U.S. patent application number 11/041797 was filed with the patent office on 2005-12-22 for method of identifying nucleic acid compositions for muting expression of a gene.
Invention is credited to Bahramian, Mohammad B., Zarbl, Helmut.
Application Number | 20050282764 11/041797 |
Document ID | / |
Family ID | 34890133 |
Filed Date | 2005-12-22 |
United States Patent
Application |
20050282764 |
Kind Code |
A1 |
Bahramian, Mohammad B. ; et
al. |
December 22, 2005 |
Method of identifying nucleic acid compositions for muting
expression of a gene
Abstract
The invention provides compositions and methods for muting
expression of an endogenous gene in an animal cell, the muting
resulting from providing a muting nucleic acid to a cell. The
muting nucleic acid comprises a transgene, which is substantially
homologous to a portion of the endogenous gene. The portion of the
endogenous gene provided on the transgene can be from the
5'-untranscribed end, from the 3' untranscribed end, from an exon
or an intron in the coding portion, or from a portion that overlaps
any of these portions. Methods are provided for obtaining muting
nucleic acid, and for screening for molecules that can mute the
gene, and for molecules that can alleviate muting of the gene.
Inventors: |
Bahramian, Mohammad B.;
(Branford, CT) ; Zarbl, Helmut; (Snoqualmie,
WA) |
Correspondence
Address: |
PETER J. KNUDSEN
KNUDSEN, ATTORNEY AT LAW LLC
13710 RIVIERA PLACE NE
SEATTLE
WA
98125
US
|
Family ID: |
34890133 |
Appl. No.: |
11/041797 |
Filed: |
January 24, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11041797 |
Jan 24, 2005 |
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09472558 |
Dec 27, 1999 |
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6939712 |
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60114107 |
Dec 29, 1998 |
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Current U.S.
Class: |
514/44A ;
536/23.1; 800/14 |
Current CPC
Class: |
C12N 15/1034
20130101 |
Class at
Publication: |
514/044 ;
800/014; 536/023.1 |
International
Class: |
A61K 048/00; A01K
067/027; C07H 021/04 |
Goverment Interests
[0002] This invention was made in part with government support
under grants IRO1-CA50378 awarded by the National Cancer Institute
and NIH-2T32-ES07020 toxicology-training grant awarded by the
National Institute of Environmental Health Science. The government
has certain rights in the invention.
Claims
What is claimed is:
1. A nucleic acid composition for muting expression of a gene with
unwanted activity in an animal cell, wherein the muting nucleic
acid includes a sequence homologous to an endogenous sequence in
the gene.
2. A nucleic acid composition according to claim 1, wherein the
gene with unwanted activity is carried on a chromosome of the
cell.
3. A nucleic acid composition according to claim 1, wherein the
cell is selected from the group consisting of a cancer cell, an
autoimmune cell, and a cell having a gene of a pathogen.
4. A nucleic acid composition according to claim 3, wherein the
pathogen is a virus.
5. A nucleic acid composition according to claim 1, wherein the
nucleic acid is selected from the group consisting of a DNA, an
RNA, and a nucleic acid analog.
6. A nucleic acid composition according to claim 5, wherein the
nucleic acid analog is selected from the group consisting of a
phosphorothioate, a 2'-o-methyl RNA, and a peptide nucleic
acid.
7. A nucleic acid composition according to claim 1, wherein the
nucleic acid is double stranded DNA.
8. A nucleic acid composition according to claim 1, wherein the
animal is a vertebrate.
9. A nucleic acid composition according to claim 8, wherein the
vertebrate is a warm-blooded animal.
10. A nucleic acid composition according to claim 9, wherein the
warm-blooded animal is a mammal.
11. A method for muting expression of an endogenous gene having
unwanted activity in a cell of an animal, the method comprising the
steps of: (a) providing a muting nucleic acid; and (b) delivering
the muting nucleic acid into the cell.
12. A method according to claim 10, wherein providing the muting
nucleic acid includes providing a nucleic acid composition having a
transgene, the transgene having a sequence that is substantially
homologous to a sequence in the endogenous gene with unwanted
activity.
13. A method according to claim 11, wherein the nucleic acid is
selected from the group consisting of DNA, RNA, and a nucleic acid
analog.
14. A method according to claim 13, wherein (a) further comprises
the step of engineering the nucleic acid into a recombinant
vector.
15. A method according to claim 14, wherein the recombinant vector
is a plasmid, a phagemid, or a virus.
16. A method according to claim 15, wherein the vector is a
double-stranded DNA plasmid.
17. A method according to claim 12, wherein the muting transgene
sequence is substantially homologous to an endogenous sequence that
extends to a portion of the endogenous gene selected from at least
one of the group of: the 5' untranscribed portion, the transcribed
coding portion including introns, the 3' untranslated portion, the
3' untranscribed portion, and a portion that overlaps adjacent ends
of at least two portion of the endogenous gene.
18. A method according to claim 17, wherein the nucleic acid
comprises a sequence that is substantially homologous to an
endogenous sequence located in the 5' portion of the endogenous
gene.
19. A method according to claim 18, wherein the endogenous sequence
located in the 5' portion comprises about 200 to about 400 bases in
length.
20. A method according to claim 18, wherein the endogenous sequence
located in the 5' portion comprises about 400 to about 600 bases in
length.
21. A method according to claim 18, wherein the endogenous sequence
located in the 5' portion comprises about 600 to about 1,000 bases
in length.
22. A method according to claim 11, wherein the muting nucleic acid
comprises a sequence that is substantially homologous to an
endogenous sequence located at the 3' portion of the gene having
unwanted activity.
23. A method according to claim 22, wherein the 3' portion of the
gene includes an untranscribed portion and a portion that overlaps
the 3' end of the coding portion.
24. A method according to claim 11, wherein the step of delivering
the muting nucleic acid in (b) is selected from the group of:
transforming, transfecting, electroporating, infecting, and
lipofecting the nucleic acid into the cell.
25. A method according to claim 24, wherein delivering the muting
nucleic comprises infecting the cell with a genetically attenuated
bacterium or virion.
26. A method according to claim 16, wherein following (b), the
plasmid is not substantially integrated into a chromosome.
27. A method according to claim 26, wherein the plasmid is
transiently maintained in the cell.
28. A method for identifying a muting nucleic acid that reduces
expression of an endogenous target gene having unwanted activity in
cells of an animal, comprising the steps of: (a) providing a set of
fragments of DNA encoding the target gene, wherein the fragments
are engineered into a plurality of vector molecules to produce a
recombinant vector library; (b) delivering the vector library into
the cells, to form a plurality of transgenic cloned fragment
recipients; and (c) comparing expression of the target gene in each
of a subset of the cloned recipients, to expression of the target
gene in the cells of the animal, to identify a cloned recipient
having a vector with the muting nucleic acid, wherein expression of
the target gene is reduced.
29. A method according to claim 28, wherein the animal is
warm-blooded.
30. A method according to claim 29, wherein the animal is a
mammal.
31. A method according to claim 28, wherein the vector carries also
a chemical resistance gene conferring a phenotype which is ability
to grow in the presence of the chemical.
32. A method according to claim 31, having an additional step of:
(a) comparing expression of the resistance gene in the cell having
the muting nucleic acid, with expression of the resistance gene in
the animal cell.
33. A method according to claim 32, wherein the resistance gene is
selected from the group consisting of AMP and CAT, encoding
encoding .beta.-lactamase and chloramphenicol acetyl transferase,
respectively.
34. A method according to claim 28, having a further step: (a)
comparing expression of a second endogenous gene which is not the
target gene in the cell having a muting nucleic acid, with
expression of the second endogenous gene in the animal cell.
35. A method according to claim 34, wherein the second endogenous
gene is GADPH, encoding glyceraldehyde-3-phosphate
dehydrogenase.
36. A method of evaluating a phenotype of animal cells engineered
to mute expression of a target endogenous gene, comprising: (a)
transforming animal cells capable of expressing the target gene
with the vector having the muting nucleic acid obtained according
to the method of claim 28; and (b) observing the transformed cells
for an altered phenotype in comparison to the parental animal cells
capable of expressing the target gene.
37. A method according to claim 36, wherein the altered phenotype
under a set of specified conditions is selected from the group
consisting of an alteration of: growth rate, nutritional
requirement, contact inhibition among confluent cells, formation of
foci, presence of a receptor for a ligand, signal transduction in
response to an effector molecule, sensitivity to a pathogen,
expression of a developmental protein, and cell cycle pattern.
38. A method according to claim 37, wherein the altered phenotype
is cessation of growth or colony formation under specified
conditions different from the conditions for growth of the parental
animal cells capable of expressing the target gene.
39. A method according to claim 37, wherein the specified
conditions different from the conditions for growth of the parental
animal cells capable of expressing the target gene comprise at
least one of the conditions selected from the group of: an elevated
temperature, a depressed temperature, a decreased serum
concentration, an elevated serum concentration, a decreased carbon
dioxide concentration, an increased carbon dioxide concentration,
an increased density of plating, and a decreased density of
plating.
40. A method according to claim 37, wherein the altered phenotype
is cessation of growth or colony formation under specified
conditions that are the same as the conditions for growth of the
parental animal cells capable of expressing the target gene.
41. A method according to claim 37, wherein the animal cells are
present in an embryonic or postnatal animal.
42. A method of screening a plurality of molecules to obtain a
composition capable of muting expression of an endogenous gene in
cells of a cell line, comprising: mixing a subset of each of the
plurality of molecules with a plurality of samples of the cells, to
produce a plurality of test cell cultures; providing a nucleic acid
capable of muting expression of the gene; transforming the nucleic
acid into a sample of the cells, to produce a positive control cell
culture having muting of expression of the endogenous gene; and
detecting an amount of expression of the endogenous gene in each of
the test cell cultures in comparison with the positive control cell
culture and with untreated cells of the cell line, such that a test
cell culture with substantially reduced expression of the gene
compared to expression in the untreated cells, and substantially
equivalent expression compared to cells in the positive control
culture, identifies the composition capable of muting expression of
the gene.
43. A method according to claim 42, wherein detecting expression of
the endogenous gene comprises analyzing cell RNA by hybridization
with a probe.
44. A method according to claim 43, wherein the hybrid of the cell
RNA and the probe is digested with RNase.
45. A method according to claim 44, the digested RNA is submitted
to gel electrophoresis to determine the size of the cell RNA
protected from RNase digestion by the probe.
46. A method according to claim 42, wherein detecting expression of
the endogenous gene comprises detecting a color change or absence
of a color change in the cells.
47. A method according to claim 46, wherein the color change in the
cells is indicative of expression of the endogenous gene which has
been fused to a second gene having a colorimetric assay.
48. A method according to claim 42, wherein the molecules are
selected from the group consisting of extracts of natural product
fermentations and synthesized organic chemicals.
49. A method according to claim 48, wherein the organic chemicals
are synthesized according to combinatorial methods.
50. A composition obtained by the method of claim 42 in a
pharmaceutically acceptable carrier.
51. A method of screening a plurality of molecules to obtain a
composition capable of alleviating muting of expression of an
endogenous gene in cells of a cell line having a muted endogenous
gene, comprising: mixing a subset of each of the plurality of
molecules with a plurality of samples of the cells having the muted
endogenous gene, to produce a plurality of test cell cultures; and
detecting amounts of expression of the endogenous gene in each of
the test cell cultures in comparison with the cells of the cell
line having the muted endogenous gene, and in untreated cells of a
parental cell line in which the endogenous gene is not muted, such
that a test cell culture with expression of the gene that is
substantially greater than the expression in the cell line having
the muted endogenous gene, and that is substantially equivalent to
expression in cells of the parental non-muted culture, identifies
the composition capable of alleviating muting of expression of the
gene.
