U.S. patent application number 10/990355 was filed with the patent office on 2005-07-21 for self-cleaving ribozymes and uses thereof.
This patent application is currently assigned to Children's Hospital. Invention is credited to Mulligan, Richard, Yen, Laising.
Application Number | 20050158741 10/990355 |
Document ID | / |
Family ID | 34619397 |
Filed Date | 2005-07-21 |
United States Patent
Application |
20050158741 |
Kind Code |
A1 |
Mulligan, Richard ; et
al. |
July 21, 2005 |
Self-cleaving ribozymes and uses thereof
Abstract
In certain embodiments, the disclosure relates to compositions
and methods relating to a ribozyme-based gene regulation system
that functions in mammalian cells. In certain specific embodiments,
the disclosure relates to schistosome self-cleaving RNA mutant
motifs.
Inventors: |
Mulligan, Richard;
(Cambridge, MA) ; Yen, Laising; (Stoughton,
MA) |
Correspondence
Address: |
FISH & NEAVE IP GROUP
ROPES & GRAY LLP
ONE INTERNATIONAL PLACE
BOSTON
MA
02110-2624
US
|
Assignee: |
Children's Hospital
Boston
MA
|
Family ID: |
34619397 |
Appl. No.: |
10/990355 |
Filed: |
November 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60519941 |
Nov 14, 2003 |
|
|
|
Current U.S.
Class: |
435/5 ; 435/456;
435/6.13; 514/44A; 536/23.1 |
Current CPC
Class: |
C12N 15/63 20130101;
C12N 2310/12 20130101; C12N 15/113 20130101; A61P 31/12 20180101;
A61P 31/18 20180101; C12N 2310/11 20130101 |
Class at
Publication: |
435/006 ;
514/044; 536/023.1; 435/456 |
International
Class: |
C12Q 001/68; C07H
021/02; A61K 048/00; C12N 005/08 |
Claims
1. A self-cleaving ribozyme, which efficiently cleaves an RNA
molecule which comprises the self-cleaving ribozyme in a mammalian
cell.
2. A schistosome ribozyme mutant, comprising a loop on stem
III.
3. The ribozyme mutant of claim 2, wherein the loop comprises at
least three nucleotides.
4. The ribozyme mutant of claim 2, wherein the loop is selected
from the group consisting of: 5'-UUCG-3',5'-CUUCGG-3', and
5'-GCUUCGGU-3'.
5. The ribozyme mutant of claim 2, further comprising at least one
nucleotide substitution in the ribozyme core sequence.
6. The ribozyme mutant of claim 2, wherein the nucleotide C at
position 7 is substituted with a nucleotide selected from the group
consisting of: U, A and G.
7. The ribozyme mutant of claim 2, comprising a schistosome
ribozyme mutant having a nucleotide sequence selected from SEQ ID
NOs: 1-63.
8. A self-cleaving RNA motif having the nucleotide sequence as set
forth in SEQ ID NO: 64, wherein, each N is independently any
nucleotide; each A, U, G and C, is adenosine, uridine, guanosine
and cytidine, respectively; n is 3-40; and X at the 5' and Y at the
3' end of said self-cleaving RNA motif are meant to indicate
additional nucleotides which may be present or absent.
9. A self-cleaving RNA motif of claim 8, having the nucleotide
sequence as set forth in SEQ ID NO: 65, wherein, N is any
nucleotide; each A, U, G and C, is adenosine, uridine, guanosine
and cytidine, respectively; and X at the 5' and Y at the 3' end of
said self-cleaving RNA motif are meant to indicate additional
nucleotides which may be present or absent.
10. The self-cleaving RNA motif of claim 9, wherein N is the
nucleotide U.
11. (canceled)
12. A nucleic acid encoding a schistosome ribozyme mutant, wherein
the schistosome ribozyme mutant comprises a loop on stem III.
13. A host cell transformed with the recombinant nucleic acid of
claim 12.
