luminescence is
developed by injection of 5 microliters of D-luciferin solution
dissolved in Reaction Buffer (25 mM Glycylglycine (pH 7.8), 15 mM
MgSO4, 4 mM EDTA, 15 mM Potassium phosphate, 1 mM DTT, 1 mM
Coenzyme A, 6.7 mM ATP and 3.35 mM D-luciferin). After 4 sec with
shaking, luminescence values were measured using the lens filter at
gains of 2000-3000.
B. Measuring a Cytotoxic Event at Fixed Time and Toxin
Concentration
[0397] Assays based upon fixed time and concentration of a
cytotoxic agent are used to identify cellular colonies/pools that
exhibit TR-mediated translation. In this example, HEK293 colonies
transformed with the CMV-EYFP, TR.sub.plp-EYFP, TR.sub.dm-EYFP,
CMV-fLUC, TR.sub.plp-fLUC or TR.sub.dm-fLUC expression cassettes
are screened for TR-specific translational responses following
incubation for 12 hr with DMEM medium supplemented with 6.7
micromolar calcium ionophore A23187 (a toxic concentration defined
by Trypan blue staining). Colonies are initially propagated in
60-100 mm dishes until 70-80% confluent, transferred to 96-well
trays and assayed using cytotoxic medium as described above.
[0398] Characteristic results for an independent set of HEK293
TR.sub.plp-EYFP colonies (FIG. 9), as well as select HEK293
TR.sub.plp-fLUC and TR.sub.dm-fLUC colonies (FIG. 9, live cell
assay) are shown. As examples of this screening assay, HEK293
TR.sub.plp-EYFP subclone #41 and HEK293 TR.sub.plp-fLUC subclone #3
displayed significant TR responses and are selected for subsequent
Western blot and microscopy validation.
C. Measuring a Cytotoxic Event as a Function of Toxin Concentration
(Dose Response)
[0399] Dose response assays are necessary to define toxic
concentrations of candidate cytotoxic agents. To establish a
TR-mediated cellular response and identify a TR effective dose,
HEK293 cells transformed with the TR.sub.plp-fLUC (subclone #3) and
TR.sub.dm-fLUC (subclone #45) expression cassettes are subjected to
a range of subtoxic to toxic concentrations of the calcium
ionophore A23187 (0, 1 nM, 10 nM, 100 nM, 2 .mu.M, 404, 604, 804,
1004) for 12 hr and analyzed as described above using a lysed cell
luciferase assay (FIG. 10).
[0400] At the 12 hr time point, using the raw luciferase numbers
(FIG. 10, panels A & B), TR-mediated translation is initially
detected from each cassette at 1 .mu.M with a peak in fLUC values
at 6 .mu.M. Lower subtoxic doses produce no apparent TR-specific
translational activity and the 8-1004 toxin concentrations exhibit
a decline in fLuc activity. However, correlating cap-dependent to
cap-independent luciferase values produce different results.
Adjusting the mean of each luciferase value to the mean of the 0 hr
timepoint (expressed as 100%) produces a dose response curve
similar to the raw data results (FIG. 10, panel C). In contrast,
adjusting the cap-independent fLuc values generated by the TR
expression cassette for the significant decline in cap-dependent
ribosomal activity exhibited by the HEK293 CMV-fLUC control cells
results in significantly higher apparent translation rates and a
less significant decline in cap-independent translation even at the
highest tested toxin dose.
D. Measuring a Cytotoxic Event as a Function of Time (Temporal
Response)
[0401] Temporal response assays are used to define the timing of
TR-regulated translation during incubation in toxic levels of a
cytotoxic agent. HEK293 TR.sub.plp-fLUC subclone #3 and HEK293
TR.sub.dm-fLUC subclone #45 are cultured in 6.7 micromolar calcium
ionophore A23187 for various times (0 hr, 1.5 hr, 3 hr, 4.5 hr, 6
hr, 7.5 hr, 9 hr, 10.5 hr, 12 hr and 13.5 hr) and analyzed as
described above using a lysed cell luciferase assay (FIGS.
11A-11D).
[0402] By 1.5 hr post-incubation, using the raw luciferase numbers,
significant TR-specific translation can be detected in each TR
transformed cell line (FIG. 11, panels A & B). After 1.5 hr,
translational activity increases linearly up to 9 hr
post-incubation, where translation exhibits an apparent plateau or
slightly lowered activity. However, as before, correlating
cap-dependent to cap-independent translation produces different
graphs. Adjusting the mean of each luciferase value to the mean of
the 0 hr timepoint (expressed as 100%) produces a dose response
curve similar to the raw data results (FIG. 11, panel C). In
contrast, adjusting the cap-independent fLuc values generated by
the TR expression cassette for the significant decrease in
cap-dependent translation observed in HEK293 CMV-fLUC cells results
in significantly higher apparent translational activity and a
linear increase in translational activity to 12 hr before an
apparent translational decline.
Example VIII
Transduction of the TR Expression Cassette into Mammalian Cells
Using Recombinant Viruses and Procedures for Assaying TR-Mediated
Translation During a Cytotoxic Event
[0403] A. Method for Transducing Mammalian Cells with rAAV-TR-ORF
Virions
[0404] This example is to show delivery of the TR.sub.plp-EYFP and
TR.sub.dm-EYFP cassettes to HEK293 and HT1080 cells using rAAV
transduction. HEK293 or HT1080 cells are plated on flame sterilized
glass slides and grown until 60-70% confluent (1-2 days), washed
with L-DMEM (DMEM containing 2% fetal bovine serum), and infected
with rAAV for 2 hr. The medium is replaced with DMEM, 10% FBS and
the cells are incubated for 24 hours. Infected cells are treated
with DMEM, 10% FBS supplemented with toxic levels of MG132 (2504)
for 24 hr prior to direct microscopic or Western analysis as
described above. Cells transduced with rAAV virions exhibit
selective translation of the fluorescent EYFP protein compared to
uninfected control cells.
B. Assaying a Cytotoxic Event Using rAAV Gene Delivery
[0405] HT1080 cells are plated in 6-well trays and allowed to grow
until 70-80% confluent. These cells are transduced with
rAAV-TR.sub.dm-EYFP virions (multiplicity of infection of 1-10
pfu/cell) for 24 hr and then treated with medium containing
compounds known to induce apoptosis/cell death. For this example,
DMEM containing 5004 MG132, 204 thapsigargin, 1 .mu.g/ml
actinomycin D, 20 .mu.g/ml cycloheximide, 1 mM
dibutrylcyclic-adenosine monophosphate (dbcAMP), 200 .mu.g/ml G418,
5 .mu.M calcium ionophore A23187, 10 .mu.g/ml mitomycin D, 5%
methanol or 10% ethanol are separately or in combination added to
transduced HT1080 cells for 24 hr. At this time, untreated and
treated cells are processed and examined by Western blot analysis,
direct microscopic analysis or plate reader analysis as described
above.
[0406] It is expected that the TR.sub.dm-EYFP cassette will be
translationally active in transduced HT1080 cells cultured in a
cytotoxic medium.
C. Transduction of Mammalian Cells with the TR-ORF Cassette Using
rBAC Virions
[0407] This example is to show delivery of the TR.sub.plp-EYFP and
TR.sub.dm-EYFP cassettes to HEK293 cells using rBAC transduction.
HEK293 cells are plated on flame sterilized glass slides and grown
until 60-70% confluent (1-2 days), washed with serum-free DMEM, and
infected with rBAC for 1-2 hr. The medium is replaced with DMEM,
10% FBS and the cells incubated for 24 hours. Infected cells were
treated with DMEM, 10% FBS supplemented with 5 .mu.M calcium
ionophore A23187 for 24 hr prior to direct microscopic examination.
Cells transduced with rBAC virions exhibit selective translation of
the fluorescent EYFP protein compared to uninfected control
cells.
D. Assaying a Cytotoxic Event Using a rBAC Gene Delivery System
[0408] HT1080 cells are plated in 6-well trays and allowed to grow
until 70-80% confluent. Two sets of HT1080 cells are transduced
with rBAC-TR.sub.dm-EYFP virions (multiplicity of infection of 10
pfu/cell and 25 pfu/cell) for 24 hr and then treated with medium
containing 5 .mu.M calcium ionophore A23187 for 13.5 hr and 23 hr.
At this time, untreated and treated cells are processed and
examined by fluorescence microscopic analysis as described
above.