52. A composition identified by the method of claim 51, in a
pharmaceutically acceptable carrier.
53. A kit for identifying a muting nucleic acid that reduces
expression of an endogenous gene, the kit comprising reagents for
assaying quantitatively both protection of a riboprobe from
ribonuclease digestion, and amount of transfected DNA.
54. A kit according to claim 53, wherein the reagents comprise
chemicals, stabilized enzymes, and buffers.
55. A kit according to claim 53, wherein the reagents comprise
diethylpyrocarbonate-treated water, placental RNase inhibitor,
tRNA, a buffer containing piperazine-N,N'-bis(2-ethanesulfonic
acid), a DNase I digestion buffer, phenylmethylsulfonyl fluoride,
and gelatin.
56. A kit according to claim 54, wherein the stabilized enzymes
comprise: an RNA polymerase selected from the group of SP6 RNA
polymerase and T7 RNA polymerase; a ribonuclease selected from the
group of RNase I and a mixture of RNases A and T.sub.1; Taq
polymerase; proteinase K; and DNase-free pancreatic RNase.
57. A method to identify a muting nucleic acid composition,
comprising the steps of: (a) synthesizing a plurality of nucleic
acid compositions homologous to all or part of the target gene, the
nucleic acid composition being double stranded, (b) introducing
said plurality of nucleic acid compositions into a cultured
population of animal cells, and (c) selecting a nucleic acid
composition that inhibits expression of the target gene in said
animal cells, wherein said muting nucleic acid composition inhibits
expression of the target gene in said animal cells.
58. A method to identify a muting nucleic acid composition
according to claim 57, comprising the additional step of
incorporating said plurality of nucleic acid compositions into a
vector comprising a promoter, wherein said nucleic acid is DNA.
59. A method to identify a muting nucleic acid composition
according to claim 58, wherein said vector causes production of a
transcript having a sequence that is homologous to a sequence in
the target gene.
60. A method to identify a muting nucleic acid composition
according to claim 59, wherein said vector is integrated into a
chromosome of the host, and wherein the structure of the target
gene is unchanged.
61. A method to identify a muting nucleic acid composition,
comprising the steps of: (a) synthesizing a nucleic acid
composition homologous to all or part of the target gene, the
nucleic acid composition being double stranded, (b) introducing
said nucleic acid composition into a cultured population of animal
cells, and (c) selecting said muting nucleic acid composition that
inhibits expression of the target gene in said animal cells,
wherein said muting nucleic acid composition inhibits expression of
the target gene over several cell divisions of said animal
cells.
62. A method to identify a muting nucleic acid composition
according to claim 61, comprising the additional step of
incorporating said nucleic acid composition into a vector
comprising a promoter, wherein said nucleic acid is DNA.
63. A method to identify a muting nucleic acid composition
according to claim 62, wherein said vector causes production of a
transcript having a sequence that is homologous to a sequence in
the target gene.
64. A method to identify a muting nucleic acid composition
according to claim 63, wherein said vector is integrated into a
chromosome of the host, and wherein the structure of the target
gene is unchanged.
65. A method to identify a muting nucleic acid composition
according to claim 63, comprising the additional step of decreasing
steady-state levels of mRNA of the target gene.
66. A method to identify a muting nucleic acid composition
according to claim 65, wherein levels of mRNA of the target gene
decrease by at least 50% within 16 hours after completing
introduction of said nucleic acid composition to said cells.
67. A method to identify a muting nucleic acid composition
according to claim 66, wherein decreased levels of mRNA of the
target gene are maintained over at least five cell cycles.
68. A muting nucleic acid composition, wherein said nucleic acid
composition is identified by: (a) synthesizing said nucleic acid
composition homologous to all or part of the target gene, the
nucleic acid composition being double stranded, (b) introducing
said nucleic acid composition into a cultured population of animal
cells, and (c) selecting said nucleic acid composition that
inhibits expression of the target gene in said animal cells,
wherein said muting nucleic acid composition inhibits expression of
the target gene over several cell divisions of said animal
cells.
69. A muting nucleic acid composition according to claim 68,
wherein a plurality of nucleic acid compositions homologous to all
or part of the target gene are synthesized, and said muting nucleic
acid is identified by transferring said plurality of nucleic acid
compositions to cells and selecting the nucleic acid composition
that inhibits expression of the target gene in said cells
70. A muting nucleic acid composition according to claim 68,
wherein said muting nucleic acid composition is DNA and is
incorporated into a vector comprising a promoter.
71. A muting nucleic acid composition according to claim 70,
wherein said vector causes production of a transcript having a
sequence that is homologous to a sequence of the target gene.
72. A muting nucleic acid composition according to claim 71,
wherein said vector is integrated into a chromosome of the host,
and wherein the structure of the target gene is unchanged.
73. A muting nucleic acid composition according to claim 71,
wherein said vector is a plasmid that does not integrate into a
chromosome of the host.
74. A muting nucleic acid composition according to claim 71,
wherein said muting nucleic acid decreases levels of mRNA of the
target gene.
75. A muting nucleic acid composition according to claim 74,
wherein said levels of mRNA of the target gene decrease by at least
50% within 16 hours after completing introduction of said nucleic
acid composition to said cells.
76. A muting nucleic acid composition according to claim 75,
wherein said decreased levels of mRNA of the target gene are
maintained over at least five cell cycles.
Description
RELATED APPLICATIONS
[0001] This application is a division (continuation) of U.S.
application Ser. No. 09/472,558, filed Dec. 27, 1999, and claims
the benefit of U.S. Provisional Application No. 60/114,107, filed
Dec. 29, 1998. Both applications are hereby incorporated by
reference.
FIELD OF THE INVENTION
[0003] The present invention relates to muting expression of a gene
in animal cells by a transgenic double-stranded (ds) nucleic acid
composition having a sequence that is homologous to a region(s) of
the targeted endogenous gene, and more particularly to muting of a
selected endogenous gene sequence which can be of genomic or
pathogenic origin.
BACKGROUND OF THE INVENTION
[0004] Gene silencing or cosuppression by homologous transgenes
introduced into the genome of plants has raised considerable
interest. A transgene can inactivate the normal (endogenous) gene
or another transgene of the same type in different genomic
locations via a variety of mechanisms (Baulcombe, D. C. et al.,
Curr. Opin. Biotech. 7:173-180 (1996)). These phenomena have
previously been observed in higher plants (Matzke, M. A. et al.,
Plant Physiol. 107: 679-685 (1995)), and related processes involved
in the silencing of duplicated genes have been observed in fungi
(Cogoni, C. et al., EMBO J. 15:3153-3163 (1996); Meyer, P., Biol.
Chem. 377: 87-95 (1996)). Cosuppression, a reciprocal function
involving interactions between the endogenous gene and the
genome-integrated transgene, has been detected in the invertebrate
insect Drosophila (Pal-Bhadra, M. et al., Cell 90:479-490
(1997)).
[0005] In genetically modified plants, transgenes that are stably
maintained can be silenced. Transgenes can in addition cause the
silencing of the endogenous plant genes if they are sufficiently
homologous, a phenomenon known as co-suppression. Silencing occurs
transcriptionally and post-transcriptionally but silencing of
endogenous genes seems predominantly post-transcriptional (Stam, M.
et al., Annals of Botany 79:3-12 (1997)). Various factors seem to
play a role, including DNA methylation (Ingelbrecht, I. et al.,
Proc. Natl. Acad. Sci. USA 91: 10502-10506 (1994)), transgene copy
number and the repetitiveness of the transgene insert (Meyer, P.,
Biol. Chem. 377: 87-95 (1996)), transgene expression level
(Vaucheret, H. et al., Plant Cell 9:1495-1504 (1997)), possible
production of aberrant RNAs (Metzlaff, M. et al., Cell 88:845-854
(1998)), and ectopic DNA-DNA interactions (Baulcombe, D. C. et al.,
Curr. Opin. Biotech. 7:173-180 (1996)).
[0006] An array of cis-acting DNA elements and trans-acting factors
are involved in regulation of expression of pro-collagen genes,
including .alpha.1(I). DNA transfection experiments have shown that
two blocks of both positive and negative regulatory elements,
located in the 5'-flanking region and the first intron, contribute
to the transcriptional regulation of the pro-.alpha.1(I) collagen
gene (Brenner, D. A. et al., Nucl. Acids Res. 17:6055-6064 (1989);
Rippe, R. A. et al., Mol. Cell. Biol. 9:2224-2227 (1989)). In
NIH3T3 mouse fibroblasts, which synthesize large amounts of
collagen (2.2% of total protein), about 220 bp of the mouse
pro-.alpha.1(I) collagen promoter carried on the construct ColCAT3
(also called pColCAT0.2) showed high transcriptional activity,
comparable to that of the highly active SV40 promoter of the
pSV2CAT construct. However, constructs carrying increasingly larger
5'-flanking sequences showed reduced amount of the reporter
chloramphenicol acetyl transferase gene (CAT) activities of between
65% to less than 20% of that of pCOlCAT0.2 (Rippe, R. A. et al.,
Mol. Cell. Biol. 9:2224-2227 (1989)). The reporter gene activity
being measured in these experiments was a fusion to the
pro-.alpha.1(I) collagen promoter carried on the transgenic plasmid
construct.
[0007] The ability to control suppression of gene expression in an
animal cell will enable several practical solutions to current
problems. For example, reducing expression of an oncogenic
transformation effector gene, a drug resistance gene, a
radioresistance gene or a viral gene, by employing an appropriate
gene delivery system, could provide improved treatment for a
variety of cancers and for infections by pathogens, for example,
viral infections. Further, determining the effects of suppression
of activity of a target gene in a cell would be a useful method for
genomic analysis, for example, as a more efficient and rapidly
available alternative to engineering a knock-out animal for
determining the phenotypes of the cells lacking expression of the
target gene. The methods of suppression and the cells thus
suppressed can provide screening tools to identify drugs capable of
reducing gene expression, and also to identify drugs that can
reverse the suppression of gene expression
SUMMARY OF THE INVENTION
[0008] Accordingly, one embodiment of the invention provides a
nucleic acid composition for muting expression of a gene with
unwanted activity in an animal cell, wherein the muting nucleic
acid includes a sequence homologous to an endogenous sequence in
the gene, or homologous to a gene of a pathogen. In this
embodiment, the gene with unwanted activity is carried on a
chromosome or on the genome of the pathogen. Further, the cell is
selected from the group consisting of a normal cell, a diseased
cell, a cell with potential to become diseased, a cancer cell, an
autoimmune cell, a cell of a pathogen, and a cell infected with a
pathogen, for example, wherein a cell infected with a virus.
[0009] A nucleic acid composition of this embodiment is selected
from the group consisting of a DNA, an RNA, and a nucleic acid
analog. Further, the nucleic acid analog is selected from the group
consisting of a phosphorothioate, a 2'-o-methyl RNA, and a
peptide-, lipid- or carbohydrate-nucleic acid. In an embodiment of
the invention, the nucleic acid is double stranded DNA.
[0010] An embodiment provides a nucleic acid composition for muting
expression of an endogenous gene with unwanted activity in an
animal cell, wherein the animal is a vertebrate, for example the
vertebrate is a warm-blooded animal, and further, wherein the
warm-blooded animal is a mammal.