14. (canceled)
15. (canceled)
16. A recombinant polynucleotide molecule comprising: (a) a
promoter; (b) a nucleic acid encoding a nucleic acid product; and
(c) a nucleic acid encoding a schistosome ribozyme mutant motif,
wherein the nucleic acid of (b) and the nucleic acid of (c) are
operably linked to the promoter and transcription of the nucleic
acid of (b) and the nucleic acid of (c) produces a RNA molecule
comprising said schistosome ribozyme mutant motif and a mRNA
encoding said nucleic acid product, wherein said schistosome
ribozyme mutant motif is capable of cleaving said RNA molecule
intramolecularly.
17. The polynucleotide molecule of claim 16, wherein the nucleic
acid of (c) further comprises a nucleic acid encoding an aptamer
which is at a position such that the cleaving activity of the
schistosome ribozyme mutant motif is regulatable by binding of an
effector to the aptamer.
18. The polynucleotide molecule of claim 16, wherein the nucleic
acid of (c) encodes at least two schistosome ribozyme mutant
motifs.
19. The polynucleotide molecule of claim 16, wherein the
self-cleaving RNA mutant motif includes a loop on stem III.
20. (canceled)
21. (canceled)
22. (canceled)
23. (canceled)
24. (canceled)
25. A host cell comprising the polynucleotide molecule of claim
16.
26. The host cell of claim 25, wherein the cell is a mammalian
cell.
27. The host cell of claim 25, wherein the cell further comprises
an inhibitor of the self-cleaving RNA mutant motif.
28. A viral vector comprising: (a) a promoter; (b) a nucleic acid
encoding a nucleic acid product; (c) a nucleic acid encoding a
schistosome ribozyme mutant motif, wherein the nucleic acid of (b)
and the nucleic acid of (c) are operably linked to the promoter and
transcription of the nucleic acid of (b) and the nucleic acid of
(c) produces a RNA molecule comprising said schistosome ribozyme
mutant motif and a mRNA encoding said nucleic acid product, wherein
said schistosome ribozyme mutant motif is capable of cleaving said
RNA molecule intramolecularly.
29. (canceled)
30. (canceled)
31. (canceled)
32. (canceled)
33. A method of inducing expression of a nucleic acid product in a
host cell comprising contacting said host cell with an agent which
inhibits cleavage of a self-cleaving schistosome RNA mutant motif,
wherein the host cell comprises: (a) a promoter; (b) a nucleic acid
encoding the nucleic acid product; and (c) a nucleic acid encoding
a self-cleaving RNA mutant motif, wherein the nucleic acid of (b)
and the nucleic acid of (c) are operably linked to the promoter,
and transcription of the nucleic acid of (b) and the nucleic acid
of (c) produces a RNA molecule comprising said self-cleaving
schistosome RNA mutant motif and mRNA encoding said nucleic acid
product, wherein said self-cleaving schistosome RNA mutant motif is
capable of cleaving said RNA molecule intramolecularly.
34. The method of claim 33, wherein the agent is an antibiotic.
35. The method of claim 33, wherein the agent is selected from the
group consisting of toyocamycin, 8-azaadenosine, sangivamycin,
tubercidin, tubercidin-cyclic monophosphate,
tubercidin-monophosphate, tubercidin-triphosphate, nebularine,
tricyclic nucleoside, 5-fluorouridine, 5-bromouridine,
5-fluorouracil, Syto-83, homidium bromide, and acridine orange.
36. The method of claim 33, wherein the nucleic acid of (c) encodes
at least two self-cleaving RNA mutant motifs.
37. The method of claim 33, wherein the self-cleaving schistosome
RNA mutant motif includes a loop on stem III.
38. (canceled)
39. (canceled)
40. (canceled)
41. (canceled)
42. A method of inducing expression of a nucleic acid product in an
individual, comprising the steps of: (a) obtaining cells from the
individual under conditions appropriate for cell growth and cell
division; (b) introducing into the cells obtained in step (a) a DNA
construct which comprises: (1) a promoter; (2) a nucleic acid
encoding a nucleic acid product; and (3) a nucleic acid encoding
said self-cleaving schistosome RNA mutant motif, wherein the
nucleic acid of (2) and the nucleic acid of (3) are operably linked
to said promoter, and transcription of the nucleic acid of (2) and
the nucleic acid of (3) produces a RNA molecule comprising said
self-cleaving schistosome RNA mutant motif and mRNA encoding said
nucleic acid product, wherein said self-cleaving schistosome RNA
mutant motif is capable of cleaving said RNA molecule
intramolecularly; (c) returning the cells produced in step (b) to
the individual; and (d) administering to the individual an agent
which is capable of inhibiting cleavage of the self-cleaving
schistosome RNA mutant motif.