[0409] As shown in FIG. 12, the TR.sub.dm-EYFP cassette is
translationally active in transduced HT1080 cells cultured in
cytotoxic medium. Significant increases in EYFP positive cells are
observed at 13.5 hr (3344% at 10 pfu/cell and 4925% at 25 pfu/cell)
and 23 hr (725% at 10 pfu/cell and 875% at 25 pfu/cell) compared to
infected (10 pfu/ml) but untreated cell samples. Total cell counts
show that increased cell detachment at 23 hr produces the apparent
decrease in the total number of positive cells, as the number of
attached cells at 23 hr had decreased by more than 50%.
Example IX
Procedure for Inducing a Cytotoxic Event Using a Pro-Drug ORF
Expressed from the TR Expression Cassette
[0410] Due to normal cytotoxic events (i.e. cell contact
inhibition, anoikis, etc), mammalian cell cultures generally
contain 1-10% nonviable cells. If these cells transcribe a pro-drug
ORF operatively linked to the TR cassette, the stressed or dying
cells should selectively translate the pro-drug ORF and sensitize
these stressed cells to pro-drugs that do not normally affect the
parental cell type.
A. Inducing a Cytotoxic Event by Varying Toxin Concentration and
Variable Time
[0411] In this example, HEK293, HEK CMV-EYFP, and HEK
TR.sub.plp-TKsr39 cells are treated with various amounts of the
pro-drug ganciclovir and tested for cell death after 4 days
incubation using the Trypan blue staining assay. Cells are plated
into 6-well trays, grown to 70-80% confluence, and treated with
DMEM, 10% FBS supplemented with ganciclovir (0, 10 nM, 100 nM, 504
or 1004) for 4 days. Viable cell counts are performed at 3 days and
4 days post-incubation (FIG. 13).
[0412] The HEK293 or HEK CMV-EYFP cells do not exhibit any
significant increase in cell death at any concentration of
ganciclovir after 3 or 4 days of culture. In these cultures,
98-100% of the cells remain Trypan blue viable during the entire
treatment period. In contrast, the HEK293 TR.sub.plp-TKsr39 cells
exhibit some microscopic cell death within 3 days as exemplified by
detached cells with deformed cellular morphology, condensed nuclei
and Trypan blue reactivity. At this time, cell viability falls from
98.5% in untreated cultures to a low of 86% cell viability in 1004
ganciclovir medium. After 3 days, the 10 nM culture does not
exhibit any decrease in cell viability compared to untreated cells,
although a slight decrease was detected in the 100 nM sample.
[0413] After an additional 24 hr, microscopic analysis establishes
that a significant fraction of HEK293 TR.sub.plp-TKsr39 cell
cultures treated with ganciclovir doses of greater than 10 nM are
detached and apparently dead (FIG. 13). This is confirmed by the
Trypan blue cell viability assay which detects a decline in cell
viability at all ganciclovir doses that ranged from 99.7% cell
viability in 10 nM cultures to 33.7% viability in 1004 ganciclovir
medium. This example establishes that translation from the TR
cassette in stressed or dying cells can be used to selectively
synthesize a pro-drug protein which can enhance cell death
following pro-drug application. The use of the TKsr39 protein
provides an example of bystander killing which is dependent upon
selective translation for initiation and underscores the use of
selective translation in supplemental gene therapy.
[0414] Having described the technology in detail, it will be
apparent that modifications and variations are possible without
departing the scope of the technology defined in the appended
claims. Furthermore, it should be appreciated that all examples in
the present disclosure, while illustrating the technology, are
provided as non-limiting examples and are, therefore, not to be
taken as limiting the various aspects of the technology so
illustrated.
Sequence CWU 1
1
631726DNAArtificial SequenceMurine derived TRdm 1ttgagtgagt
tagagtagtg agctagttgt ctggtagggg ccccctttgc ttccctggtg 60gccactggat
tgtgtttctt tggagtggca ctgttctgtg gatgtggaca tgaagctctc
120actggtacag aaaagctaat tgagacctat ttctccaaaa actaccagga
ctatgagtat 180ctcattaatg tgattcatgc tttccagtat gtcatctatg
gaactgcctc tttcttcttc 240ctttatgggg ccctcctgct ggctgagggc
ttctacacca ccggcgctgt caggcagatc 300tttggcgact acaagaccac
catctgcggc aagggcctga gcgcaacgtt tgtgggcatc 360acctatgccc
tgactgttgt atggctcctg gtgtttgcct gctcggctgt acctgtgtac
420atttacttca atacctggac cacctgtcag tctattgcct tccctagcaa
gacctctgcc 480agtataggca gtctctgcgc tgatgccaga ttgtatggtg
ttctcccatg gaatgctttc 540cctggcaagg tttgtggctc caaccttctg
tccatctgca aaacagctga gttccaattg 600accttccacc tgtttattgc
tgcgtttgtg ggtgctgcgg ccacactagt ttccctgctc 660accttcatga
ttgctgccac ttacaacttc gccgtcctta aactcatggg ccgaggcacc 720aagttc
7262831DNAArtificial SequenceMurine derived TRplp 2ttgagtgagt
tagagtagtg agctagttgt ctggtagggg ccccctttgc ttccctggtg 60gccactggat
tgtgtttctt tggagtggca ctgttctgtg gatgtggaca tgaagctctc
120actggtacag aaaagctaat tgagacctat ttctccaaaa actaccagga
ctatgagtat 180ctcattaatg tgattcatgc tttccagtat gtcatctatg
gaactgcctc tttcttcttc 240ctttatgggg ccctcctgct ggctgagggc
ttctacacca ccggcgctgt caggcagatc 300tttggcgact acaagaccac
catctgcggc aagggcctga gcgcaacggt aacagggggc 360cagaagggga
ggggttccag aggccaacat caagctcatt ctttggagcg ggtgtgtcat
420tgtttgggaa aatggctagg acatcccgac aagtttgtgg gcatcaccta
tgccctgact 480gttgtatggc tcctggtgtt tgcctgctcg gctgtacctg
tgtacattta cttcaatacc 540tggaccacct gtcagtctat tgccttccct
agcaagacct ctgccagtat aggcagtctc 600tgcgctgatg ccagattgta
tggtgttctc ccatggaatg ctttccctgg caaggtttgt 660ggctccaacc
ttctgtccat ctgcaaaaca gctgagttcc aattgacctt ccacctgttt
720attgctgcgt ttgtgggtgc tgcggccaca ctagtttccc tgctcacctt
catgattgct 780gccacttaca acttcgccgt ccttaaactc atgggccgag
gcaccaagtt c 83133223DNAArtificial sequenceMonocistronic TRdm
luciferase expression cassette 3tagttattaa tagtaatcaa ttacggggtc
attagttcat agcccatata tggagttccg 60cgttacataa cttacggtaa atggcccgcc
tggctgaccg cccaacgacc cccgcccatt 120gacgtcaata atgacgtatg
ttcccatagt aacgccaata gggactttcc attgacgtca 180atgggtggag
tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc
240aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
atgcccagta 300catgacctta tgggactttc ctacttggca gtacatctac