[0011] In another embodiment, the invention provides a method for
muting expression of an endogenous gene having unwanted activity in
a cell of an animal, the method comprising the steps of: (a)
providing a muting nucleic acid; (b) delivering the muting nucleic
acid into the cell; and (c) muting expression of the endogenous
gene. According to this embodiment, the step of providing the
muting nucleic acid includes providing a nucleic acid composition
having a transgene, the transgene having a sequence that is
substantially homologous to a sequence of the endogenous gene with
unwanted activity. In a further embodiment, the transgene sequence
is substantially homologous to an endogenous sequence that is
located within a portion of the endogenous gene selected from at
least one of the group of: the 5' untranscribed portion, the coding
portion including introns, the 3' untranslated portion, the 3'
untranscribed portion, and a portion that overlaps the ends of the
coding portion of the endogenous gene. The endogenous sequence
within the transgene sequence can comprise about 50 to 300 bases in
length, or can comprise about 300 to 600 bases in length, can
comprise about 600 to 1,000 bases in length, or can comprise about
1,000 to 5,000 bases in length.
[0012] In a further embodiment, the invention provides a method
wherein the step of delivering the muting nucleic acid in (b) is
selected from the group of: transforming, transfecting,
electroporating, infecting, and lipofecting the nucleic acid into
the cell. For example, delivering the muting nucleic acid can
comprise infecting the cell with a genetically attenuated bacterium
or virion. Another embodiment is a process whereby the muting
nucleic acid is not substantially integrated into a chromosome,
e.g., in which the muting nucleic acid is located on a plasmid that
is transiently maintained in the cell.
[0013] The invention in one embodiment provides a method for
identifying a muting nucleic acid that reduces expression of an
endogenous target gene having unwanted activity in cells of an
animal, comprising the steps of: (a) providing a set of fragments
of DNA encoding the target gene, wherein the fragments are
engineered into a plurality of vector molecules to produce a
recombinant vector library; (b) delivering the vector library into
the cells, to form a plurality of transgenic cloned fragment
recipients; and (c) comparing expression of the target gene in each
of a subset of the cloned recipients, to expression of the target
gene in the cells of the animal, to identify a cloned recipient
having a vector with the muting nucleic acid, wherein expression of
the target gene is reduced. According to this method, the animal is
warm-blooded, for example, the animal is a mammal. The vector in
one embodiment of the method carries also a chemical resistance
gene conferring a phenotype which is ability to grow in the
presence of the chemical. A method which is an example of the
embodiment can have an additional step of: (a) comparing expression
of the resistance gene in the cell having the muting nucleic acid,
with expression of the resistance gene in the animal cell, wherein
the resistance gene is selected from the group consisting of genes
encoding .beta.-lactamase (AMP) and chloramphenicol acetyl
transferase (CAT), respectively. A method according to this
embodiment can have a further step: (a) comparing expression of a
second endogenous gene which is not the target gene in the cell
having a muting nucleic acid, with expression of the second
endogenous gene in the animal cell, for example in which the second
endogenous gene encodes glyceraldehyde-3-phosphate dehydrogenase
(GADPH).
[0014] In another embodiment, a method is provided of evaluating a
phenotype of animal cells engineered to mute expression of a target
endogenous gene, comprising: (a) transforming animal cells capable
of expressing the target gene with the vector having the muting
nucleic acid obtained according to a method of above; and (b)
observing the transformed cells for an altered phenotype in
comparison to the parental animal cells capable of expressing the
target gene. Thus the altered phenotype under a set of specified
conditions is selected from the group consisting of an alteration
of: growth rate, nutritional requirement, contact inhibition among
confluent cells, formation of foci, tumorigenicity in nude mice or
in a syngeneic rodent strain, presence of a receptor for a ligand,
signal transduction in response to an effector molecule,
sensitivity to a pathogen, expression of a developmental protein,
and cell cycle pattern. The specified conditions different from the
conditions for growth of the parental animal cells capable of
expressing the target gene comprise at least one of the conditions
selected from the group of: an elevated temperature, a depressed
temperature, a decreased serum concentration, an elevated serum
concentration, a decreased carbon dioxide concentration, an
increased carbon dioxide concentration, an increased density of
plating, and a decreased density of plating. The animal cells can
be present in an embryonic or a postnatal animal.
[0015] Yet another embodiment provides a method of screening a
plurality of molecules to obtain a composition capable of muting
expression of an endogenous gene in cells of a cell line,
comprising: mixing a subset of each of the plurality of molecules
with a plurality of samples of the cells, to produce a plurality of
test cell cultures; providing a nucleic acid capable of muting
expression of the gene; transforming the nucleic acid into a sample
of the cells, to produce a positive control cell culture having
muting of expression of the endogenous gene; and detecting an
amount of expression of the endogenous gene in each of the test
cell cultures in comparison with the positive control cell culture
and with untreated cells of the cell line, such that a test cell
culture with substantially reduced expression of the gene compared
to expression in the untreated cells, and substantially equivalent
expression compared to cells in the positive control culture,
identifies the composition capable of muting expression of the
gene. An embodiment of this invention provides that detecting
expression of the endogenous gene comprises analyzing cell RNA by
hybridization with a probe, for example the hybrid of the cell RNA
and the probe is digested with RNase, and further, the digested RNA
is submitted to gel electrophoresis to determine the size of the
cell RNA protected from RNase digestion by the probe.
[0016] Another embodiment of the method provides detecting
expression of the endogenous gene comprises detecting a color
change or absence of a color change in the cells, for example,
wherein the color change in the cells is indicative of expression
of the endogenous gene, where the transgene has been fused to a
second gene having a colorimetric assay. The molecules can be
selected from the group consisting of extracts of natural product
fermentations and synthesized organic chemicals, for example,
organic chemicals that are synthesized according to combinatorial
methods. An embodiment of the invention is a composition obtained
by these methods in a pharmaceutically acceptable carrier.
[0017] A method of screening a plurality of molecules to obtain a
composition capable of alleviating muting of expression of an
endogenous gene in cells of a cell line having a muted endogenous
gene is provided, comprising: mixing a subset of each of the
plurality of molecules with a plurality of samples of the cells
having the muted endogenous gene, to produce a plurality of test
cell cultures; and detecting amounts of expression of the
endogenous gene in each of the test cell cultures in comparison
with the cells of the cell line having the muted endogenous gene,
and in untreated cells of a parental cell line in which the
endogenous gene is not muted, such that a test cell culture with
expression of the gene that is substantially greater than the
expression in the cell line having the muted endogenous gene, and
that is substantially equivalent to expression in cells of the
parental non-muted culture, identifies the composition capable of
alleviating muting of expression of the gene. A composition
identified by this method can be provided in a pharmaceutically
acceptable carrier.
[0018] In another embodiment, the invention provides a kit for
identifying a muting nucleic acid that reduces expression of an
endogenous gene, the kit comprising reagents for assaying
quantitatively both protection of a riboprobe from ribonuclease
digestion, and amount of transfected DNA. A kit provides reagents
which comprise chemicals, stabilized enzymes, and buffers. The
reagents can comprise diethylpyrocarbonate-treated water, placental
RNase inhibitor, tRNA, a buffer containing
piperazine-N,N'-bis(2-ethanesulfonic acid), a DNase I digestion
buffer, phenylmethylsulfonyl fluoride, and gelatin. The stabilized
enzymes can comprise: an RNA polymerase selected from the group of
SP6 RNA polymerase and T7 RNA polymerase; a ribonuclease selected
from the group of RNase I and a mixture of RNases A and T.sub.1;
Taq polymerase; proteinase K; and DNase-free pancreatic RNase.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1 shows a map of pro-.alpha.1(I)-collagen plasmids and
the structure of the RNase protection riboprobe. Positions of the
first five mRNA exons are indicated by open boxes. The vertical
insert marked X/B/X indicates the position of the insertion of the
BamHI linker within an XbaI site in the 5' untranslated portion of
the mRNA. Horizontal solid or dotted lines represent the
procollagen gene sequences. Arrow shows the transcription start
position and direction. Relevant restriction sites for the enzymes
SalI (S), XbaI (X), HindIII (H), PvuII (Pv), BglII (Bg), BamHI (B),
PstI (P), and EcoRI (E) are indicated. The position of the
pro-.alpha.1(I) collagen gene probe transcribed in vitro by T7 RNA
polymerase from pSTBB0.7 (EcoRI digested) is shown. This antisense
riboprobe of about 850 nucleotides (nt) protects the 194 nt
endogenous mouse or rat .alpha.1(I) mRNA corresponding to exon
1.
[0020] FIG. 2 shows RNase protection assays for analysis of
endogenous pro-.alpha.1(I)-collagen mRNA levels in Rat-1, v-fos
transformed 1302-4-1, and revertant EMS-1-19 cells, untransfected
or transiently transfected with pWTC1. The 850 nt antisense
riboprobes transcribed by T7 RNA polymerase from a mouse
pro-.alpha.1(I)-collagen fragment (HindIII/EcoRI) of pSTBB0.7 were
hybridized with total RNA extracted from equal numbers of cultured
cells (about 10.sup.6), of cells either untransfected or
transfected by pWTC1, and harvested at the indicated times after
electroporation. In panel (A), cells were harvested and RNA
extracted 24 h after electroporation. In panel (B), cells were
harvested and RNA extracted 48 h after electroporation.
[0021] FIG. 3 shows specific suppression of
pro-.alpha.1(I)-collagen mRNA by transiently transfected
.alpha.1(I)-collagen genes. Panel (A) is a photograph of RNase
protection results using total RNA from Rat-1, v-fos transformed
and revertant cells, either transfected with plasmid DNA or not.
The .alpha.1(I) antisense riboprobe and the expected protected
bands are as described in FIG. 2. The 473 nt antisense riboprobe
transcribed by T7 RNA polymerase from a rat GAPDH fragment
(SmaI/HindIII) of pLS-1 protects a 361 nt GAPDH mRNA fragment which
serves as an internal standard. Phosphorimager units determined for
each set of GAPDH and .alpha.1(I) bands and the corresponding
.alpha.1(I)/GAPDH ratio are indicated. (a) Cells were harvested 24
h post-electroporation with pWTC1. (b) Cells were electroporated
without any DNA 16 h before harvesting. (c) Cells were harvested 48
h post-electroporation with plasmid. (d) DEAE-dextran transfection
was used in these samples. Viability of cells after this
transfection was less than 20% of that obtained by electroporation,
therefore, less RNA was available for use. (e) A rat 3-actin sense
riboprobe, described in Materials and Methods, was used as a
negative internal control (in addition to tRNA) for RNase
protection assays. Cells used for RNA preparations 17-19 were
harvested at the indicated number of days post-electroporation
(P.E.) with no DNA. Panel (B) shows a determination of plasmid (or
.beta.-lactamase gene) copy numbers corresponding to the
transfected samples in A, using quantitative PCR amplification of a
223 bp fragment of the AMP gene. Lane numbers in A and B are
related. (f) Numbers refer to the observed total number of plasmids
in the total number of transfected cells (10.sup.6-10.sup.7 cells
per determination).
[0022] FIG. 4 shows RNase protection assays that were used to
determine the endogenous pro-.alpha.1(I)-collagen mRNA levels in
Rat-1 cells, untransfected or transiently transfected with either
pColCAT0.2 or pBR322. RNase protection assays and determination of
the corresponding .alpha.1(I)/GAPDH ratio were determined as
described in FIG. 3, at 24 hours after transfection.
[0023] FIG. 5 shows an RNase 1 protection assay-based deletion
mapping of the pro-.alpha.1(I)-collagen promoter region, to
identify collagen mRNA suppressive elements in Rat-1, v-fos
transformed 1302-4-1 and revertant EMS-1-19 cells. The .alpha.1(I)
and GAPDH-rat antisense riboprobes and the expected protected bands
are as described in FIGS. 2 and 3. The .alpha.1(I)/GAPDH
protected-bands ratios, quantitated by PhosphorImager, are
shown.