43. The method of claim 42, wherein the agent is an antibiotic.
44. A method of inducing expression of a nucleic acid product in a
host cell comprising contacting said host cell with an antisense
oligonucleotide of a self-cleaving schistosome RNA mutant motif,
wherein the host cell comprises: (a) a promoter; (b) a nucleic acid
encoding the nucleic acid product; and (c) a nucleic acid encoding
a self-cleaving RNA mutant motif, wherein the nucleic acid of (b)
and the nucleic acid of (c) are operably linked to the promoter,
and transcription of the nucleic acid of (b) and the nucleic acid
of (c) produces a RNA molecule comprising said self-cleaving
schistosome RNA mutant motif and mRNA encoding said nucleic acid
product, wherein said self-cleaving schistosome RNA mutant motif is
capable of cleaving said RNA molecule intramolecularly.
45. The method of claim 44, wherein the self-cleaving schistosome
RNA mutant motif includes a loop on stem III.
46. The method of claim 44, wherein the antisense oligonucleotide
base pairs with a region of the self-cleaving schistosome RNA
mutant motif as set forth in SEQ ID NO: 67.
47. The method of claim 44, wherein the antisense oligonucleotide
is a modified oligonucleotide selected from the group consisting
of: morpholino, phosphorothioate RNA, 2'-O-methyl RNA, and
phosphorothioate 2'-O-methoxyethyl RNA.
48. (canceled)
49. (canceled)
50. The modified antisense oligonucleotide of claim 48, which
oligonucleotide base pairs with a region of the self-cleaving
schistosome RNA mutant motif as set forth in SEQ ID NO: 67.
51. A method of screening for an agent which is capable of
inhibiting the catalytic activity of a schistosome ribozyme mutant
comprising: (a) introducing into host cells a DNA construct which
comprises: (1) a promoter; (2) a nucleic acid encoding a reporter;
and (3) a nucleic acid encoding a schistosome ribozyme mutant,
wherein the nucleic acid of (2) and the nucleic acid of (3) are
downstream of the promoter and operably linked to said promoter;
(b) contacting said host cells with an agent to be assessed for its
ability to inhibit catalytic activity of said schistosome ribozyme
mutant under conditions appropriate for expression of said
reporter; and (c) assaying reporter activity, wherein detection of
reporter activity in the presence of said agent greater than
reporter activity in the absence of the said agent identifies the
agent as one which inhibits the catalytic activity of said
schistosome ribozyme mutant.
52. A method for producing a transgenic nonhuman animal comprising
introducing a DNA construct into a germ cell of a nonhuman animal
or a germ cell of an ancestor of said animal, wherein said DNA
construct comprises: (a) a promoter; (b) a nucleic acid encoding a
desired nucleic acid product; and (c) a nucleic acid encoding a
schistosome ribozyme mutant, wherein the nucleic acid of (b) and
the nucleic acid of (c) are operably linked to said promoter and
transcription of the nucleic acid of (b) and the nucleic acid of
(c) produces a RNA molecule comprising said schistosome ribozyme
mutant and a mRNA encoding said nucleic acid product, wherein said
schistosome ribozyme mutant is capable of cleaving said RNA
molecule intramolecularly.
53. The method of claim 52, wherein the nucleic acid of (3) further
comprises nucleic acid encoding an aptamer which is at a position
such that the cleaving activity of said schistosome ribozyme mutant
is regulatable by binding of an effector to said aptamer.
54. A method for determining the level of an inhibitor of a
schistosome ribozyme mutant in a cell, comprising: (a) introducing
into a cell a DNA construct which comprises: (1) a promoter; (2) a
nucleic acid encoding a reporter; and (3) a nucleic acid encoding a
schistosome ribozyme mutant, wherein the nucleic acid of (2) and
the nucleic acid of (3) are downstream of the promoter and operably
linked to said promoter, under conditions which result in
inhibition of the ribozyme mutant and expression of the reporter;
and (b) assaying reporter activity in the cell produced by (a),
wherein the level of said inhibitor in the cell is identified by
comparing the reporter activity with an appropriate control.