gtattagtca tcgctattac 360catggtgatg cggttttggc agtacatcaa
tgggcgtgga tagcggtttg actcacgggg 420atttccaagt ctccacccca
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480ggactttcca
aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt
540acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc
gctagcgcta 600ccggactcag atcctcagag tgccgaattc ttgagtgagt
tagagtagtg agctagttgt 660ctggtagggg ccccctttgc ttccctggtg
gccactggat tgtgtttctt tggagtggca 720ctgttctgtg gatgtggaca
tgaagctctc actggtacag aaaagctaat tgagacctat 780ttctccaaaa
actaccagga ctatgagtat ctcattaatg tgattcatgc tttccagtat
840gtcatctatg gaactgcctc tttcttcttc ctttatgggg ccctcctgct
ggctgagggc 900ttctacacca ccggcgctgt caggcagatc tttggcgact
acaagaccac catctgcggc 960aagggcctga gcgcaacgtt tgtgggcatc
acctatgccc tgactgttgt atggctcctg 1020gtgtttgcct gctcggctgt
acctgtgtac atttacttca atacctggac cacctgtcag 1080tctattgcct
tccctagcaa gacctctgcc agtataggca gtctctgcgc tgatgccaga
1140ttgtatggtg ttctcccatg gaatgctttc cctggcaagg tttgtggctc
caaccttctg 1200tccatctgca aaacagctga gttccaattg accttccacc
tgtttattgc tgcgtttgtg 1260ggtgctgcgg ccacactagt ttccctgctc
accttcatga ttgctgccac ttacaacttc 1320gccgtcctta aactcatggg
ccgaggcacc aagttcgaaa gcttcgccac catggaagac 1380gccaaaaaca
taaagaaagg cccggcgcca ttctatccgc tggaagatgg aaccgctgga
1440gagcaactgc ataaggctat gaagagatac gccctggttc ctggaacaat
tgcttttaca 1500gatgcacata tcgaggtgga catcacttac gctgagtact
tcgaaatgtc cgttcggttg 1560gcagaagcta tgaaacgata tgggctgaat
acaaatcaca gaatcgtcgt atgcagtgaa 1620aactctcttc aattctttat
gccggtgttg ggcgcgttat ttatcggagt tgcagttgcg 1680cccgcgaacg
acatttataa tgaacgtgaa ttgctcaaca gtatgggcat ttcgcagcct
1740accgtggtgt tcgtttccaa aaaggggttg caaaaaattt tgaacgtgca
aaaaaagctc 1800ccaatcatcc aaaaaattat tatcatggat tctaaaacgg
attaccaggg atttcagtcg 1860atgtacacgt tcgtcacatc tcatctacct
cccggtttta atgaatacga ttttgtgcca 1920gagtccttcg atagggacaa
gacaattgca ctgatcatga actcctctgg atctactggt 1980ctgcctaaag
gtgtcgctct gcctcataga actgcctgcg tgagattctc gcatgccaga
2040gatcctattt ttggcaatca aatcattccg gatactgcga ttttaagtgt
tgttccattc 2100catcacggtt ttggaatgtt tactacactc ggatatttga
tatgtggatt tcgagtcgtc 2160ttaatgtata gatttgaaga agagctgttt
ctgaggagcc ttcaggatta caagattcaa 2220agtgcgctgc tggtgccaac
cctattctcc ttcttcgcca aaagcactct gattgacaaa 2280tacgatttat
ctaatttaca cgaaattgct tctggtggcg ctcccctctc taaggaagtc
2340ggggaagcgg ttgccaagag gttccatctg ccaggtatca ggcaaggata
tgggctcact 2400gagactacat cagctattct gattacaccc gagggggatg
ataaaccggg cgcggtcggt 2460aaagttgttc cattttttga agcgaaggtt
gtggatctgg ataccgggaa aacgctgggc 2520gttaatcaaa gaggcgaact
gtgtgtgaga ggtcctatga ttatgtccgg ttatgtaaac 2580aatccggaag
cgaccaacgc cttgattgac aaggatggat ggctacattc tggagacata
2640gcttactggg acgaagacga acacttcttc atcgttgacc gcctgaagtc
tctgattaag 2700tacaaaggct atcaggtggc tcccgctgaa ttggaatcca
tcttgctcca acaccccaac 2760atcttcgacg caggtgtcgc aggtcttccc
gacgatgacg ccggtgaact tcccgccgcc 2820gttgttgttt tggagcacgg
aaagacgatg acggaaaaag agatcgtgga ttacgtcgcc 2880agtcaagtaa
caaccgcgaa aaagttgcgc ggaggagttg tgtttgtgga cgaagtaccg
2940aaaggtctta ccggaaaact cgacgcaaga aaaatcagag agatcctcat
aaaggccaag 3000aagggcggaa agatcgccgt gtaactcgag atctagatca
taatcagcca taccacattt 3060gtagaggttt tacttgcttt aaaaaacctc
ccacacctcc ccctgaacct gaaacataaa 3120atgaatgcaa ttgttgttgt
taacttgttt attgcagctt ataatggtta caaataaagc 3180aatagcatca
caaatttcac aaataaagca tttttttcac tgc 322343328DNAArtificial
SequenceMonocistronic TRplp luciferase expression cassette
4tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
60cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
120gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
attgacgtca 180atgggtggag tatttacggt aaactgccca cttggcagta
catcaagtgt atcatatgcc 240aagtacgccc cctattgacg tcaatgacgg
taaatggccc gcctggcatt atgcccagta 300catgacctta tgggactttc
ctacttggca gtacatctac gtattagtca tcgctattac 360catggtgatg
cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg
420atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
aaaatcaacg 480ggactttcca aaatgtcgta acaactccgc cccattgacg
caaatgggcg gtaggcgtgt 540acggtgggag gtctatataa gcagagctgg
tttagtgaac cgtcagatcc gctagcgcta 600ccggactcag atcctcagag
tgccgaattc ttgagtgagt tagagtagtg agctagttgt 660ctggtagggg
ccccctttgc ttccctggtg gccactggat tgtgtttctt tggagtggca
720ctgttctgtg gatgtggaca tgaagctctc actggtacag aaaagctaat
tgagacctat 780ttctccaaaa actaccagga ctatgagtat ctcattaatg
tgattcatgc tttccagtat 840gtcatctatg gaactgcctc tttcttcttc
ctttatgggg ccctcctgct ggctgagggc 900ttctacacca ccggcgctgt
caggcagatc tttggcgact acaagaccac catctgcggc 960aagggcctga
gcgcaacggt aacagggggc cagaagggga ggggttccag aggccaacat
1020caagctcatt ctttggagcg ggtgtgtcat tgtttgggaa aatggctagg
acatcccgac 1080aagtttgtgg gcatcaccta tgccctgact gttgtatggc
tcctggtgtt tgcctgctcg 1140gctgtacctg tgtacattta cttcaatacc
tggaccacct gtcagtctat tgccttccct 1200agcaagacct ctgccagtat
aggcagtctc tgcgctgatg ccagattgta tggtgttctc 1260ccatggaatg
ctttccctgg caaggtttgt ggctccaacc ttctgtccat ctgcaaaaca
1320gctgagttcc aattgacctt ccacctgttt attgctgcgt ttgtgggtgc
tgcggccaca 1380ctagtttccc tgctcacctt catgattgct gccacttaca
acttcgccgt ccttaaactc 1440atgggccgag gcaccaagtt cgaaagcttc
gccaccatgg aagacgccaa aaacataaag 1500aaaggcccgg cgccattcta
tccgctggaa gatggaaccg ctggagagca actgcataag 1560gctatgaaga
gatacgccct ggttcctgga acaattgctt ttacagatgc acatatcgag
1620gtggacatca cttacgctga gtacttcgaa atgtccgttc ggttggcaga
agctatgaaa 1680cgatatgggc tgaatacaaa tcacagaatc gtcgtatgca
gtgaaaactc tcttcaattc 1740tttatgccgg tgttgggcgc gttatttatc
ggagttgcag ttgcgcccgc gaacgacatt 1800tataatgaac gtgaattgct
caacagtatg ggcatttcgc agcctaccgt ggtgttcgtt 1860tccaaaaagg
ggttgcaaaa aattttgaac gtgcaaaaaa agctcccaat catccaaaaa
1920attattatca tggattctaa aacggattac cagggatttc agtcgatgta
cacgttcgtc 1980acatctcatc tacctcccgg ttttaatgaa tacgattttg
tgccagagtc cttcgatagg 2040gacaagacaa ttgcactgat catgaactcc
tctggatcta ctggtctgcc taaaggtgtc 2100gctctgcctc atagaactgc
ctgcgtgaga ttctcgcatg ccagagatcc tatttttggc 2160aatcaaatca
ttccggatac tgcgatttta agtgttgttc cattccatca cggttttgga
2220atgtttacta cactcggata tttgatatgt ggatttcgag tcgtcttaat
gtatagattt 2280gaagaagagc tgtttctgag gagccttcag gattacaaga
ttcaaagtgc gctgctggtg 2340ccaaccctat tctccttctt cgccaaaagc