[0024] FIG. 6 shows ribonuclease A/T1 protection assay used to
identify collagen mRNA suppressive elements in the first five
exon/intron regions in various rodent fibroblast cell lines. Panel
(A) shows an RNase protection assay using total RNA from Rat-1 and
v-fos transformed 1302-4-1 cells untransfected or transiently
transfected with either pSTBB2.6 or pSTBB0.7. Panel (B) shows a
determination of plasmid (or .beta.-lactamase gene) copy numbers
corresponding to the transfected samples in panel A, using
quantitative PCR amplification of a 223-bp fragment of the amp
gene. The .alpha.1(I) and GAPDH-rat antisense riboprobes and the
expected protected bands are as described in FIGS. 2 and 3. The 406
nt antisense GAPDH-mouse riboprobe transcribed from the
pTRI-GAPDH-mouse protects a 316 nt GAPDH-mouse mRNA fragment. Panel
(C) shows a determination of the number of copies of various
plasmids transfected into different rodent cell lines shown in
Panels A and B, using quantitative PCR amplification of the Amp
gene and PhosphorImager analyses.
[0025] FIG. 7 shows an RNase T1 protection assay demonstrating the
absence of antisense pro-.alpha.1(I) collagen mRNA in various rat
fibroblast lines, either untransfected or transiently transfected
by constructs carrying different lengths of the collagen gene. The
GAPDH-rat antisense riboprobe and the expected protected band are
as described in FIG. 3. The 850 nt .alpha.1(I) sense riboprobes
transcribed in vitro by SP6 RNA polymerase from pSTBB0.7 were
predicted to protect 5'-end .alpha.1(I) antisense transcripts of up
to 585 nt. Evaluation of transfections by different plasmids is
shown in FIG. 4C.
DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS
[0026] Unless the context otherwise requires, the terms and phrases
defined below as well as throughout this description, shall be
understood to have the meanings set forth, for purposes of both
this description and the following claims.
[0027] Homology-dependent gene silencing (also known as quelling
and co-suppression) was discovered in plants, fungi, and Drosophila
melanogaster. These terms refer to the phenomenon of reciprocal
silencing among genome-integrated dispersed homologous genes.
Mechanisms may have evolved in eukaryotic organisms to inactivate
expression of multiple copies of genes, gene overexpression, or
abnormal transcription. In fungi and plants, there is evidence that
mechanisms involve DNA-DNA association (Matzke, M. et al. Plant
Phys. 107:679-685 (1995)) or turnover of RNA (Cogoni, C. et al.,
Proc. Natl. Acad. Sci. USA 94:10233-10238 (1997)).
[0028] The genetic regulation observed in embodiments of the
invention herein differs from previously described silencing
phenomena. The term "muting" means a method of using a muting
nucleic acid to efficiently reduce expression of an endogenous
gene, for example located on the genome of a cell, the endogenous
gene having a portion of substantial homology to the transgene.
Muting of the endogenous gene embodied in the examples herein can
be independent of expression of the transgene and integration of
the transgene into the genome, unlike previously described
silencing phenomena.
[0029] The term "transgene" means a gene or gene fragment that is
or has been exogenously supplied to a recipient cell by any of
several procedures known to one of ordinary skill in the art of
recombinant DNA methodologies. A "muting nucleic acid" is a nucleic
acid composition for muting expression of a gene in an animal cell,
wherein the muting nucleic acid includes a double stranded sequence
homologous to an endogenous sequence in the gene. The muting
nucleic acid is comprised of DNA, RNA, a nucleic acid analog, or a
mixture of these. The recipient cell has been transformed into a
transgenic cell. Cells from the same cell line as the recipient
cell which have not been engineered to carry the transgene are
referred to as "parental" or "untreated" cells. In prior reports
describing gene silencing, observations were restricted to cells in
which the transgene was integrated into the genome of the recipient
cell and stably maintained at one or more sites on one or more
chromosomes of the cell.
[0030] An "endogenous" gene or a "target" gene as used herein
generally means a gene or gene fragment that is normally found
indigenous to the genome of the organism, and is therefore
replicatively maintained by the normal mitotic process of cell
division and distributed to gametes by normal meiotic processes. An
endogenous gene indigenous to the cell and having unwanted
expression can be a gene encoding a protein associated with
inflammation, such as a gene encoding TNF-.alpha., for example, or
a gene encoding an MHC class II protein associated with an
autoimmune disease and expressed in an autoimmune cell. An
"autoimmune cell" means an immune cell which has acquired ability
to attach an auto antigen.
[0031] However in certain embodiments an endogenous gene can mean a
gene or gene fragment of a pathogen, such as a virus, bacterium,
fungus, protozoan, or helminth, which can be found in a cell or in
an animal prior to treatment by introduction of a transgene by a
method of the invention herein. An endogenous gene, whether
indigenous to the genome or found in a pathogen, is a target for
the methods of muting as described herein.
[0032] The term "plasmid" means a covalently closed circular DNA
molecule. The plasmids of the present invention can replicate in
microorganisms but not in animal cells. Therefore the plasmids in
transformed recipient animal cells are maintained in the cells into
which they have been introduced for a limited number of cell
divisions, that is, in a substantially transient condition in the
majority of the transformed cells. A plasmid of the invention can
be engineered to carry a eukaryotic origin of replication, enabling
a greater period of maintenance of the plasmid in the recipient
cell.
[0033] The term "transformation" means the genetic process of
causing a muting nucleic acid to enter a cell.
[0034] The term "transfection" means cellular transformation by a
nucleic acid comprising a genetic element from a virus, e.g., the
cohesive ends (cos) of bacteriophage lambda, to enter a cell. The
transformation of a cell can be achieved by the process of
transfection, for example by methods that are chemical in nature
(use of calcium phosphate, DEAE-dextran, lipofection by use of
liposomes) or physical. The term "electroporation" means a physical
process of applying an electric voltage to cells in the presence of
a nucleic acid, causing transient pores in the cell membrane such
that the nucleic acid enters the cell.
[0035] The term "infection" means a biological process of causing a
nucleic acid carried on a cell pathogen (such as a virus or a
bacterium), to enter a cell. The virus or bacterium can be
engineered to deliver a transgene to the recipient cell.
[0036] The methods and compositions of various embodiments of the
present invention can be used to mute an endogenous gene in an
animal cell, for example an oncogene such as ras, or can be used to
mute a viral gene such as a gene encoding a coat protein from
HSV-II or from HIV.
[0037] A composition described herein can be administered in an
effective dose, in a pharmaceutically effective carrier. The term
"effective dose" means that amount of a composition such as a
muting nucleic acid, or a drug having a muting effect, or a drug
capable of reversing a muting effect that is provided to achieve a
therapeutic end point of altering expression of an endogenous gene
in an animal cell. An effective dose can be determined by one of
ordinary skill in the pharmacological art.
[0038] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, e.g., human
albumin or cross-linked gelatin polypeptides, coatings,
antibacterial and antifungal agents, isotonic agents, e.g., sodium
chloride or sodium glutamate, and absorption delaying agents, and
the like that are physiologically compatible. The use of such media
and agents for pharmaceutically active substances is well known in
the art. Preferably, the carrier is suitable for oral, intravenous,
intramuscular, subcutaneous, parenteral, spinal or epidermal
administration (e.g., by injection or infusion). Depending on the
route of administration, the active compound can be coated in a
material to protect the compound from the action of acids and other
natural conditions that can inactivate the compound.
[0039] Dosage regimens are adjusted to provide the optimum desired
response, e.g., a therapeutic response, such as muting of an
endogenous gene. For example, a single bolus can be administered,
several divided doses can be administered over time or the dose can
be proportionally reduced and administered over a time period by
infusion, or increased, as indicated by the exigencies of the
therapeutic situation.
[0040] One of ordinary skill in the medical and pharmacological
arts can determine and prescribe the effective amount of the
pharmaceutical composition required. For example, one could start
doses at levels lower than that required in order to achieve the
desired therapeutic effect and gradually increase the dosage until
the desired effect is achieved. In general, a suitable daily dose
of a composition of the invention will be that amount of the
composition which is the lowest dose effective to produce a
therapeutic effect. Such an effective dose will generally depend
upon the factors described above. It is preferred that
administration be intravenous, intracoronary, intramuscular,
intraperitoneal, or subcutaneous.
[0041] There have been no prior reported phenomena in animal cells
of down regulation of gene expression caused by transgenes which
had not integrated into at least one site on the chromosomes of a
cell's genome. In the transiently transformed cells of the present
invention, in which exogenously added genetic material was not
generally integrated into the chromosome, the phenomenon of "muting
of expression" was observed in several different types of mammalian
cells. Muting of expression observed in the embodiments of the
present invention was non-reciprocal, i.e., expression of the
target endogenous gene was specifically reduced by the presence of
the homologous transgene, however the transgene was self-silent or
was expressed in a dose-dependent manner. All traces of expression
that could be detected in the most highly muted cells were found to
have the physical characteristics of the endogenous gene. Muting as
the term is here defined has not previously been detected in an
animal cell.
[0042] Expression of the pWTC1, a plasmid that carries the entire
pro-.alpha.1(I) collagen gene including 3.7 kb of the 5'-promoter
and 4 kb of the 3'-untranslated sequences, was analyzed in
transient transfection experiments. This plasmid is marked by the
insertion of a linker in the 5'-untranslated region, to enable
distinguishing its transcripts from those of the endogenous
pro-.alpha.1(I) collagen gene (Barker, D. D. et al., Mol. Cell.
Biol. 11:5154-5163 (1991)). The ability to distinguish transcripts
of the endogenous pro-.alpha.1(I) collagen gene from that of the
transgene is exploited herein to monitor expression of each of the
exogenously supplied transgenic gene and the native endogenous
pro-.alpha.1(I) collagen gene. This comparison led to the
surprising finding that the transgene, as well as the endogenous
gene, remained muted in the recipient cells even after several days
of growth of cells and dilution of the plasmid number by the cell
replication process, which is characteristic of a catalytic rather
than a dose-dependent phenomenon. Previous pro-.alpha.1(I) collagen
gene plasmid constructs all were found to express their reporter
genes to some extent.
[0043] The embodiments of the invention herein are described in
examples by which extra-chromosomal pro-.alpha.1(I) collagen genes,
encoded by exogenous plasmids shown in FIG. 1, greatly reduce the
steady-state level of procollagen mRNA transcribed from the
endogenous gene, and completely mute the expression of the
exogenous transgene. The present examples were conducted in
different mammalian cell types, normal (Rat-1 and mouse 3T3)
fibroblasts, FBJ v-fos transformed Rat-1 fibroblasts (1302-4-1),
and a revertant of v-fos-transformed cells (EMS-1-19). The examples
herein show that within hours following cellular transfection by
multiple copies of pWTC1, a set of events were found to occur. The
endogenous pool of pro-.alpha.1(I) collagen mRNA existing prior to
transfection was rapidly degraded, and a much-reduced muted
steady-state level of RNA was established. The same reduced
steady-state level of this mRNA was maintained for several days (up
to at least a period of 4 days as shown by examples herein). The
transgenes also remained transcriptionally muted (FIGS. 2 and 3).
The data in examples herein showed that these events are not
stress-related, but are induced by procollagen-specific DNA
sequences, and manifest equally well in rat and mouse fibroblast
lines (FIGS. 3-6). Evidence for degradation of the endogenous
collagen mRNA following transfection by pWTC1 was shown by the
observation that within 16 hours post-electroporation, the
steady-state level of mRNA for this endogenous gene decreased to
less than 10% in Rat-1 and v-fos transformed cells. Considering
that the half-life of this mRNA is longer than 8 h, the residual
mRNA level 16 hours after transfection would be expected to be no
less than 25%, even assuming no new transcription from this gene
during the experiment. The steady-state mRNA is comprised of
processed cytoplasmic and unprocessed nuclear fractions and a delay
in processing of nuclear RNA could result in its degradation.