55. The method of claim 54, wherein the inhibitor is selected from
the group consisting of: 5-fluorouracil and 5-fluorouridine.
56. The method of claim 54, wherein the cell is a cancer cell.
57. A method for determining the level of an inhibitor of a
schistosome ribozyme mutant in a biological sample, comprising: (a)
contacting a cell with the biological sample, wherein the cell
expresses a DNA construct which comprises: (1) a promoter; (2) a
nucleic acid encoding a reporter; and (3) a nucleic acid encoding a
schistosome ribozyme mutant, wherein the nucleic acid of (2) and
the nucleic acid of (3) are downstream of the promoter and operably
linked to said promoter, under conditions which result in
inhibition of the ribozyme mutant and expression of the reporter;
and (b) assaying reporter activity in the presence of the
biological sample, wherein the level of said inhibitor in the
biological sample is identified by comparing the reporter activity
with an appropriate control.
58. A method of inhibiting activity of a catalytic RNA in a cell,
comprising contacting a cell with an inhibitor of a schistosome
ribozyme mutant.
59. The method of claim 58, wherein the cell is infected with a
virus or a pathogenic microorganism.
60. The method of claim 58, wherein the inhibitor is selected from
the group consisting of toyocamycin, 8-azaadenosine, sangivamycin,
tubercidin, tubercidin-cyclic monophosphate,
tubercidin-monophosphate, tubercidin-triphosphate, nebularine,
tricyclic nucleoside, 5-fluorouridine, 5-bromouridine,
5-fluorouracil, Syto-83, homidium bromide, and acridine orange.
61. The method of claim 58, wherein the inhibitor is an antisense
oligonucleotide.
62. A method of inhibiting infection by a virus or a pathogenic
microorganism in a cell, comprising contacting a cell with an
inhibitor of a schistosome ribozyme mutant.
63. The method of claim 62, wherein the infection is caused by a
virus selected from the group consisting of a human
immunodeficiency virus, a herpes virus, a hepatitis virus, and a
human papillomavirus.
64. The method of claim 62, wherein the infection is caused by a
pathogenic microorganism selected from the group consisting of
Notophthalmus viddescens, Ambystoma talpoideum, Amphiuma
tridactylum, and Schistosoma mansoni.
65. The method of claim 62, wherein the cell is a mammalian
cell.
66. The method of claim 62, wherein the cell is a plant cell.
67. A kit for regulating gene expression, comprising a nucleic acid
comprising: (a) a schistosome ribozyme mutant sequence; and (b) a
cloning site for introduction of a target nucleotide sequence to be
transcribed operatively linked to the schistosome ribozyme mutant
sequence.
68. The kit of claim 67, further comprising an inhibitor of the
schistosome ribozyme mutant, wherein transcription of the target
gene is inhibited in the absence of the inhibitor.
69. The kit of claim 67, wherein the schistosome ribozyme mutant
sequence comprises a nucleotide sequence selected from SEQ ID NOs:
1-63.
70. The kit of claim 67, wherein the schistosome ribozyme mutant
comprises a loop on stem III.
71. The kit of claim 67, wherein the nucleic acid comprises at
least two self-cleaving RNA mutant motifs.
Description
RELATED APPLICATIONS
[0001] This application claims the benefit of priority of U.S.
Provisional Application No. 60/519,941, entitled "Self-Cleaving
Schistosome RNA Mutant Motifs" and filed Nov. 14, 2003. The
teachings of the referenced Provisional Application are
incorporated herein by reference in their entirety.
BACKGROUND OF THE INVENTION
[0002] RNA enzymes (ribozymes) are being developed as treatments
for a variety of diseases ranging from inborn metabolic disorders
to viral infections and acquired diseases such as cancer. Ribozymes
can be used both to down-regulate and to repair pathogenic genes.