actctgattg acaaatacga tttatctaat 2400ttacacgaaa ttgcttctgg
tggcgctccc ctctctaagg aagtcgggga agcggttgcc 2460aagaggttcc
atctgccagg tatcaggcaa ggatatgggc tcactgagac tacatcagct
2520attctgatta cacccgaggg ggatgataaa ccgggcgcgg tcggtaaagt
tgttccattt 2580tttgaagcga aggttgtgga tctggatacc gggaaaacgc
tgggcgttaa tcaaagaggc 2640gaactgtgtg tgagaggtcc tatgattatg
tccggttatg taaacaatcc ggaagcgacc 2700aacgccttga ttgacaagga
tggatggcta cattctggag acatagctta ctgggacgaa 2760gacgaacact
tcttcatcgt tgaccgcctg aagtctctga ttaagtacaa aggctatcag
2820gtggctcccg ctgaattgga atccatcttg ctccaacacc ccaacatctt
cgacgcaggt 2880gtcgcaggtc ttcccgacga tgacgccggt gaacttcccg
ccgccgttgt tgttttggag 2940cacggaaaga cgatgacgga aaaagagatc
gtggattacg tcgccagtca agtaacaacc 3000gcgaaaaagt tgcgcggagg
agttgtgttt gtggacgaag taccgaaagg tcttaccgga 3060aaactcgacg
caagaaaaat cagagagatc ctcataaagg ccaagaaggg cggaaagatc
3120gccgtgtaac tcgagatcta gatcataatc agccatacca catttgtaga
ggttttactt 3180gctttaaaaa acctcccaca cctccccctg aacctgaaac
ataaaatgaa tgcaattgtt 3240gttgttaact tgtttattgc agcttataat
ggttacaaat aaagcaatag catcacaaat 3300ttcacaaata aagcattttt ttcactgc
332853964DNAArtificial sequenceBicistronic luciferase TRdm EYFP
expression cassette 5tagttattaa tagtaatcaa ttacggggtc attagttcat
agcccatata tggagttccg 60cgttacataa cttacggtaa atggcccgcc tggctgaccg
cccaacgacc cccgcccatt 120gacgtcaata atgacgtatg ttcccatagt
aacgccaata gggactttcc attgacgtca 180atgggtggag tatttacggt
aaactgccca cttggcagta catcaagtgt atcatatgcc 240aagtacgccc
cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta
300catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
tcgctattac 360catggtgatg cggttttggc agtacatcaa tgggcgtgga
tagcggtttg actcacgggg 420atttccaagt ctccacccca ttgacgtcaa
tgggagtttg ttttggcacc aaaatcaacg 480ggactttcca aaatgtcgta
acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540acggtgggag
gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta
600ccggactcag atcctcagag tgccgaattc atggaagacg ccaaaaacat
aaagaaaggc 660ccggcgccat tctatccgct ggaagatgga accgctggag
agcaactgca taaggctatg 720aagagatacg ccctggttcc tggaacaatt
gcttttacag atgcacatat cgaggtggac 780atcacttacg ctgagtactt
cgaaatgtcc gttcggttgg cagaagctat gaaacgatat 840gggctgaata
caaatcacag aatcgtcgta tgcagtgaaa actctcttca attctttatg
900ccggtgttgg gcgcgttatt tatcggagtt gcagttgcgc ccgcgaacga
catttataat 960gaacgtgaat tgctcaacag tatgggcatt tcgcagccta
ccgtggtgtt cgtttccaaa 1020aaggggttgc aaaaaatttt gaacgtgcaa
aaaaagctcc caatcatcca aaaaattatt 1080atcatggatt ctaaaacgga
ttaccaggga tttcagtcga tgtacacgtt cgtcacatct 1140catctacctc
ccggttttaa tgaatacgat tttgtgccag agtccttcga tagggacaag
1200acaattgcac tgatcatgaa ctcctctgga tctactggtc tgcctaaagg
tgtcgctctg 1260cctcatagaa ctgcctgcgt gagattctcg catgccagag
atcctatttt tggcaatcaa 1320atcattccgg atactgcgat tttaagtgtt
gttccattcc atcacggttt tggaatgttt 1380actacactcg gatatttgat
atgtggattt cgagtcgtct taatgtatag atttgaagaa 1440gagctgtttc
tgaggagcct tcaggattac aagattcaaa gtgcgctgct ggtgccaacc
1500ctattctcct tcttcgccaa aagcactctg attgacaaat acgatttatc
taatttacac 1560gaaattgctt ctggtggcgc tcccctctct aaggaagtcg
gggaagcggt tgccaagagg 1620ttccatctgc caggtatcag gcaaggatat
gggctcactg agactacatc agctattctg 1680attacacccg agggggatga
taaaccgggc gcggtcggta aagttgttcc attttttgaa 1740gcgaaggttg
tggatctgga taccgggaaa acgctgggcg ttaatcaaag aggcgaactg
1800tgtgtgagag gtcctatgat tatgtccggt tatgtaaaca atccggaagc
gaccaacgcc 1860ttgattgaca aggatggatg gctacattct ggagacatag
cttactggga cgaagacgaa 1920cacttcttca tcgttgaccg cctgaagtct
ctgattaagt acaaaggcta tcaggtggct 1980cccgctgaat tggaatccat
cttgctccaa caccccaaca tcttcgacgc aggtgtcgca 2040ggtcttcccg
acgatgacgc cggtgaactt cccgccgccg ttgttgtttt ggagcacgga
2100aagacgatga cggaaaaaga gatcgtggat tacgtcgcca gtcaagtaac
aaccgcgaaa 2160aagttgcgcg gaggagttgt gtttgtggac gaagtaccga
aaggtcttac cggaaaactc 2220gacgcaagaa aaatcagaga gatcctcata
aaggccaaga agggcggaaa gatcgccgtg 2280taagaattct tgagtgagtt
agagtagtga gctagttgtc tggtaggggc cccctttgct 2340tccctggtgg
ccactggatt gtgtttcttt ggagtggcac tgttctgtgg atgtggacat
2400gaagctctca ctggtacaga aaagctaatt gagacctatt tctccaaaaa
ctaccaggac 2460tatgagtatc tcattaatgt gattcatgct ttccagtatg
tcatctatgg aactgcctct 2520ttcttcttcc tttatggggc cctcctgctg
gctgagggct tctacaccac cggcgctgtc 2580aggcagatct ttggcgacta
caagaccacc atctgcggca agggcctgag cgcaacgttt 2640gtgggcatca
cctatgccct gactgttgta tggctcctgg tgtttgcctg ctcggctgta
2700cctgtgtaca tttacttcaa tacctggacc acctgtcagt ctattgcctt
ccctagcaag 2760acctctgcca gtataggcag tctctgcgct gatgccagat
tgtatggtgt tctcccatgg 2820aatgctttcc ctggcaaggt ttgtggctcc
aaccttctgt ccatctgcaa aacagctgag 2880ttccaattga ccttccacct
gtttattgct gcgtttgtgg gtgctgcggc cacactagtt 2940tccctgctca
ccttcatgat tgctgccact tacaacttcg ccgtccttaa actcatgggc
3000cgaggcacca agttcccgcg ggcccgggat ccaccggtcg ccaccatggt
gagcaagggc 3060gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
tggacggcga cgtaaacggc 3120cacaagttca gcgtgtccgg cgagggcgag
ggcgatgcca cctacggcaa gctgaccctg 3180aagttcatct gcaccaccgg
caagctgccc gtgccctggc ccaccctcgt gaccaccttc 3240ggctacggcc
tgcagtgctt cgcccgctac ccacatgaag cagcacgact tcttcaagtc
3300cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
acggcaacta 3360caagacccgc gccgaggtga agttcgaggg cgacaccctg
gtgaaccgca tcgagctgaa 3420gggcatcgac ttcaaggagg acggcaacat
cctggggcac aagctggagt acaactacaa 3480cagccacaac gtctatatca
tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa 3540gatccgccac
aacatcgagg acggcagcgt gcagctcgcc gaccactacc agcagaacac
3600ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct
accagtccgc 3660cctgagcaaa gaccccaacg agaagcgcga tcacatggtc
ctgctggagt tcgtgaccgc 3720cgccgggatc actctcggca tggacgagct
gtacaagtaa agcggccgcg actctagatc 3780ataatcagcc ataccacatt
tgtagaggtt ttacttgctt taaaaaacct cccacacctc 3840cccctgaacc
tgaaacataa aatgaatgca attgttgttg ttaacttgtt tattgcagct
3900tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc
atttttttca 3960ctgc 396464069DNAArtificial sequenceBicistronic
luciferase TRplp EYFP expression cassette 6tagttattaa tagtaatcaa
ttacggggtc attagttcat agcccatata tggagttccg 60cgttacataa cttacggtaa
atggcccgcc tggctgaccg cccaacgacc cccgcccatt 120gacgtcaata
atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
180atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
atcatatgcc 240aagtacgccc cctattgacg tcaatgacgg taaatggccc
gcctggcatt atgcccagta 300catgacctta tgggactttc ctacttggca
gtacatctac gtattagtca tcgctattac 360catggtgatg cggttttggc
agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420atttccaagt
ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg
480ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
gtaggcgtgt 540acggtgggag gtctatataa gcagagctgg tttagtgaac
cgtcagatcc gctagcgcta 600ccggactcag atcctcagag tgccgaattc
atggaagacg ccaaaaacat aaagaaaggc 660ccggcgccat tctatccgct
ggaagatgga accgctggag agcaactgca taaggctatg 720aagagatacg
ccctggttcc tggaacaatt gcttttacag atgcacatat cgaggtggac
780atcacttacg ctgagtactt cgaaatgtcc gttcggttgg cagaagctat
gaaacgatat 840gggctgaata caaatcacag aatcgtcgta tgcagtgaaa
actctcttca attctttatg 900ccggtgttgg gcgcgttatt tatcggagtt
gcagttgcgc ccgcgaacga catttataat 960gaacgtgaat tgctcaacag
tatgggcatt tcgcagccta ccgtggtgtt cgtttccaaa 1020aaggggttgc
aaaaaatttt gaacgtgcaa aaaaagctcc caatcatcca aaaaattatt
1080atcatggatt ctaaaacgga ttaccaggga tttcagtcga tgtacacgtt
cgtcacatct 1140catctacctc ccggttttaa tgaatacgat tttgtgccag
agtccttcga tagggacaag 1200acaattgcac tgatcatgaa ctcctctgga
tctactggtc tgcctaaagg tgtcgctctg 1260cctcatagaa ctgcctgcgt
gagattctcg catgccagag atcctatttt tggcaatcaa 1320atcattccgg
atactgcgat tttaagtgtt gttccattcc atcacggttt tggaatgttt
1380actacactcg gatatttgat atgtggattt cgagtcgtct taatgtatag
atttgaagaa 1440gagctgtttc tgaggagcct tcaggattac aagattcaaa
gtgcgctgct ggtgccaacc 1500ctattctcct tcttcgccaa aagcactctg
attgacaaat acgatttatc taatttacac 1560gaaattgctt ctggtggcgc
tcccctctct aaggaagtcg gggaagcggt tgccaagagg 1620ttccatctgc
caggtatcag gcaaggatat gggctcactg agactacatc agctattctg
1680attacacccg agggggatga taaaccgggc gcggtcggta aagttgttcc
attttttgaa 1740gcgaaggttg tggatctgga taccgggaaa acgctgggcg
ttaatcaaag aggcgaactg 1800tgtgtgagag gtcctatgat tatgtccggt
tatgtaaaca atccggaagc gaccaacgcc 1860ttgattgaca aggatggatg
gctacattct ggagacatag cttactggga cgaagacgaa 1920cacttcttca
tcgttgaccg cctgaagtct ctgattaagt acaaaggcta tcaggtggct
1980cccgctgaat tggaatccat cttgctccaa caccccaaca tcttcgacgc
aggtgtcgca 2040ggtcttcccg acgatgacgc cggtgaactt cccgccgccg
ttgttgtttt ggagcacgga 2100aagacgatga cggaaaaaga gatcgtggat
tacgtcgcca gtcaagtaac aaccgcgaaa 2160aagttgcgcg gaggagttgt
gtttgtggac gaagtaccga aaggtcttac cggaaaactc 2220gacgcaagaa
aaatcagaga gatcctcata aaggccaaga agggcggaaa gatcgccgtg
2280taagaattct tgagtgagtt agagtagtga gctagttgtc tggtaggggc
cccctttgct 2340tccctggtgg ccactggatt gtgtttcttt ggagtggcac
tgttctgtgg atgtggacat 2400gaagctctca ctggtacaga
aaagctaatt gagacctatt tctccaaaaa ctaccaggac 2460tatgagtatc
tcattaatgt gattcatgct ttccagtatg tcatctatgg aactgcctct
2520ttcttcttcc tttatggggc cctcctgctg gctgagggct tctacaccac
cggcgctgtc 2580aggcagatct ttggcgacta caagaccacc atctgcggca
agggcctgag cgcaacggta 2640acagggggcc agaaggggag gggttccaga
ggccaacatc aagctcattc tttggagcgg 2700gtgtgtcatt gtttgggaaa
atggctagga catcccgaca agtttgtggg catcacctat 2760gccctgactg
ttgtatggct cctggtgttt gcctgctcgg ctgtacctgt gtacatttac
2820ttcaatacct ggaccacctg tcagtctatt gccttcccta gcaagacctc
tgccagtata 2880ggcagtctct gcgctgatgc cagattgtat ggtgttctcc
catggaatgc tttccctggc 2940aaggtttgtg gctccaacct tctgtccatc
tgcaaaacag ctgagttcca attgaccttc 3000cacctgttta ttgctgcgtt
tgtgggtgct gcggccacac tagtttccct gctcaccttc 3060atgattgctg
ccacttacaa cttcgccgtc cttaaactca tgggccgagg caccaagttc
3120ccgcgggccc gggatccacc ggtcgccacc atggtgagca agggcgagga
gctgttcacc 3180ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
acggccacaa gttcagcgtg 3240tccggcgagg gcgagggcga tgccacctac
ggcaagctga ccctgaagtt catctgcacc 3300accggcaagc tgcccgtgcc
ctggcccacc ctcgtgacca ccttcggcta cggcctgcag 3360tgcttcgccc
gctacccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
3420ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga
cccgcgccga 3480ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
ctgaagggca tcgacttcaa 3540ggaggacggc aacatcctgg ggcacaagct
ggagtacaac tacaacagcc acaacgtcta 3600tatcatggcc gacaagcaga
agaacggcat caaggtgaac ttcaagatcc gccacaacat 3660cgaggacggc
agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
3720ccccgtgctg ctgcccgaca accactacct gagctaccag tccgccctga
gcaaagaccc 3780caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
accgccgccg ggatcactct 3840cggcatggac gagctgtaca agtaaagcgg
ccgcgactct agatcataat cagccatacc 3900acatttgtag aggttttact
tgctttaaaa aacctcccac acctccccct gaacctgaaa 3960cataaaatga
atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa
4020taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgc
4069733DNAArtificial sequenceL205.5w primer 7ctgcgctgat gccagattgt
atggtgttct ccc 33833DNAArtificial sequenceL205.3w primer
8gggagaacac catacaatct ggcatcagcg cag 33937DNAArtificial
sequenceL235.5 primer 9ctgcaaaaca gctgagttcc aattgacctt ccacctg
371037DNAArtificial sequenceL235.3 primer 10caggtggaag gtcaattgga
actcagctgt tttgcag 371166DNAArtificial sequenceRI-stop_s primer
11cagatcctca gagtgccgaa ttcttgagtg agttagagta gtgagctagt tgtctggtag
60gggccc 661266DNAArtificial sequenceRI-stop_a primer 12gggcccctac
cagacaacta gctcactact ctaactcact caagaattcg gcactctgag 60gatctg
661360DNAArtificial sequenceH-Xh-Xb_s primer 13ggccgaggca
ccaagttcaa gcttgctcga gatctagagg tcgccaccat ggtgagcaag
601460DNAArtificial sequenceH-Xh-Xb_a primer 14cttgctcacc
atggtggcga cctctagatc tcgagcaagc ttgaacttgg tgcctcggcc
601524DNAArtificial sequenceSK-15 primer 15tgggcggtag gcgtgtacgg
tggg 241624DNAArtificial sequenceSK-16 primer 16cagcttgccg
gtggtgcaga tgaa 241723DNAArtificial sequenceSK-17 primer
17cagatccgct agcgctaccg gac 231822DNAArtificial sequenceEYFP(-)1
primer 18ctgctcacct tcttgattgc tg 221921DNAArtificial
sequenceEYFP(-)2 primer 19gttcaggggg aggtgtggga g
212018DNAArtificial sequenceBAC-1 primer 20gccattgtaa tgagacgc
182122DNAArtificial sequenceBAC-2 primer 21ccatctcgca aataaataag ta
222220DNAArtificial sequenceBAC-3 primer 22tgtaaatcaa caacgcacag
202382DNAArtificial sequenceAUGback_s primer 23gctcaccttc
atgattgctg ccacttacaa cttcgccgtc cttaaactca tgggccgagg 60caccaagttc
gaaagcttcg cc 822482DNAArtificial sequenceAUGback_a primer
24ggcgaagctt tcgaacttgg tgcctcggcc catgagttta aggacggcga agttgtaagt