[0044] The present invention shows that two distinct and adjacent
portions of the transgenes (-220 to +115 bp and +115 to +585 bp,
with respect to transcription start) contribute to transcriptional
muting of the endogenous procollagen gene in normal and v-fos
transformed rodent fibroblasts, but not in a revertant of
v-fos-transformed Rat-1 cells. Other DNA sequences, from 390 bp
past the first exon/intron boundary to the end of exon-5, and from
-3500 to -220 bp of the 5'-promoter, do not contribute to muting of
this gene. The 3' portion of .alpha.1(I) procollagen gene present
in pWTC1 carries some additional regulatory elements which effect
post-transcriptional muting of the endogenous procollagen gene in
all fibroblast lines, including the revertants. The collagen
transgenes present in pWTC1 remain transcriptionally muted in all
cell lines used in this study. These results indicate that genome
integration and activation of this self-silenced gene by cis-acting
chromosomal factors, not present in pWTC1, are necessary for its
expression. Further examples herein indicate that the muting
phenomena are not regulated by synthesis of antisense
pro-.alpha.1(I) collagen mRNA synthesis complementary to the 5'
portion of the gene.
[0045] Homologous transgene-induced gene muting has significant
potential in gene therapy for viral diseases and for pathological
cell proliferative diseases, and for characterization of phenotypes
of animal cells lacking expression of a target gene. Developing a
transgenic or knock-out animal is an expensive and labor intensive
procedure (Sedlack, B. J., Gen. Eng. News 19 (19):14 (1999)).
Embodiments of the present invention provide methods and
compositions for engineering transgenic animals and animal cells
with a muted endogenous gene, and for evaluating the cells so
engineered. In this manner, the functional genomic purpose of
knocking out a gene can be evaluated. Further, the effect of muting
cells of a tissue or an organ in vivo in a whole animal can
likewise be determined, by providing those cells or tissue or
organs with a muting nucleic acid for an endogenous gene, and
determining the effect on a potential resulting altered
phenotype.
[0046] Embodiments of the invention provide also methods for
screening mixtures of compounds present in extracts of natural
products, or arising from organic synthetic methods including
combinational methods, to obtain a composition capable of causing
muting of an endogenous gene in an animal cell. Similarly, the
extracts or the synthesized compounds can be screened to obtain a
composition capable of reversing muting of an endogenous gene in an
animal cell. Methods of recovery of products from fermentation
broths are known to ordinarily skilled artisans of the science of
natural products, as described in Carlton, G. J. et al., Ch. 30 in
Demain, A. L. et al., "Manual of Industrial Microbiology and
Biotechnology," Washington, D.C.; American Society for
Microbiology, 1986, p. 436; and in Sitrin, R. D. et al., in
"Developments in Industrial Microbiology, Vol. 27," New York;
Elsevier Science Publishing Co., 1987, p. 65.
[0047] Methods of obtaining libraries of compounds are exemplified
by U.S. Pat. No. 5,908,960 and the published patent application
WO97/01560, which are incorporated by reference herein. Additional
enabling patents for construction of particular libraries and
methodologies therefore are found in Caldwell, J. W., Biotech. and
Bioeng. (Combin. Chem.) 61 (1):69-75 (1998).
EXAMPLES
Example 1
Cell Lines, Cell Culture and Transfection
[0048] The generation of FBJ v-fos transformed Rat-1 fibroblasts,
1302-4-1, and revertant EMS-1-19 have been described (Zarbl, H. et
al., Cell 51:357-369 (1987)). Cell culture medium, and
electroporation conditions for transient gene expression have been
described (Bahramian, M. B. et al., PCR Methods Appl. 4:145-153
(1994)).
[0049] Transfection by DEAE-dextran according to an "extended
protocol" was performed as follows: cultured cells were washed
twice with phosphate-buffered saline (PBS). DNA (10 .mu.g) was
applied in a DEAE-dextran solution to the cells, followed by
incubation for 8 h. The DEAE-dextran stock solution is 2 mg/ml
dissolved in PBS, filter sterilized and stored at 4.degree. C. A
working solution contains 10 ml of DEAE-dextran stock, 10 ml of 1 M
Tris HCl, pH 7.3, and 80 ml of serum-free medium, which can be
stored and is stable for several weeks at 4.degree. C. To a culture
dish of cells was added 3-4 ml of DNA in the DEAE-dextran working
solution, to cover a 10 cm plate. After removing the DNA solution,
cells were washed gently twice with PBS, and chloroquine (100 .mu.M
in medium+serum) freshly prepared from stock solution (10 mM
chloroquine in PBS, filtered and stored in the dark at 4.degree.
C.) was applied for 4 h. After removing the chloroquine solution,
cells were gently washed twice with PBS, then complete medium with
serum was added, and cells were incubated for 48 h.
Example 2
Plasmids and Probes for the Pro-.alpha.1(I) Collagen Gene
[0050] Plasmid pWTC1 (Schnieke, A. et al., Proc. Natl. Acad. Sci.
USA 84:764-769 (1987); Slack, J. L. et al., Mol. Cell. Biol. 12:
4714-4723 (1992)) contains the entire wild type mouse
pro-.alpha.1(I) collagen gene, including 3.7 kb of the 5'-flanking
promoter portion and 4 kb of the 3'-flanking DNA. This gene has
been marked by the insertion of a 21-bp XbaI-BamHI-XbaI linker in
the 5'-untranslated portion of the procollagen transcript, which
allows the user to distinguish between endogenous and
transgene-specific .alpha.1(I) mRNAs in an assay of gene
expression, for example, by use of a ribonuclease protection assay.
Plasmid pSTBB2.6 comprises a 2.6 kb BglII DNA fragment containing
the mouse pro-.alpha.1(I) collagen basal promoter, exons 1-5 and
introns 1-4, cloned into the BamHI site of pSP6/T7-19 (GIBCO/BRL
Life Technologies, Inc., Bethesda, Md.). Plasmid pSTBB0.7, used for
riboprobe synthesis in RNase protection studies, was derived from
pSTBB2.6.
[0051] Plasmid pSTBB2.6 was cut by PstI (which cuts at positions
+585 of the pro-.alpha.1(I) collagen gene in the first intron, at
+2067 in the third intron, and in the polylinker), and the 3.5 kb
fragment containing the 5'-end of the .alpha.1(I) gene plus the
vector sequences was isolated and ligated. Digested by EcoRI and
transcribed in vitro by T7 RNA polymerase, this plasmid produces an
antisense transcript of about 850 nt long, which protect 194 nt of
endogenous mouse or rat .alpha.1(I) mRNA.
[0052] Digestion of pSTBB0.7 with PstI and in vitro transcription
by SP6 RNA polymerase, generates sense riboprobes of about 850 nt
long, which could potentially protect antisense-.alpha.1(I) mRNA of
about 600 nt, including the first exon and the 5'-end of the first
intron. Plasmids pColCAT3.5 and pColCAT0.9 (Lichtler, A. et al., J.
Biol. Chem. 264:3072-3077 (1989)) contain respectively 3.6 kb
(-3521 to +115) and 1.0 kb (-947 to +115) of the 5'-untranslated
portion of rat pro-.alpha.1(I) collagen gene fused to the CAT
reporter gene and the simian virus 40 splice and polyadenylation
sequences. Plasmids pColCAT2.3 and pColCAT0.2 contain the mouse
pro-.alpha.1(I) collagen promoters, -2296 to +115 and -220 to +115,
respectively (Rippe, R. A. et al., Mol. Cell. Biol. 9:2224-2227
(1989)).
[0053] The RNA Century Markers, a mixture of five linearized
plasmids, were used as templates for in vitro transcription
reactions for synthesis of labeled molecular size standards
(Ambion, Inc., Austin, Tex.), and pTR1-GAPDH-mouse (Ambion, Inc), a
plasmid that can express RNA complementary to a portion of the
internal control gene, GAPDH mouse, which gives a protected
fragment of 316 bp, was used as an internal standard. The positive
internal control plasmid used in the RNase protection experiments,
pLS-1, was constructed by cloning a Klenow-blunted 361-bp XbaI-NcoI
fragment from pGAPDH-rat into the SmaI site of pGem 3Z.
HindIII-linearized plasmid transcribed by T7 RNA polymerase
produced a riboprobe of 473-nucleotides which protected a DNA of
361 bp. Plasmid pGract, a rat .beta.-actin probe in pGem 3Z,
carries a 637 bp PCR fragment obtained using primers derived from
the human .beta.-actin gene sequence and rat DNA, cloned into the
SmaI site. This plasmid was linearized with EcoRI and transcribed
by SP6 RNA polymerase in vitro. The antisense riboprobe obtained by
this procedure was calculated to be 749 nt, and capable of
protecting a 612 nt fragment. This plasmid was linearized also with
HindIII and transcribed by T7 RNA polymerase, and the sense
riboprobe obtained was used as a negative control in the RNase
protection experiments.
Example 3
RNA Purification
[0054] Cells were harvested and plasmids were purified and
quantitated as described in Bahramian, M. B. et al., PCR Methods
Appl. 4:145-153 (1994), which is hereby incorporated by reference
herein. A sample having one half of the cells was saved for
preparation of DNA from nuclei and determination of transgene copy
number by quantitative PCR, while the other half was used for
isolation of total RNA (procedure adapted from Chomczynski, P. et
al., Anal. Biochem. 162:156-159 (1987)). Since RNA prepared by this
method is contaminated with organic chemicals and plasmid DNA, it
was further purified as follows. The RNA pellet was dissolved in 50
.mu.l of diethyl pyrocarbonate-treated water (DEPC-water), then
precipitated by the addition of 200 .mu.l of 2.5 M ammonium acetate
and 750 .mu.l of ethanol, and incubated at -20.degree. C. for 1 h.
The RNA precipitate was collected by centrifugation at 12,000 g for
5 min at 4.degree. C., redissolved in water and precipitated as
above. The RNA pellet was rinsed with 0.5 ml of 75% ethanol/25% 0.1
M sodium acetate, pH 5.2, and centrifuged for 2 min at 4.degree. C.
The supernatant was decanted, and the RNA pellet was allowed to dry
by incubation at room temperature for a few minutes, and was then
dissolved in 100 .mu.l of DNase I digestion buffer (40 mM Tris HCl,
pH 7.8, 10 mM NaCl, 6 mM MgCl.sub.2, 0.1 mM CaCl.sub.2, and 0.1 mM
dithiothreitol) containing 100 units of placental ribonuclease
inhibitor (RNAguard, Pharmacia LKB) and 1 Kunitz unit of RNase-free
DNase I (Boehringer Mannheim Biochemicals, Indianapolis, Ind.). The
sample was incubated at 37.degree. C. for 15 min, and the DNase
digestion was stopped by the addition of EDTA solution, pH 8.0, to
final concentration of 6 mM.
[0055] The sample was extracted once with an equal volume of
phenol/chloroform/isoamyl alcohol, and once with chloroform/isoamyl
alcohol. The aqueous and organic phases were separated by
centrifugation for 5-10 min at room temperature. The aqueous phase
was transferred to a fresh tube, and the RNA precipitated from the
aqueous phase with 0.3 M sodium acetate, pH 5.2, plus 2.5 volumes
of ice-cold ethanol, and the mixture was incubated on ice for 2 h.