In some instances, short-term exogenous delivery of stabilized RNA
is desirable, but many treatments will require viral-mediated
delivery to provide long-term expression of the therapeutic
catalyst. Although some variations on naturally occurring ribozymes
are available, they have not been very effective in mammalian
cells. There is a need to develop modified ribozymes that show
improved activity and function in mammalian cells with high
efficiency. These ribozymes are useful for developing regulated
gene expression systems and have great therapeutic values.
SUMMARY OF THE INVENTION
[0003] The present invention relates to compositions and methods
for novel ribozyme-based gene regulation systems that function in
mammalian cells. In certain aspects, the present invention provides
a self-cleaving ribozyme, which efficiently cleaves an RNA molecule
that comprises the self-cleaving ribozyme in a mammalian cell. The
term "ribozyme" as used herein, include naturally-occurring
(wildtype) ribozymes and modified ribozymes (referred to as mutants
or variants). An exemplary ribozyme of the invention is a
schistosome ribozyme and mutants thereof.
[0004] In certain embodiments, the present invention relates to
self-cleaving schistosome RNA mutant motifs, also referred to as
schistosome RNA mutant motifs, schistosome ribozyme mutants and
modified schistosome ribozymes. These terms are used herein
interchangeably. The self-cleaving schistosome RNA mutant motifs of
the invention include a modification at a position in a
self-cleaving schistosome RNA motif that results in modulation or
alteration of the cleaving activity of the self-cleaving
schistosome RNA motif. Self-cleaving schistosome RNA motifs or
schistosome ribozymes are members of the hammerhead ribozyme family
and are characterized by their secondary structure. Hammerhead
ribozymes are composed of structural elements including three
helices, referred to as stem I, stem II and stem III, and joined at
a central core of 11-12 single strand nucleotides. Hammerhead
ribozymes may also contain loop structures extending from some or
all of the helices. These loops are numbered according to the stem
from which they extend (e.g., loop I, loop II, and loop III).
Schistosome RNA mutants of the present invention differ from a
naturally occurring self-cleaving schistosome RNA by one or more
modifications, which can be addition, deletion, substitution and/or
alteration of at least one (one or more) nucleotide. Such
modifications can result in addition of structural elements, such
as addition of a loop or stem; lengthening or shortening of an
existing stem or loop; changes in the composition or structure of a
loop(s) or a stem(s); or any combination of these.
[0005] In one embodiment, the present invention relates to a
self-cleaving schistosome RNA motif modified to include a loop on
stem III. A loop on stem III is also referred to herein as a loop
III. A self-cleaving schistosome RNA motif including a loop on stem
III is also referred to herein as a self-cleaving schistosome RNA
motif including a loop III. A self-cleaving schistosome RNA motif
including a loop III is an example of a self-cleaving schistosome
RNA mutant motif of the invention.
[0006] The naturally occurring self-cleaving schistosome RNA motif
does not contain a loop on stem III. As described herein, addition
of a loop III increases the cleaving activity of the self-cleaving
schistosome RNA motif. In a particular embodiment, the loop on stem
III (loop III) comprises at least three nucleotides. In one
embodiment, loop III comprises 5'-UUCG-3'. In another embodiment,
loop III comprises 5'-CUUCGG-3'. In another particular embodiment,
loop III comprises 5'-GCUUCGGU-3'. Loops can comprise nucleotides
that can base pair and result in non-loop structures. The present
invention relates to self-cleaving schistosome RNA mutant motifs
illustrated in the working examples.
[0007] The present invention further relates to self-cleaving
schistosome RNA mutant motifs including additional structural
elements or nucleotide sequences which modulate the cleaving
activity the self-cleaving schistosome RNA mutant motifs described
herein, such as aptamer moieties. The self-cleaving schistosome RNA
mutant motifs of the present invention can comprise one or more of
these additional structural elements and nucleotide sequences.