60ggcagcaatc atgaaggtga gc 822546DNAArtificial sequenceLuc-1 primer
25gagtaagctt cgccaccatg gaagacgcca aaaacataaa gaaagg
462635DNAArtificial sequenceLuc-2 primer 26ctgactcgag ttacacggcg
atctttccgc ccttc 352742DNAArtificial sequenceTK-1 primer
27gagtaagctt cgccaccatg gcttcgtacc ccggccatca ac
422837DNAArtificial sequenceTK-2 primer 28ctgactcgag gtctccttcc
gtgtttcagt tagcctc 372948DNAArtificial sequenceCAT-1 primer
29gagtaagctt cgccaccatg gagaaaaaaa tcactggata taccaccg
483031DNAArtificial sequenceCAT-2 primer 30ctgactcgag ttacgccccg
ccctgccact c 313147DNAArtificial sequenceLacZ-1 primer 31gagtaagctt
cgccaccatg tcgtttactt tgaccaacaa gaacgtg 473246DNAArtificial
sequenceLacZ-2 primer 32ctgactcgag ttatttttga caccagacca actggtaatg
gtagcg 463353DNAArtificial sequenceSr39-1 primer 33ggccctcacc
atcttcctcg accgccatcc catcgccttc atgctgtgct acc 533453DNAArtificial
sequenceSr39-2 primer 34ggtagcacag catgaaggcg atgggatggc ggtcgaggaa
gatggtgagg gcc 533532DNAArtificial sequenceCMV-1 primer
35gagtaggcct ccgtattacc gccatgcatt ag 323623DNAArtificial
sequenceCMV-2 primer 36ctcaagaatt cggcactctg agg
2337100DNAArtificial sequencePolyA-1 primer 37ctaggtgttg ttgttaactt
gtttattgca gcttataatg gttacaaata aagcaatagc 60atcacaaatt tcacaaataa
agcatttttt tcactgcatg 1003892DNAArtificial sequencePolyA-2 primer
38cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
60ataagctgca ataaacaagt taacaacaac ac 92391977DNAArtificial
sequencenucleotides 1-846 represent TRplp and a linker sequence;
nucleotides 847-1977 represent TKsr39 39ttgagtgagt tagagtagtg
agctagttgt ctggtagggg ccccctttgc ttccctggtg 60gccactggat tgtgtttctt
tggagtggca ctgttctgtg gatgtggaca tgaagctctc 120actggtacag
aaaagctaat tgagacctat ttctccaaaa actaccagga ctatgagtat
180ctcattaatg tgattcatgc tttccagtat gtcatctatg gaactgcctc
tttcttcttc 240ctttatgggg ccctcctgct ggctgagggc ttctacacca
ccggcgctgt caggcagatc 300tttggcgact acaagaccac catctgcggc
aagggcctga gcgcaacggt aacagggggc 360cagaagggga ggggttccag
aggccaacat caagctcatt ctttggagcg ggtgtgtcat 420tgtttgggaa
aatggctagg acatcccgac aagtttgtgg gcatcaccta tgccctgact
480gttgtatggc tcctggtgtt tgcctgctcg gctgtacctg tgtacattta
cttcaatacc 540tggaccacct gtcagtctat tgccttccct agcaagacct
ctgccagtat aggcagtctc 600tgcgctgatg ccagattgta tggtgttctc
ccatggaatg ctttccctgg caaggtttgt 660ggctccaacc ttctgtccat
ctgcaaaaca gctgagttcc aattgacctt ccacctgttt 720attgctgcgt
ttgtgggtgc tgcggccaca ctagtttccc tgctcacctt catgattgct
780gccacttaca acttcgccgt ccttaaactc atgggccgag gcaccaagtt
cgaaagcttc 840gccaccatgg cttcgtaccc cggccatcaa cacgcgtctg
cgttcgacca ggctgcgcgt 900tctcgcggcc atagcaaccg acgtacggcg
ttgcgccctc gccggcagca agaagccacg 960gaagtccgcc cggagcagaa
aatgcccacg ctactgcggg tttatataga cggtccccac 1020gggatgggga
aaaccaccac cacgcaactg ctggtggccc tgggttcgcg cgacgatatc
1080gtctacgtac ccgagccgat gacttactgg cgggtgctgg gggcttccga
gacaatcgcg 1140aacatctaca ccacacaaca ccgcctcgac cagggtgaga
tatcggccgg ggacgcggcg 1200gtggtaatga caagcgccca gataacaatg
ggcatgcctt atgccgtgac cgacgccgtt 1260ctggctcctc atatcggggg
ggaggctggg agctcacatg ccccgccccc ggccctcacc 1320atcttcctcg
accgccatcc catcgccttc atgctgtgct acccggccgc gcggtacctt
1380atgggcagca tgacccccca ggccgtgctg gcgttcgtgg ccctcatccc
gccgaccttg 1440cccggcacca acatcgtgct tggggccctt ccggaggaca
gacacatcga ccgcctggcc 1500aaacgccagc gccccggcga gcggctggac
ctggctatgc tggctgcgat tcgccgcgtt 1560tacgggctac ttgccaatac
ggtgcggtat ctgcagtgcg gcgggtcgtg gcgggaggac 1620tggggacagc
tttcggggac ggccgtgccg ccccagggtg ccgagcccca gagcaacgcg
1680ggcccacgac cccatatcgg ggacacgtta tttaccctgt ttcgggcccc
cgagttgctg 1740gcccccaacg gcgacctgta taacgtgttt gcctgggcct
tggacgtctt ggccaaacgc 1800ctccgttcca tgcacgtctt tatcctggat
tacgaccaat cgcccgccgg ctgccgggac 1860gccctgctgc aacttacctc
cgggatggtc cagacccacg tcaccacccc cggctccata 1920ccgacgatat
gcgacctggc gcgcacgttt gcccgggaga tgggggaggc taactga
1977401872DNAArtificial sequencenucleotides 1-741 represent TRdm
and a linker sequence; nucleotides 742-1872 represent TKsr39
40ttgagtgagt tagagtagtg agctagttgt ctggtagggg ccccctttgc ttccctggtg
60gccactggat tgtgtttctt tggagtggca ctgttctgtg gatgtggaca tgaagctctc
120actggtacag aaaagctaat tgagacctat ttctccaaaa actaccagga
ctatgagtat 180ctcattaatg tgattcatgc tttccagtat gtcatctatg
gaactgcctc tttcttcttc 240ctttatgggg ccctcctgct ggctgagggc
ttctacacca ccggcgctgt caggcagatc 300tttggcgact acaagaccac
catctgcggc aagggcctga gcgcaacgtt tgtgggcatc 360acctatgccc
tgactgttgt atggctcctg gtgtttgcct gctcggctgt acctgtgtac
420atttacttca atacctggac cacctgtcag tctattgcct tccctagcaa
gacctctgcc 480agtataggca gtctctgcgc tgatgccaga ttgtatggtg
ttctcccatg gaatgctttc 540cctggcaagg tttgtggctc caaccttctg
tccatctgca aaacagctga gttccaattg 600accttccacc tgtttattgc
tgcgtttgtg ggtgctgcgg ccacactagt ttccctgctc 660accttcatga
ttgctgccac ttacaacttc gccgtcctta aactcatggg ccgaggcacc
720aagttcgaaa gcttcgccac catggcttcg taccccggcc atcaacacgc
gtctgcgttc 780gaccaggctg cgcgttctcg cggccatagc aaccgacgta
cggcgttgcg ccctcgccgg 840cagcaagaag ccacggaagt ccgcccggag
cagaaaatgc ccacgctact gcgggtttat 900atagacggtc cccacgggat
ggggaaaacc accaccacgc aactgctggt ggccctgggt 960tcgcgcgacg
atatcgtcta cgtacccgag ccgatgactt actggcgggt gctgggggct
1020tccgagacaa tcgcgaacat ctacaccaca caacaccgcc tcgaccaggg
tgagatatcg 1080gccggggacg cggcggtggt aatgacaagc gcccagataa
caatgggcat gccttatgcc 1140gtgaccgacg ccgttctggc tcctcatatc
gggggggagg ctgggagctc acatgccccg 1200cccccggccc tcaccatctt
cctcgaccgc catcccatcg ccttcatgct gtgctacccg 1260gccgcgcggt
accttatggg cagcatgacc ccccaggccg tgctggcgtt cgtggccctc
1320atcccgccga ccttgcccgg caccaacatc gtgcttgggg cccttccgga
ggacagacac 1380atcgaccgcc tggccaaacg ccagcgcccc ggcgagcggc
tggacctggc tatgctggct 1440gcgattcgcc gcgtttacgg gctacttgcc
aatacggtgc ggtatctgca gtgcggcggg 1500tcgtggcggg aggactgggg
acagctttcg gggacggccg tgccgcccca gggtgccgag 1560ccccagagca
acgcgggccc acgaccccat atcggggaca cgttatttac cctgtttcgg
1620gcccccgagt tgctggcccc caacggcgac ctgtataacg tgtttgcctg
ggccttggac 1680gtcttggcca aacgcctccg ttccatgcac gtctttatcc
tggattacga ccaatcgccc 1740gccggctgcc gggacgccct gctgcaactt
acctccggga tggtccagac ccacgtcacc 1800acccccggct ccataccgac
gatatgcgac ctggcgcgca cgtttgcccg ggagatgggg 1860gaggctaact ga
18724172PRTHomo sapiens 41Met Tyr Gly Val Leu Pro Trp Asn Ala Phe