The RNA pellet was collected by centrifugation at 12,000 g for 5
min at 4.degree. C., and rinsed with 75% ethanol/25% 0.1 M sodium
acetate, pH 5.2. The ethanol supernatant was removed completely,
and the open tube was left on the bench for a few minutes to allow
the remaining to evaporate. The RNA pellet was dissolved in 200
.mu.l of TE (Tris HCl 10 mM, EDTA 1 mM), pH 7.6, then 500 .mu.l of
ethanol was added and the preparation was stored at -70.degree. C.
until use.
[0056] To recover RNA for ribonuclease protection assay, 2 .mu.l of
a 10 mg/ml tRNA solution (type V from wheat germ, Sigma-Aldrich,
St. Louis, Mo.) and 22 .mu.l of 3 M sodium acetate, pH 5.2, were
added to the sample, mixed, incubated at -20.degree. C. for 30 min,
and centrifuged at 12,000 g for 5 min at 4.degree. C. The RNA
pellet was dissolved in 200 .mu.l of DEPC-treated water; an aliquot
of one-fifth of the RNA solution was used for the RNase protection
assay. Thus, to the 40 .mu.l aliquot of the RNA solution were added
20 .mu.g of tRNA, 5 .mu.l of 3 M sodium acetate, pH 5.2, and 120
.mu.l of ethanol. RNA was precipitated at -20.degree. C. for 30
min, and pelleted by centrifugation at 12,000 g for 5 min at
4.degree. C. The RNA pellet was dissolved in 30 .mu.l of
hybridization buffer (40 mM PIPES, pH 6.4, 400 mM sodium acetate,
pH 7.0, 1 mM EDTA, and 80% deionized formamide) containing
5.times.10.sup.5 CPM of riboprobe.
Example 4
RNase Protection Assay
[0057] RNase protection analysis (modified from Bornstein, P. et
al., J. Biol. Chem. 263:1603-1606 (1988)) was performed as follows.
.sup.32P-labeled riboprobes were synthesized by in vitro
transcription from appropriate plasmids with either SP6 RNA
polymerase (GIBCO/BRL) or T7 RNA polymerase (Promega, Madison,
Wis.), respectively, with the manufacturer's reagents, buffers and
reaction conditions, in the presence of 50 .mu.Ci of .sup.32P-CTP
(DuPont/NEN, Boston, Mass.; 800 Ci/mmol). Labeled riboprobe
transcripts were treated for 15 min at 37.degree. C. with
RNase-free DNase I (Boehringer-Mannheim), followed by the addition
of 20 .mu.g of tRNA and purification of RNA by
phenol/chloroform/isoamyl alcohol extractions and chromatography on
RNase-free G-50 Quick Spin column (Boehringer-Mannheim). A 0.5
volume of 7.5 M ammonium acetate and 2.5 volumes of ethanol were
added to the column eluate, mixed, and the mixture was placed at
-70.degree. C. for 30 min. The riboprobe was collected by
centrifugation for 10 min at 12,000 g at 4.degree. C. The
supernatant was removed, and the pellet containing the riboprobe
was dissolved in hybridization buffer at 5.times.10.sup.5 CPM/30
.mu.l.
[0058] Ribonuclease 1.TM. (Promega) the preferred enzyme, was used
in the protection experiments according to the manufacturer's
instructions. However, in certain experiments designated in the
Examples, a mixture of ribonuclease A/T1 (or T1 alone) was
substituted for RNase 1 when this enzyme was unavailable. In those
occasions, RNase A and T1 (Ambion, Inc.) were used according to the
manufacturer's protocols. Because the RNase A/T1 mixture is not
highly specific for single-stranded RNA unlike RNase 1, an
internal-control comprising protected RNA was used in each
experiment involving RNase A/T1 as a control to assure that the
proper extent of digestion was achieved.
Example 5
Quantitative Determination of Transfected DNA and Phosphorimage
Analysis
[0059] Quantitation of transiently transfected DNA inside the
nuclei of cells was achieved by a polymerase chain reaction (PCR)
as described previously (Bahramian, M. B. et al., PCR Methods Appl.
4:145-153 (1994)), using a pair of primers
(5'-GTAGTTCGCCAGTTAATAGT, SEQ ID NO. 1; and
5'-GCTGCCATAACCATGAGTGA, SEQ ID NO. 2). These primers amplified a
specific 223 bp DNA fragment from the .alpha.-lactamase gene
(Soberon, X. et al., Gene 9:287-305 (1980)).
[0060] Radioactivity in each of the .sup.32P-labeled bands in dried
polyacrylamide gels containing the results of RNase protection
assays, or in PCR products of transfected DNA, was quantitated by
using a Molecular Dynamics PhosphorImager and the computer software
(Sunnyvale, Calif.). The ratio of pro-.alpha.1(I) collagen major
protected bands to an internal standard RNA, transcribed from the
rat- or mouse-GAPDH gene
(glyceraldehyde-3-phosphate-dehydrogenase), was taken as a measure
of gene expression. The GAPDH gene was found to be expressed
uniformly in the cell lines herein.
Example 6
Effect of Transient Transfection with pWTC1 on Reduction in
Steady-State Level of Endogenous Pro-.alpha.1(I) Collagen mRNA
Caused by Transcriptional and Post-Transcriptional Muting, and
Muting of Transgenes
[0061] To enable determination of transgenic pro-.alpha.1(I)
collagen gene expression in the presence of the endogenous gene
expression, mouse riboprobe vector pSTBB0.7 from plasmid pSTBB2.6
was constructed (see FIG. 1). The antisense in vitro transcripts
from pSTBB0.7 were found to protect a 194 nt endogenous RNA
fragment corresponding to exon-1 of rat pro-.alpha.1(I) collagen
gene (rat and mouse DNA sequences are highly homologous in this
region). However, additional minor protected bands were expected
due to the presence of some nucleotide mismatches between rat and
mouse DNA. The probe was expected to protect a 118 nt and a 76 nt
band from pWTC1, which carries a 21 bp insert in the
5'-untranslated region of the gene.
[0062] FIG. 2 shows data from the rat fibroblast lines as
indicated, which were each electroporated with 10 .mu.g of pWTC1.
After a designated period of cell culture, total RNA was extracted.
RNA from equal number of cells in each sample was hybridized to the
.sup.32P-labeled riboprobe, and was subsequently treated with RNase
1 and analyzed on denaturing polyacrylamide gels.
[0063] The result of the protection assay for each cell line
harvested 24 h post-electroporation is shown in FIG. 2A. Endogenous
rat collagen mRNA protected a 194 nt major band (shown by the
arrow) and some smaller minor bands of mouse .alpha.1(I) probe
after treatment with RNase 1. Mouse .alpha.1(I)-transcripts from
pWTC1 were predicted to protect 118 nt and 76 nt bands. The data
from RNase protection experiment are representative of four
independent assays with similar results. In three cell lines
electroporated with pWTC1 (Rat-1, v-fos transformed and the
revertant), the level of endogenous procollagen mRNA was
surprisingly greatly reduced. Further, expression of the transgenic
procollagen gene as determined by synthesis of mRNA was
undetectable.
[0064] Muting of the ectopic pWTC1-collagen genes appeared
concomitantly with initiation of transcription. Since
post-transcriptional processing and stability of the endogenous RNA
and the exogenous pWTC1-collagen transgene mRNA were observed
previously to be similar in stable tranfectants, and because each
transfected cell contains two copies of the endogenous gene and
hundreds of copies of the transgene, one of ordinary skill in the
art of regulation of gene expression would have expected to detect
more transcription of the transgene and less of the endogenous
gene. Surprisingly, the contrary is true: pWTC1-collagen mRNA was
undetectable even after an extended period of time, and transcripts
of the endogenous gene, although greatly reduced in amount, were
clearly visible. Therefore, suppression of transcription rather
than post-transcriptional mRNA degradation was responsible for the
absence of pWTC1-transcripts. In mouse fibroblast cell lines stably
transfected with pWTC1 so that the gene was integrated into the
cellular genome, the transgenic pro-.alpha.1(I) collagen mRNA was
expressed distinct from and equivalent to the endogenous
.alpha.1(I) mRNA (Barker, D. D. et al., Mol. Cell. Biol. 11:
5154-5163 (1991); Chan, H. et al., Mol. Cell. Biol. 11:47-54
(1991); Slack, J. L. et al., Mol. Cell. Biol. 12: 4714-4723 (1992);
Stacey, A. et al., Nature (London) 332:131-136 (1988)).
[0065] Analysis of the contrasting findings of muting of endogenous
genes and the non-expressed state of the transient transgenes as
shown herein, and those findings reported by others, indicates that
integration of pWTC1-collagen transgene into the chromosome was
necessary for its expression in those studies.
Example 7
Transcriptional Muting as a Function of Post-Electroporation Time
and Comparison to Expression by a Control Endogenous Gene
[0066] The substantial reduction in the steady-state levels of the
endogenous transcripts following transfection by pWTC1 shown herein
can be the result of, without being bound by any particular theory
or mechanism, increased procollagen mRNA turnover rate, or
decreased transcription rate of the endogenous gene, or both.
Evidence exists for degradation of the pre-transfection population
of the procollagen mRNA shortly after ectopic transfection by
pWTC1, and subsequent establishment of a much-reduced steady-state
level of this RNA. Several reports have shown that in most systems
investigated, pro-.alpha.1(I) collagen mRNA is a long-lived
molecule with a half-life of >8 h in adherent cells, whether
growing, quiescent, or replated (Dhawan, J. et al., J. Biol. Chem.
266:8470-8475 (1991)).
[0067] FIG. 2 shows results obtained using RNA prepared only 16 h
after electroporation of Rat-1 cells with pWTC1. This result was
similar to the result obtained in 24 h post-electroporation cells
(FIG. 2A): the level of endogenous collagen mRNA was about 7% that
of the control cells. Assuming a half-life for this mRNA of 8 h and
that RNA turnover was the sole factor in loss of this species, the
residual pro-.alpha.1(I) collagen mRNA prepared at a time point of
16 h after electroporation would be expected to be found at a
minimum only as low as 25% of the control cells, even in the
absence of any de novo transcription. Thus the gene muting observed
here was found to be partly due to a post-transcriptional
component. The results of a 48 h post-electroporation (FIG. 2B) RNA
preparation were also similar to the 24 h point, in spite of
ongoing dilution of the transfected DNA by an additional round of
cell division. To achieve precise quantitation of data showing
relative transcription of an endogenous gene in a cell line from
various experiments, and to compare expression among different cell
lines, the level of endogenous GAPDH mRNA was determined for each
data point. GAPDH was found to be expressed equally in Rat-1, v-fos
transformed and revertant cell lines. Thus, GAPDH mRNA could be
employed as a reliable internal control for the subsequent
ribonuclease protection experiments and for computations of
specific pro-.alpha.1(I) collagen gene expression levels. The data
shown in FIG. 3 illustrate such data with the ratio of
.alpha.1(I)collagen/GAPDH indicated for each lane. This ratio
represents the specific expression of the endogenous collagen gene
under the designated condition, normalized to the GAPDH internal
standard. The specific expression of pro-.alpha.(I) collagen for
samples electroporated with pWTC1 followed by cell culture for
24-48 h, was dramatically reduced in all cells electroporated with
pWTC1, to a level of less than 10% in Rat-1 and v-fos transformed
cells, and to about 30% in the revertant cells (FIG. 3A, lanes
2-13). The average number of transfected plasmids per cell was
estimated by PCR. Expression of the GAPDH gene was unaffected in
these cells.
[0068] Since all of the plasmids carry the .beta.-lactamase gene
(Amp), a fragment of the Amp gene was amplified and quantitated by
phosphorimaging, to determine the plasmid copy number in each of
the recipient cell lines. The data presented in FIG. 3B indicate
the number of plasmids in 10.sup.6-10.sup.7 cells harvested
(generally, several thousand copies per cell) and show that the
extent of gene muting was unaffected by fluctuations in the average
plasmid copy number per cell.