[0008] The present invention further relates to the use of aptamer
sequences to control the cleaving activity of the self-cleaving
schistosome RNA mutant motifs of the invention. An aptamer is a
nucleotide sequence which can be bound by an effector molecule; an
effector molecule is a ligand which binds the aptamer. Aptamer
sequences can be grafted onto a self-cleaving schistosome RNA
mutant motif at a location such that the cleaving activity of the
self-cleaving schistosome RNA mutant motif can be controlled by
binding of an effector to the aptamer sequence. Cleaving activity
of the self-cleaving schistosome RNA mutant motif can be modulated
by binding of an effector to an aptamer sequence that is grafted
onto the self-cleaving schistosome RNA mutant motif at a location
such that the cleaving activity is controlled by binding of the
effector to the aptamer sequence. Grafting, as used herein, refers
to the incorporation or addition of the aptamer sequence into the
self-cleaving schistosome RNA mutant motif. Grafting can be within
the self-cleaving schistosome RNA mutant sequence, such as, for
example, an insertion within stem I sequences. Alternatively,
grafting can be outside of the normal secondary structure of the
self-cleaving schistosome RNA mutant motif in a manner similar to
the loop III modifications. Additionally, an aptamer can be grafted
onto stem II, stem III, loop I, loop II, loop III, the nucleotide
core or a combination of two or more of the aforementioned
structural elements of the self-cleaving schistosome RNA mutant
motif. The present invention relates to nucleic acids, constructs
(DNA or RNA) which encode the novel self-cleaving schistosome RNA
mutant motifs and schistosome RNA, described and illustrated
herein. Constructs, such as DNA constructs, can be used alone or in
a vector, such as a plasmid or a viral vector.
[0009] The present invention provides DNA constructs comprising:
(a) a promoter; (b) nucleic acid encoding a nucleic acid product
and operably linked to the promoter; and (c) nucleic acid encoding
a self-cleaving schistosome RNA mutant motif as described herein.
The nucleic acid encoding the self-cleaving schistosome RNA mutant
motif can be 5' of the nucleic acid encoding the nucleic acid
product or 3' of the nucleic acid encoding the nucleic acid
product, and is operably linked to the promoter. The term
"promoter" refers to a nucleic acid which, when operably linked to
nucleic acid encoding a nucleic acid product, is sufficient for
initiation of transcription of the nucleic acid encoding the
nucleic acid product to be expressed. Transcription of the nucleic
acid encoding the nucleic acid product and the nucleic acid
encoding the self-cleaving schistosome RNA mutant motif produces a
mRNA comprising the self-cleaving schistosome RNA mutant motif and
mRNA encoding the nucleic acid product. The cleaving activity of
the self-cleaving schistosome RNA mutant motif controls cleavage of
the mRNA and, as a result, expression of the nucleic acid product;
the self-cleaving schistosome RNA mutant motif is located in the
mRNA at a position such that the nucleic acid product is not
expressed when the mRNA is cleaved. As used herein, a "nucleic acid
product" is a protein or polypeptide, DNA or RNA other than a
self-cleaving schistosome RNA mutant motif of the invention. In a
particular embodiment, the nucleic acid product is a therapeutic
protein.
[0010] Under conditions appropriate for transcription of the
nucleic acid encoding the nucleic acid product and the nucleic acid
encoding the self-cleaving schistosome RNA mutant motif, an mRNA of
the nucleic acid product and the self-cleaving schistosome RNA
mutant motif are produced. Self-cleavage of the mRNA by the
self-cleaving schistosome RNA mutant motif prevents expression of
the nucleic acid product. Treatment of a cell or an individual, in
which the instant DNA constructs are present, with an agent such as
a drug (e.g., an antibiotic) or other molecule or composition,
which inhibits (totally or partially) cleaving activity of the
self-cleaving schistosome RNA mutant motif, results in expression
of the nucleic acid product encoded by the mRNA.