Pro Gly Lys Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser Ile Cys Lys
Thr Ala Glu Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala Ala
Phe Val Gly Ala Ala Ala Thr Leu Val Ser 35 40 45 Leu Leu Thr Phe
Met Ile Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys 50 55 60 Leu Met
Gly Arg Gly Thr Lys Phe 65 70 4272PRTPongo pygmaeus 42Met Tyr Gly
Val Leu Pro Trp Asn Ala Phe Pro Gly Lys Val Cys Gly 1 5 10 15 Ser
Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu Phe Gln Met Thr Phe 20 25
30 His Leu Phe Ile Ala Ala Phe Val Gly Ala Ala Ala Thr Leu Val Ser
35 40 45 Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn Phe Ala Val
Leu Lys 50 55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70 4372PRTPan
troglodytes 43Met Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro Gly Lys
Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu
Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala Ala Phe Val Gly
Ala Ala Ala Thr Leu Val Ser 35 40 45 Leu Leu Thr Phe Met Ile Ala
Ala Thr Tyr Asn Phe Ala Val Leu Lys 50 55 60 Leu Met Gly Arg Gly
Thr Lys Phe 65 70 4472PRTMacaca mulatta 44Met Tyr Gly Val Leu Pro
Trp Asn Ala Phe Pro Gly Lys Val Cys Gly 1 5 10 15 Ser Asn Leu Leu
Ser Ile Cys Lys Thr Ala Glu Phe Gln Met Thr Phe 20 25 30 His Leu
Phe Ile Ala Ala Phe Val Gly Ala Ala Ala Thr Leu Ile Ser 35 40 45
Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys 50
55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70 4572PRTMacaca
fascicularis 45Met Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro Gly Lys
Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu
Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala Ala Phe Val Gly
Ala Ala Ala Thr Leu Ile Ser 35 40 45 Leu Leu Thr Phe Met Ile Ala
Ala Thr Tyr Asn Phe Ala Val Leu Lys 50 55 60 Leu Met Gly Arg Gly
Thr Lys Phe 65 70 4672PRTSus scrofa 46Met Tyr Gly Val Leu Pro Trp
Asn Ala Phe Pro Gly Lys Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser
Ile Cys Lys Thr Ala Glu Phe Gln Met Thr Phe 20 25 30 His Leu Phe
Ile Ala Ala Phe Val Gly Ala Ala Ala Thr Leu Val Ser 35 40 45 Leu
Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys 50 55
60 Leu Met Gly Arg Gly Thr Lys Phe 65 70 4772PRTMus musculus 47Met
Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro Gly Lys Val Cys Gly 1 5 10
15 Ser Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu Phe Gln Met Thr Phe
20 25 30 His Leu Phe Ile Ala Ala Phe Val Gly Ala Ala Ala Thr Leu
Val Ser 35 40 45 Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn Phe
Ala Val Leu Lys 50 55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70
4872PRTRattus norvegicus 48Met Tyr Gly Val Leu Pro Trp Asn Ala Phe
Pro Gly Lys Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser Ile Cys Lys
Thr Ala Glu Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala Ala
Phe Val Gly Ala Ala Ala Thr Leu Val Ser 35 40 45 Leu Leu Thr Phe
Met Ile Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys 50 55 60 Leu Met
Gly Arg Gly Thr Lys Phe 65 70 4972PRTMonodelphis domestica 49Met
Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro Gly Lys Val Cys Gly 1 5 10
15 Ser Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu Phe Gln Met Thr Phe
20 25 30 His Leu Phe Ile Ala Ala Phe Ile Gly Ala Ala Ala Thr Leu
Val Ser 35 40 45 Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn Phe
Ala Val Leu Lys 50 55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70
5072PRTOryctolagus cuniculus 50Met Tyr Gly Val Leu Pro Trp Asn Ala
Phe Pro Gly Lys Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser Ile Cys
Lys Thr Ala Glu Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala
Ala Phe Val Gly Ala Ala Ala Thr Leu Val Ser 35 40 45 Leu Leu Thr
Phe Met Ile Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys 50 55 60 Leu
Met Gly Arg Gly Thr Lys Phe 65 70 5172PRTBos taurus 51Met Tyr Gly
Val Leu Pro Trp Asn Ala Phe Pro Gly Lys Val Cys Gly 1 5 10 15 Ser
Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu Phe Gln Met Thr Phe 20 25
30 His Leu Phe Ile Ala Ala Phe Val Gly Ala Ala Ala Thr Leu Val Ser
35 40 45 Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn Phe Ala Val
Leu Lys 50 55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70 5272PRTCanis
familiaris 52Met Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro Gly Lys
Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser Ile Cys Lys Thr Ala Glu
Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala Ala Phe Val Gly
Ala Ala Ala Thr Leu Val Ser 35 40
45 Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn Phe Ala Val Leu Lys
50 55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70 5372PRTGallus gallus
53Met Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro Gly Lys Val Cys Gly 1
5 10 15 Ser Asn Leu Leu Ser Ile Cys Lys Thr Ser Glu Phe Gln Met Thr
Phe 20 25 30 His Leu Phe Ile Ala Ala Phe Val Gly Ala Ala Ala Thr
Leu Val Ser 35 40 45 Leu Leu Thr Phe Met Ile Ala Ala Thr Tyr Asn
Phe Ala Val Leu Lys 50 55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70
5472PRTTaeniopygia guttata 54Met Tyr Gly Ile Leu Pro Trp Asn Ala
Phe Pro Gly Lys Val Cys Gly 1 5 10 15 Ser Asn Leu Leu Ser Ile Cys
Lys Thr Ser Glu Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala
Ala Phe Val Gly Ala Ala Ala Thr Leu Val Ser 35 40 45 Leu Val Thr
Phe Ile Ile Ala Thr Thr Tyr Asn Phe Ala Val Leu Arg 50 55 60 Leu
Met Gly Arg Gly Thr Lys Phe 65 70 5572PRTGekko gecko 55Met Tyr Gly
Val Leu Pro Trp Asn Ala Ser Pro Gly Arg Val Cys Gly 1 5 10 15 Gln
Ser Leu Leu Ser Ile Cys Lys Thr Ala Glu Phe Gln Met Thr Phe 20 25
30 His Leu Phe Ile Ala Ala Phe Val Gly Ala Ala Ile Thr Leu Val Ala
35 40 45 Leu Leu Thr Phe Ile Ile Ala Ala Thr Tyr Asn Phe Ala Val
Leu Lys 50 55 60 Leu Met Gly Arg Gly Thr Lys Phe 65 70
5674PRTXenopus laevis 56Met Tyr Gly Val Leu Pro Trp Asn Ala Phe Pro
Gly Lys Val Cys Gly 1 5 10 15 Thr Ser Leu Leu Ala Ile Cys Lys Thr
Ser Glu Phe Gln Met Thr Phe 20 25 30 His Leu Phe Ile Ala Ala Phe
Val Gly Ala Ala Ala Thr Leu Val Ala 35 40 45 Leu Leu Thr Tyr Met
Val Gly Ala Ser Phe Asn Tyr Ala Val Leu Arg 50 55 60 Val Thr Gly
Arg Ser Asp Arg Ser Lys Phe 65 70 5779PRTLatimeria chalumnae 57Met