[0069] The above experiments were repeated also with RNA isolated
from cells up to 4 days post-electroporation; the results were
essentially similar to those shown here for 16-48 h. These results
indicate that, shortly after ectopic transfection of the rat
fibroblast cell lines by pWTC1, the endogenous pre-transfection
population of procollagen mRNA was degraded, and a much-reduced
steady-state level of this mRNA was maintained for at least several
days. Absence of the protected RNA bands corresponding to
pWTC1-collagen transgenes in the above experiments indicates that
the transgenes were not transcribed. Since the endogenous and the
transgenic procollagen transcripts have equal stabilities (Slack,
J. L. et al., Mol. Cell. Biol. 12:4714-4723 (1992)), and there are
many more copies of the transgene per cell than the endogenous gene
in the transfectant cells, lack of transcription rather than mRNA
instability was determined to be the primary basis for the absence
of the transgenic pWTC1-collagen transcripts.
Example 8
Transgene-Induced .alpha.1(I) Gene Muting and mRNA Instability are
not Stress Related
[0070] Shock or stress applied to the cells by a process, for
example, by electroporation or trypsin-EDTA treatment for
suspension of the cells, might induce the observed muting
phenomenon. A stress mechanism of gene muting would predict that
extending the post-electroporation incubation time to 48 h and
longer instead of the 24 h used supra, would provide a greater
recovery period and relieve some of the observed suppression.
However this result was not obtained (see, for example, FIGS. 2 and
3). Changing the transfection method to a gentler one, such as
treatment with DEAE-dextran rather than by electroporation, should,
according to this model also reduce the muting, however this result
also was not obtained (FIG. 3, lanes 21-24). Further, to examine
the effect of this enzymatic and mechanical treatment on the
expression of the endogenous collagen gene, control
non-electroporated cells in the 48 h experiment (FIG. 3), were
trypsinized and replated 16 h prior to harvesting. FIG. 3 lanes
16-19 shows RNase-I protection assays on RNA samples from equal
numbers of cells harvested 2, 4, and 6 days after electroporation
in the absence of DNA, respectively, and non-electroporated cells.
The results (lanes 8-13) show that trypsin-EDTA treatment did not
mute endogenous gene expression.
[0071] The data from this experiment show that the shock of
electroporation per se did not alter the pattern of gene expression
for either the internal standard or for the procollagen gene, since
both the absolute values and the ratios of .alpha.1(I)/GAPDH mRNA
were similar, regardless of cell treatment. These data do not
support the hypothesis that specific gene muting is due to cell
shock or stress, but rather these data point to a molecular
intracellular mechanism.
Example 9
Gene Muting and Transcript Destabilization are Mediated by Specific
DNA Sequences
[0072] The construct pColCAT0.2 contains 220 bp of the
pro-.alpha.1(I) collagen promoter plus 115 bp of the untranslated
portion of exon-1 genetically fused to the CAT gene. This construct
has been shown to express the CAT protein efficiently in a number
of different cell systems.
[0073] In Rat-1 cells transfected with this plasmid, the endogenous
procollagen gene was suppressed by 50%, compared to untransfected
cells (FIG. 3, compare lanes 15 and 16). However, the transgenic
mRNA was not detectable, presumably, because it was rapidly turned
over. Transfection of Rat-1 cells by control plasmid pBR322
(carrying only prokaryotic genes), did not alter the level of
expression of the endogenous pro-collagen gene (FIG. 4), but
pColCAT0.2 and pWTC1 both reduced the steady state levels of this
mRNA. These data show that suppression and destabilization of the
endogenous procollagen transcripts are mediated by a sequence
specific mechanism.
[0074] To determine whether negative regulatory sequences in the
transgenes would decrease the transcription activity of the
endogenous .alpha.1(I) promoter, ribonuclease protection
experiments were performed. Following electroporation with various
constructs carrying different lengths of rat or mouse .alpha.1(I)
promoter attached to the CAT gene, the specific expression of the
endogenous collagen gene, and the specific expression of the CAT
gene in different cell lines were determined. Enzyme immunoassay
for the quantitative determination of Escherichia coli CAT protein
in transfected eukaryotic cells was performed by CAT ELISA
(Boehringer-Mannheim), according to the protocols and with the
materials provided by the manufacturer.
[0075] Cells carrying each of the four promoter constructs, rat
pColCAT3.5, mouse pColCAT2.3, rat pColCAT0.9, and mouse pColCAT0.2
(containing, respectively, 3521, 2296, 947, and 222 bp of the
5'-flanking promoter sequences), were found to produce the same
result in the same cell line, compared to untransfected cells of
that cell line (FIG. 5). In Rat-1 and v-fos transformed fibroblasts
transfected with any of these plasmids, the endogenous collagen
mRNA was muted to 50% (FIG. 5, lanes 2-11) compared to the
untransfected parent cells. The plasmid bearing a length of
sequence including the 222 bp proximal promoter and 115 bp of the
beginning of the first exon sequences was sufficient to achieve 50%
muting of the endogenous collagen gene transcription, and the
promoter sequences upstream of -222 were found not to additionally
contribute to muting of expression. These results showed that there
was no relationship between the level of muting of the endogenous
gene and the activity of different promoter constructs.
[0076] In the revertant EMS-1-19 cell line transfected with each of
the various .alpha.1(I) promoter constructs, expression of the
endogenous procollagen genes was not significantly different from
the control, untransfected cells (FIG. 5, lanes 12-16, i.e., no
muting was observed). Transfection efficiencies of different
constructs into each of the different cell lines were comparable.
Taken together, these data show that one or more gene-specific
transcription enhancing factors which interact with the procollagen
proximal promoter were titrated by the presence of the multiple
copies of the exogenous transgenes electroporated into Rat-1 and
v-fos-transformed cells, resulting in decreased transcription from
the endogenous .alpha.1(I) gene.
[0077] Revertant cells, expressing endogenous procollagen
independent of this factor(s), remained unaffected by transfection
with the plasmids carrying different portions of the procollagen
promoter. However, the transcription-start-proximal promoter
sequences cannot account for all of the endogenous gene muting
observed when cells were transfected by pWTC1. This plasmid
contains additional procollagen regulatory elements at the 3'-end,
which caused additional muting of transcription and/or the mRNA
instability in all of the three cell lines. Muting of endogenous
procollagen mRNA by the combined 5' and 3' elements present in
pWTC1 was 70% for the revertant, and greater than 90% for Rat-1 and
v-fos-transformed cells.
[0078] These data show that in transient transfection of
.alpha.1(I)-5'-promoter constructs of various lengths, employing
sensitive techniques of ribonuclease protection and
quantitative-PCR for determinations of mRNA steady-state level and
plasmid copy number in cell nuclei, respectively, the sequences
-222 to +115 caused the endogenous gene muting by 50% in Rat-1 and
v-fos transformed cells. Further upstream sequences, to -3521,
showed no additional muting effect (FIG. 5).
Example 10
Sequences from the Middle of Exon-1 to the Initial Quarter of
Intron-1 Contribute to the Endogenous .alpha.1(I) Procollagen Gene
Muting
[0079] The construct pSTBB2.6 carries a 2.6 kb Bgl II fragment
containing transcription-start-proximal 222 bp upstream of the
mouse .alpha.1(I) promoter, and also exons 1-5 and introns 1-4,
cloned into the Bam HI site of the vector pSP6/T7-19 (GIBCO-BRL).
The plasmid pSTBB0.7 is a deletion construct derived from pSTBB2.6,
which contains the promoter, the first exon and the initial 390 bp
of the first intron (FIG. 1). These constructs were investigated in
Rat-1 and v-fos transformed 1302-4-1 cells for the ability to
further suppress the endogenous collagen gene. Both constructs were
predicted to express the encoded truncated mRNA poorly, by virtue
of two features: they carry the first-intronic sequences which
contain sequences that are inhibitory to the transcription, and
they are unstable because they lack the 3'-end sequences. Since all
of the .alpha.1(I) DNA sequences carried by pColCAT0.2 are present
in constructs pSTBB2.6 and pSTBB0.7, at minimum 50% suppression of
the endogenous .alpha.1(I) gene would be predicted following
transfection of the cells by either of these constructs. Any
additional inhibition would be attributed to the extra exon/intron
sequences carried by these constructs.
[0080] The results (FIG. 6A) show that there was about 70%
reduction of the level of endogenous .alpha.1(I) protected bands in
Rat-1 or v-fos-transformed cells transfected with either pSTBB2.6
or pSTBB0.7. Since these constructs performed similarly in
suppression of the procollagen gene in the protection assays, only
the DNA sequences from +115 to +585, and no other sequences to the
end of exon-5, contributed to the collagen gene muting.
[0081] Additional muting was observed with the construct carrying
the basal promoter and the initial part of exon 1 (-222 to +115),
and further extending to the rest of the exon 1 and 390 nucleotides
of the initial portion of intron 1 (+116 to +585). The two regions
combined resulted in 70% transcriptional muting of the endogenous
collagen gene in Rat-1 and v-fos transformed fibroblasts. Further
downstream sequences, from +586 to the end of exon 5, did not cause
additional decrease of the extent of the endogenous transcripts and
therefore do not carry muting elements (FIG. 6A).
Example 11
Muting of the Procollagen Genes in Fibroblasts of Mouse and Rat
Origin Occurs to the Same Extent
[0082] In order to examine whether the endogenous and exogenous
collagen gene-muting phenomena were unique to the rat fibroblasts,
or were due to transfection of rat cell lines by mouse constructs,
mouse NIH3T3 cells were transfected with each of plasmids pSTBB2.6,
pSTBB0.7, and pWTC1. The purified RNA samples were analyzed by
RNase protection assays, using the mouse .alpha.1(I) and the mouse
internal standard (GAPDH gene) riboprobes. The results obtained
(FIG. 6B) were similar to those observed with rat fibroblasts;
about 70% suppression of the endogenous gene by pSTBB2.6 and
pSTBB0.7, was observed. A dramatic reduction by pWTC1 was found,
although the computation of the specific expression of the latter
was complicated by high noise to signal ratio in the corresponding
lane. No protected band corresponding to pWTC1-.alpha.1(I)
procollagen transcripts was detected, even after prolonged film
exposures, indicating total transcriptional muting of the exogenous
transgenes. Transfection efficiencies of various cells
(1-3.times.10.sup.6 cells recovered after transfection) by
different constructs were comparable (FIG. 5C).
Example 12
Gene Muting is Not Dependent on the Level of Expression of the
Transgene
[0083] Although muting of the endogenous gene was observed using
constructs that carried either the pro-.alpha.(I) collagen gene or
the CAT reporter gene, each transcript included the 5'-untranslated
region of the collagen transcript. If these sequences were involved
in muting, the level of the transcripts present in the recipient
cells might determine the extent of gene muting. CAT assays were
routinely conducted during all transfection experiments, as a
relatively easy control assay to provide a second level of
demonstration of successful transfection of the cells.
[0084] The results in Table 1 are from CAT assays performed on cell
extracts from the same transfection experiments shown in FIG. 5. A
comparison of these two data sets clearly demonstrates that
different promoter constructs with very different rates of gene
expression were equally effective at gene muting. While these data
indicate that there is no relationship between the rate of
expression of the various constructs and the level of the
endogenous gene muting, it is not possible to rule out that a low
undetectable level of transgene expression is required for
muting.