[0011] In a specific embodiment, the invention provides DNA
constructs comprising: (a) a promoter; (b) nucleic acid encoding a
nucleic acid product operably linked to the promoter; and (c)
nucleic acid encoding a self-cleaving schistosome RNA mutant motif
of the invention which includes an aptamer grafted onto the
self-cleaving schistosome RNA mutant motif at a location such that
the cleaving activity of the self-cleaving schistosome RNA mutant
motif can be controlled (is regulatable) by binding of an effector
to the aptamer. Binding of an effector to the aptamer results in
modulation (induction, enhancement, reduction, inhibition (total or
partial) or regulation) of the cleaving activity of the
self-cleaving schistosome RNA mutant motif. If the binding of the
effector to the aptamer reduces or inhibits the cleaving activity
of the self-cleaving schistosome RNA mutant motif, cleavage of the
mRNA does not occur or is reduced, and the nucleic acid product is
expressed. If the binding of the effector to the aptamer does not
inhibit the cleaving activity of the self-cleaving schistosome RNA
mutant motif, the mRNA is cleaved and as a result, the nucleic acid
product is not expressed (not produced).
[0012] In another specific embodiment, the DNA constructs of the
invention comprise nucleic acid encoding two or more (multiple)
self-cleaving schistosome RNA mutant motifs of the invention.
Multiple self-cleaving schistosome RNA mutant motifs can comprise
the same or different self-cleaving schistosome RNA mutant motifs.
Multiple self-cleaving schistosome RNA mutant motifs encoded by a
nucleic acid are joined 5' to 3'. For example, the 3' terminus of
the nucleic acid encoding the first self-cleaving schistosome RNA
mutant motif is joined to the 5' terminus of the nucleic acid
encoding the next self-cleaving schistosome RNA mutant motif.
Optionally, the two self-cleaving schistosome RNA mutant motifs can
be separated by a nucleic acid linker. Generally, the nucleic acid
encoding the self-cleaving schistosome RNA mutant motif (one or
more) is upstream of the nucleic acid encoding the nucleic acid
product. Thus, the order of the components (5' to 3') in the
present invention can be promoter--nucleic acid encoding a
self-cleaving schistosome RNA mutant motif--nucleic acid encoding a
nucleic acid product.
[0013] In addition to nucleic acid encoding a nucleic acid product
to be expressed, vectors of the present invention can further
comprise additional components, such as an enhancer, targeting
sequences, transcriptional binding sites, and backbone nucleic
acids.
[0014] The present invention relates to host cells comprising a DNA
construct of the present invention. The construct comprises: (a) a
promoter; (b) nucleic acid encoding a self-cleaving schistosome RNA
mutant motif of the invention; and (c) nucleic acid encoding a
nucleic acid product operably linked to the promoter. The nucleic
acid encoding the self-cleaving schistosome RNA mutant motif and
the nucleic acid encoding the nucleic acid product are downstream
of the promoter. Transcription of the nucleic acid of (b) and the
nucleic acid of (c) produces a mRNA comprising the self-cleaving
schistosome RNA mutant motif and mRNA encoding the nucleic acid
product. The cleaving activity of the self-cleaving schistosome RNA
mutant motif controls cleavage of the mRNA and, as a result,
expression of the nucleic acid product. In a specific embodiment,
host cells of the invention comprise a nucleic acid encoding an
aptamer which is grafted onto a self-cleaving schistosome RNA
mutant motif, as described above. Optionally, host cells can also
comprise a nucleic acid encoding two or more (e.g., multiple)
schistosome self-cleaving schistosome RNA mutant motifs of the
invention.
[0015] In certain embodiments, the invention relates to packaging
cell lines useful for generating recombinant viral vectors and
viruses of the invention. It also relates to construction of such
cell lines and to methods of using the recombinant viral vectors
and viruses to modulate production of a nucleic acid product in
vitro, in vivo and ex vivo. Cell lines useful for generating
recombinant viral vectors and viruses of the invention are produced
by transfecting host cells, such as mammalian host cells, with a
viral vector or virus of the invention.
[0016] The present invention relates to the use of the
self-cleaving schistosome RNA mutant motifs described herein in
methods of modulating expression of a nucleic acid product in a
host cell or an individual. Expression of the nucleic acid product
is modulated through the control of the cis-cleavage of a mRNA
which encodes for the nucleic acid product. By modulating is meant
inducing, enhancing (increasing), reducing, inhibiting (total or
partial) or regulating a process. In a particular embodiment,
modulating expression refers to the ability to increasing
(enhancing) expression of the nucleic acid product. In another
particular embodiment, modulating expression refers to reducing
expression of the nucleic acid product. Thus, modulation can be
positive (increase) or negative (decrease). By regulate is meant
the ability to control the rate and/or extent to which a process
occurs. For example, regulating activity of a self cleaving RNA
motif refers to controling the rate and extent to which activity of
the self cleaving RNA motif occurs. Regulating expression of a
nucleic acid refers to controling the rate and extent to which
expression of the nucleic acid product occurs.