Asp Pro Arg Gln Tyr Gly Val Leu Pro Trp Thr Ala Thr Pro Gly 1 5 10
15 Lys Val Cys Gly Met Ala Leu Val Ser Ile Cys Asn Lys Pro Glu Phe
20 25 30 Asn Met Thr Tyr His Leu Phe Ile Thr Ala Phe Thr Gly Ala
Ala Ala 35 40 45 Thr Leu Val Ser Leu Leu Thr Tyr Met Met Ser Thr
Thr Tyr Asn Phe 50 55 60 Ala Val Leu Arg Phe Leu Gly Arg Glu Asp
Phe Cys Thr Lys Phe 65 70 75 58729DNAMus musculus 58atgggcttgt
tagagtgttg tgctagatgt ctggtagggg ccccctttgc ttccctggtg 60gccactggat
tgtgtttctt tggagtggca ctgttctgtg gatgtggaca tgaagctctc
120actggtacag aaaagctaat tgagacctat ttctccaaaa actaccagga
ctatgagtat 180ctcattaatg tgattcatgc tttccagtat gtcatctatg
gaactgcctc tttcttcttc 240ctttatgggg ccctcctgct ggctgagggc
ttctacacca ccggcgctgt caggcagatc 300tttggcgact acaagaccac
catctgcggc aagggcctga gcgcaacgtt tgtgggcatc 360acctatgccc
tgactgttgt atggctcctg gtgtttgcct gctcggctgt acctgtgtac
420atttacttca atacctggac cacctgtcag tctattgcct tccctagcaa
gacctctgcc 480agtataggca gtctctgcgc tgatgccaga atgtatggtg
ttctcccatg gaatgctttc 540cctggcaagg tttgtggctc caaccttctg
tccatctgca aaacagctga gttccaaatg 600accttccacc tgtttattgc
tgcgtttgtg ggtgctgcgg ccacactagt ttccctgctc 660accttcatga
ttgctgccac ttacaacttc gccgtcctta aactcatggg ccgaggcacc 720aagttctga
72959834DNAMus musculus 59atgggcttgt tagagtgttg tgctagatgt
ctggtagggg ccccctttgc ttccctggtg 60gccactggat tgtgtttctt tggagtggca
ctgttctgtg gatgtggaca tgaagctctc 120actggtacag aaaagctaat
tgagacctat ttctccaaaa actaccagga ctatgagtat 180ctcattaatg
tgattcatgc tttccagtat gtcatctatg gaactgcctc tttcttcttc
240ctttatgggg ccctcctgct ggctgagggc ttctacacca ccggcgctgt
caggcagatc 300tttggcgact acaagaccac catctgcggc aagggcctga
gcgcaacggt aacagggggc 360cagaagggga ggggttccag aggccaacat
caagctcatt ctttggagcg ggtgtgtcat 420tgtttgggaa aatggctagg
acatcccgac aagtttgtgg gcatcaccta tgccctgact 480gttgtatggc
tcctggtgtt tgcctgctcg gctgtacctg tgtacattta cttcaatacc
540tggaccacct gtcagtctat tgccttccct agcaagacct ctgccagtat
aggcagtctc 600tgcgctgatg ccagaatgta tggtgttctc ccatggaatg
ctttccctgg caaggtttgt 660ggctccaacc ttctgtccat ctgcaaaaca
gctgagttcc aaatgacctt ccacctgttt 720attgctgcgt ttgtgggtgc
tgcggccaca ctagtttccc tgctcacctt catgattgct 780gccacttaca
acttcgccgt ccttaaactc atgggccgag gcaccaagtt ctga 83460729DNAHomo
sapiens 60atgggcttgt tagagtgctg tgcaagatgt ctggtagggg ccccctttgc
ttccctggtg 60gccactggat tgtgtttctt tggggtggca ctgttctgtg gctgtggaca
tgaagccctc 120actggcacag aaaagctaat tgagacctat ttctccaaaa
actaccaaga ctatgagtat 180ctcatcaatg tgatccatgc cttccagtat
gtcatctatg gaactgcctc tttcttcttc 240ctttatgggg ccctcctgct
ggctgagggc ttctacacca ccggcgcagt caggcagatc 300tttggcgact
acaagaccac catctgcggc aagggcctga gcgcaacgtt tgtgggcatc
360acctatgccc tgaccgttgt gtggctcctg gtgtttgcct gctctgctgt
gcccgtgtac 420atttacttca acacctggac cacctgcgac tctattgcct
tccccagcaa gacctctgcc 480agtataggca gtctctgtgc tgacgccaga
atgtatggtg ttctcccatg gaatgctttc 540cctggcaagg tttgtggctc
caaccttctg tccatctgca aaacagctga gttccaaatg 600accttccacc
tgtttattgc tgcatttgtg ggggctgcag ctacactggt ttccctgctc
660accttcatga ttgctgccac ttacaacttt gccgtcctta aactcatggg
ccgaggcacc 720aagttctga 72961850DNAHomo sapiens 61atgggcttgt
tagagtgctg tgcaagatgt ctggtagggg ccccctttgc ttccctggtg 60gccactggat
tgtgtttctt tggggtggca ctgttctgtg gctgtggaca tgaagccctc
120actggcacag aaaagctaat tgagacctat ttctccaaaa actaccaaga
ctatgagtat 180ctcatcaatg tgatccatgc cttccagtat gtcatctatg
gaactgcctc tttcttcttc 240ctttatgggg ccctcctgct ggctgagggc
ttctacacca ccggcgcagt caggcagatc 300tttggcgact acaagaccac
catctgcggc aagggcctga gcgcaacggt aacagggggc 360cagaagggga
ggggttccag aggccaacat caagctcatt ctttggagcg ggtgtgtcat
420tgtttgggaa aatggctagg acatcccgac aagtttgtgg gcatcaccta
tgccctgacc 480gttgtgtggc tcctggtgtt tgcctgctct gctgtgcccg
tgtacattta cttcaacacc 540tggaccacct gcgactctat tgccttcccc
agcaagacct ctgccagtat aggcagtctc 600tgtgctgacg ccagaatgta
tggtgttctc ccatggaatg ctttccctgg caaggtttgt 660ggctccaacc
ttctgtccat ctgcaaaaca gctgagttcc aaatgacctt ccacctgttt
720attgctgcat ttgtgggggc tgcagctaca ctggtttccc tgctcacctt
catgattgct 780gccacttaca actttgccgt ccttaaactc atgggccgag
gcaccaagtt ctgatacact 840ggtttccctg 85062834DNAArtificial
SequenceVertebrate PLP/DM20 consensus sequence 62atgggyykgy
wdgakkgytg yrynmgmtgy mtbrtwgggg ymccmttygc ytchbtsrtb 60gccacwgkvy
tvtgyttyky tggrgtsgcv ctvttctgyg gmtgyggrca ygargchytv
120asygghacmg armagytvat ygagacmtay ttytccaara aytaccaaga
mtaygartay 180ctcatyvayg tsatymaygc yttycagtay gtcatctatg
gaaywgccwy yttcttctty 240cthtwyggrr ycctvctkyt ggcygarggm
ttctacacca cmrsygchrt cargcavatc 300ythggsgast wcmrrmccmc
mryywkmrrs rrkggsctga kykcwacrgt racwggrggm 360cmkaarggga
grrghdcsmg rggmmvvcak cvagyycayw cywtrsagck srtstgtcrb
420tgyttgggaa artggctmgg acayccygay aagtttgtsg gyrtyacyta
tryyhtsacy 480rtyktvtggm tmctrrystt ygcctgctcd gcygtdccyg
tvtacatyta yttyaayacc 540tggrycacyt gycagtctat ygcckyccch
rssaagacyw cwrccagyrt mrgyasbcts 600tgykcdgayg symgvatgta
yggtgtycts ccmtggaayg cbttycchgg saargtktgy 660ggswccarcc
tkctbkccat ctgcaaracm rsygagttcc aratgacntt ycayctbttt
720atygckgcvt tygtgggkgc wgcngchacw ctdgtbkcmc tgctcacytw
yatgrthgsy 780gcmwcwtwca actwygcygt sctbmrastb aykggccgrr
gcwcmaagtt ytga 83463834DNAArtificial SequenceMammalian PLP/DM20
consensus sequence 63atgggcytgt tagagtgytg ygcnagatgy ctsgtagggg
ccccctttgc ttccytggtg 60gccactggat trtgtttctt tggrgtggca ctsttctgtg
gmtgtggaca tgaagchytm 120actggyacag aaaagytaat tgagacmtat
ttctccaaaa aytaccaaga ctaygagtat 180ctcatyaatg tgatycatgc
yttccagtat gtcatctatg gaactgcctc tttcttcttc 240ctttatgggg
ccctcctgct ggcygagggc ttctacacca ccggygcwgt caggcagatc
300tttggcgact acaagaccac catctgcggs aagggcctga gygcaacggt
aacagggggc 360cagaagggga ggggttccag aggccaacat caagctcatt
ctttggagcg ggtgtgtcat 420tgtttgggaa aatggctagg acatcccgac
aagtttgtgg gcatcaccta tgccytgacy 480gttgtrtggc tcctrgtgtt
tgcctgctck gctgtrcctg tgtacattta yttcaayacc 540tggaccacyt
gycagtctat tgcckycccy agcaagacyt ctgccagyat aggcastctc
600tgygctgatg ccagaatgta tggtgttctc ccatggaatg ctttyccwgg
caargtktgt 660ggctccaacc ttctgtccat ctgcaaaaca gctgagttcc
aaatgacstt ccayctgttt 720attgctgcvt tygtgggkgc tgcrgcyaca
ctrgtktccc tgctcacctt catgattgct 780gccacttaca acttygccgt
cctkaaactc atgggccgag gcaccaagtt ctga 834
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