Example 13
Pro-.alpha.1(I) Collagen Gene Muting is Not Regulated by
Differential Antisense mRNA Synthesis Complementary to the Initial
585 bp of the Gene
[0085] Down-regulation of the .alpha.1(I) collagen gene in chick
embryo chondrocytes is accompanied by the presence of large
antisense transcripts of moderate stability that span both ends of
the gene (Farrell. C. M. et al., J. Biol. Chem. 270: 3400-3408
(1995)). To investigate possible involvement of antisense RNA in
regulation of the rat fibroblast pro-.alpha.1(I) collagen gene, and
differential antisense RNA synthesis in the procollagen gene muting
phenomena, RNA obtained from the untransfected and transfected cell
lines was analyzed by RNase protection experiments.
[0086] FIG. 7 shows the results of RNase T1 protection experiments
using total RNA extracted from various rat fibroblast lines either
untransfected, or transfected by one of constructs pWTC1, pSTBB2.6,
and pSTBB0.7. The 850 nt sense riboprobes originate in the vector
upstream of the position -221 bp of the rat .alpha.1(I) promoter
and extend to +585 in the first intron. These probes could anneal
to and protect any antisense RNA of up to 806 nt long in the 5'-end
of the gene. The transfected cell lines would be expected to show
increased intensity of this protected antisense RNA in comparison
to control cells, if antisense RNA were present.
[0087] The significant amount of radioactive probe with increased
mobility over that of the full length probe in FIG. 7 indicates
that at least half of the probe was digested during the experiment.
Since in this experiment, protection by antisense RNA would have
been detected had protected product in fact been present, and as it
was not detected, then in the cell lines of the present invention,
the gene muting of .alpha.1(I) was not mediated by synthesis of
antisense RNA.
[0088] Without being bound by any particular mechanism of the
muting of gene expression, it is likely that both the rate of
transcription and the post-transcriptional stability of the
endogenous procollagen mRNA were decreased in the normal and v-fos
transformed cells transfected with pWTC1. Further, in the revertant
cell line, the endogenous procollagen gene which is partially
liberated from the mechanisms of v-fos-induced suppression was
liberated also from the transgene-induced transcription muting, but
not from pWTC1-induced post-transcriptional degradation. Data here
show that the transcriptionally active 220 bp procollagen basal
promoter construct, present in pColCAT0.2 and in promoter
constructs of greater length, was transiently transfected into
Rat-1 or v-fos transformed cells, and inhibited transcription of
the endogenous collagen gene by at least 50%, presumably by
competition for at least one transcription enhancing factor.
However, introduction of the same transgenes into the revertant
cells had no effect on the transcription rate (as determined by the
steady-state level) of the endogenous collagen mRNA. Since pWTC1
transfection of the revertant cells reduced the steady-state level
of the endogenous transcripts by 70%, the regulatory element(s)
present at the 3' region of this gene (which are not present in the
5'-promoter constructs) can effect post-transcriptional muting of
this gene. This mechanism could also explain the rapidity with
which a low level steady-state mRNA was obtained in all cell lines
analyzed herein following pWTC1-transfection.
[0089] To investigate whether differential antisense RNA synthesis
plays a part in muting of the procollagen gene in rat fibroblast
cell lines, the examples here analyzed RNA from transfected and
untransfected cells by ribonuclease protection assays. No antisense
RNA corresponding to the first five exons and four introns of the
gene could be detected (FIG. 7).
Example 14
Muting by a Transgene can be Obtained Independent of Expression
Level
[0090] In the Examples above, substantial muting of an endogenous
gene in the absence of expression of the transgene supplied on an
exogenous nucleic acid was observed. In addition, substantial
muting of an endogenous target gene may be obtained even in the
presence of some transcription and translation of the transgene.
With a transgene which is a gene fragment rather than an entire
gene, the transgene can be transcribed, however as the resulting
RNA product lacks a proper 3' terminus, the half-life of the RNA in
a cell will be substantially reduced compared to that of a full
length transcript. Further, the translation product is an
incomplete peptide fragment translated from a 5' RNA fragment,
which is physiologically unstable in vivo. The amount and stability
of the peptide fragment can be further reduced by engineering
translation stop codons (UAG, UAA and UGA) into the sequence in the
correct reading frame.
[0091] These considerations indicate that in a method to obtain a
muting gene fragment, a successful outcome can be achieved, and a
muting nucleic acid can be obtained, in the presence of some level
of expression of the transgene. Once the muting nucleic acid is
obtained, it can be further engineered by recombinant and nucleic
acid synthetic methods to be used to reduce the amount of
expression of the target gene.
Example 15
Muting of Endogenous Genes Encoding an Unwanted Growth Factor, an
Autoimmune Gene, or a Viral Gene
[0092] The embodiments of the present invention include methods for
muting one or more endogenous genes associated with various disease
states, such as a gene encoding TNF-.alpha., overexpression of
which is associated with inflammation and wasting, a gene for an
autoimmune-disease associated antibody, and a gene of a pathogenic
organism such as the tat gene of a strain of human immunodeficiency
virus, HIV.
[0093] The tat gene encodes a positive transacting regulatory 86
amino acid protein that is required for extension of HIV
transcription initiated in the 5'-LTR promoter (U.S. Pat. No.
5,804,604; Daelemans, D. et al., Antivir. Chem. Chemother. 10:1-14
(1999)). This protein also regulates expression of genes encoding
TNF.alpha. and TGF.beta.-1 in CNS cells (Sawaya, B. et al., J.
Neuroimm. 87:33-42 (1998)). In the condition of integration in the
genome of an infected cell, HIV-1 is transcriptionally silent. The
transition to a stage of viral expression and replication requires
tat expression and subsequent tat transactivation of other HIV
genes. A nucleosome binding site, nuc-1, is positioned between -5
and +155 of this gene, and activation of HIV-infected T cells
results in disruption of this nucleosome and increased HIV-1
transcription (Widlak, P. et al., Acta Biochim. Polon 45
(1):209-219 (1998)).
[0094] Isolation of a nucleic acid capable of muting the tat gene
is desirable for use in preventing intracellular HIV replication
and maintenance of the HIV genome in a quiescent condition.
Previous approaches to inhibition of HIV using a tat-inducible
vector show that HIV infection is related to levels of expression
of an exogenously provided gene encoding an inhibitor (Fraisier, C.
et al., Gene Ther. 5: 1665-1676 (1998)). In contrast to this
previous approach, muting of the tat gene as provided herein should
not require expression of the exogenous transgene.
[0095] U.S. Pat. No. 5,837,512 shows vectors carrying various
portions of the HIV genome, and vectors carrying portions with
mutations at each of several sites. Muting DNA having HIV-1 genes
or gene fragments that carry one or more binding sites for cellular
transcription factors NF-.kappa.B and Sp1, or lack each one or both
of these sites, is provided to an infected cell by transformation
of such nucleic acids on a non-integrating vector, which is
maintained in a transient non-integrated state. RNA from the
treated and untreated cells is prepared, and the level of
tat-specific RNA is measured by RNase protection using a probe
having a sequence from the tat gene. Useful restriction sites for
construction of these vectors and examples of these vectors are
shown in U.S. Pat. No. 5,837,512, which is hereby incorporated by
reference herein.
[0096] Isolation of the smallest effective length of the muting
nucleic acid can be achieved by purification and subcloning of
different fragments of HIV, starting from within the 5'-LTR (long
terminal repeat having the promoter), and extending into the tat
gene. Initially, large fragments (up to 2 kb) are tested for muting
nucleic acid activity. Upon obtaining a positive muting response,
the active portion can be isolated by subsequent restriction enzyme
digestion, purification of fragments, cloning of each fragment into
the vector, and testing each for having a muting activity.
[0097] TNF.alpha. regulates expression of several receptors in
vascular endothelial cells (Giraudo, E. et al., J. Biol. Chem.
273:22128-35 (1998)) and TNF promoter polymorphisms affect
transcriptional activation (Wilson, A. G., Proc. Natl. Acad. Sci.
USA 94:3195-3199 (1997)). One polymorphism is associated with
susceptibility to alcoholic steatohepatitis (Grove, J. et al.,
Hepatology 26:143-146 (1997)). Cell specific regulation of the
human TNF.alpha. gene has been shown with cell transcription
factors NFATp and AFT-2/JUN (Tsai, E. et al., Mol. Cell Biol.
16:5232-5244 (1996)).
[0098] Down regulation by expression of the TNF.alpha. gene has
utility for a number of conditions, for example, it can activate
transcription factor 2, which increases UVC-induced apoptosis of
late-stage melanoma cells (Ivanov, V. et al., J. Biol. Chem.
274:14079-14089 (1999)). A muting nucleic acid for the endogenous
TNF.alpha. gene can be provided by the methods herein, using a
vector which is transiently maintained, the vector carrying each of
a variety of 5'-fragments of this gene or the entire gene.
[0099] Muting nucleic acids can be provided to turn off expression
of a gene encoding an immunoglobulin which is associated with an
autoimmune disease. Autoimmune diseases include multiple sclerosis,
systemic lupus erythematosus, and rheumatoid arthritis. Antibody
molecules generally have a heterotetrameric quaternary structure,
and include two copies of each of a heavy (H) chain and light (L)
chain, so that any single antibody species is encoded by an H gene
and an L gene. Substantial information is available on
transcriptional regulation of expression of immunoglobin genes
(Currie, R. A. Nucl. Acids Res. 18:2987-2992 (1990)): Lefranc, G.
et al., Biochimie 72:7-17 (1990)); Staudt, L. et al., Ann. Rev.
Immun. 9:373-398 (1991)); and Wang, J. et al. Mol. Cell Bio.
11:75-83 (1991)).
[0100] To suppress expression of that molecule, a muting nucleic
acid for the H gene or the L gene or both can be provided on a
vector which is transiently maintained in the cell. Since antibody
synthesis occurs primarily in leukocytes, for example in B cells,
these cells can be isolated by methods known in the art from blood
of a subject having an autoimmune disease (autoimmune cells), and
the muting nucleic acid can be provided ex vivo. Alternatively, in
vivo delivery of the muting nucleic acid can be achieved by use of
methods that direct the nucleic acid to the leukocytes. The muting
nucleic acid can be isolated from DNA fragments of upstream 5'
portions of a gene encoding an H or an L chain, and extending into
the gene. Further subcloning of the active portion can be achieved
as described above.
1TABLE 1 Specific Transient Expressions of Various Rodent
pro-.alpha.1 (I) collagen Promoter-CAT Constructs in Rat-1, v-fos
Transformed 1302-4-1 and Revertant EMS-1-19 Cell Lines. Promoter
Activity.sup.a (pg CAT/plasmid copy number .times. 10.sup.-7) Cell
Lines Constructs Rat-1 v-fos transformants Revertant
pColCAT3.5.sup.b 0.7 .+-. 0.2 ND.sup.d ND.sup.d pColCAT2.3.sup.c
9.8 .+-. 0.5 1.7 .+-. 0.2 5.0 .+-. 0.3 pColCAT0.9.sup.b 4.7 .+-.
0.4 1.0 .+-. 0.2 2.3 .+-. 0.2 pColCAT0.2.sup.c 18.1 .+-. 2.5 3.2
.+-. 0.3 21.2 .+-. 3.6 .sup.aData are obtained from the same
samples used in FIG. 4. Transfected DNA and CAT enzyme
determinations are described in Examples 5 and 9, respectively.
Data are expressed as the mean of three determinations plus and
minus the standard error of the mean. .sup.bRat promoter.
.sup.cMouse promoter. .sup.dND: not detected.
[0101]
Sequence CWU 1
1
2 1 20 DNA Artificial Sequence PCR Primer 1 gtagttcgcc agttaatagt
20 2 20 DNA Artificial Sequence PCR Primer 2 gctgccataa ccatgagtga
20
* * * * *