[0017] The present invention relates to a method of modulating
expression of a nucleic acid product in a host cell comprising
introducing into the host cell a DNA construct of the invention
comprising: (a) a promoter; (b) a nucleic acid encoding a nucleic
acid product operably linked to the promoter; and (c) a nucleic
acid encoding a self-cleaving schistosome RNA mutant motif, such
that transcription of the nucleic acids produces a RNA molecule
comprising the self-cleaving schistosome RNA mutant motif and a
mRNA encoding the nucleic acid product, wherein the self-cleaving
schistosome RNA mutant motif is capable of cleaving the RNA
intramolecularly. Expression in cells in accordance with the
present invention is modulated through control of the cleavage of a
mRNA encoding the nucleic acid product. Cleavage of the mRNA is
controlled through activity of a self-cleaving schistosome RNA
mutant motif, which is located in the mRNA at a position such that
the nucleic acid product is not expressed when the self-cleaving
schistosome RNA mutant motif is expressed. Under conditions which
permit expression of the self-cleaving schistosome RNA mutant
motif, the mRNA is cleaved and, as a result, the nucleic acid
product encoded is not produced. In this method, the host cell
comprising the DNA construct is cultured in the presence of an
agent, such as a drug (e.g., antibiotic) or other molecule or
composition, which inhibits or reduces cleaving activity of the
self-cleaving schistosome RNA mutant motif such that the nucleic
acid product encoded by the mRNA is expressed. The DNA constructs
used in this method can further comprise nucleic acid encoding an
aptamer which is at a position such that the cleaving activity of
the self-cleaving schistosome RNA motif is regulatable by the
binding of an effector to the aptamer and/or nucleic acid encoding
multiple self-cleaving schistosome RNA mutant motifs as described
herein.
[0018] The present invention also relates to a method of expressing
or modulating expression of a nucleic acid product in an individual
(e.g., a human or other mammal or vertebrate). The method comprises
modulating expression of a nucleic acid product from a DNA
construct of the invention which is present in (contained in) cells
in the individual. The DNA construct comprises nucleic acid
encoding the nucleic acid product and nucleic acid encoding a
self-cleaving schistosome RNA mutant motif of the invention whose
activity can, in turn, be modulated by an agent when the nucleic
acid product is to be expressed. Transcription of the nucleic acid
encoding the nucleic acid product and the nucleic acid encoding the
self-cleaving schistosome RNA mutant motif produces a mRNA
comprising the self-cleaving schistosome RNA mutant motif and mRNA
encoding the nucleic acid product.
[0019] In one embodiment, expression of a nucleic acid product is
effected by administering an antibiotic to an individual, some of
whose cells contain a DNA construct of the present invention. The
DNA construct comprises: (a) a promoter; (b) nucleic acid encoding
the nucleic acid product operably linked to the promoter; and (c)
nucleic acid encoding a self-cleaving schistosome RNA mutant motif
of the invention. Transcription of the nucleic acid of (b) and the
nucleic acid of (c) produces mRNA comprising the self-cleaving
schistosome RNA mutant motif and mRNA encoding the nucleic acid
product. As a result, activity of the encoded self-cleaving
schistosome RNA mutant motif is inhibited (partially or totally),
with the result that the nucleic acid product of interest is
expressed in the individual. The DNA construct can be introduced
into cells in the individual in vivo (e.g., by introducing the DNA
construct) into a tissue or body fluid of the individual) or ex
vivo (e.g., by introducing the DNA construct into cells obtained
from the individual or from another (different) individual or
source and then introducing the resulting cells into the
individual). In either case, administration of an antibiotic
results in inhibition of the activity of the self-cleaving
schistosome RNA mutant motif and, as a result, the mRNA coding for
the nucleic acid product of interest is not cleaved and the nucleic
acid product of interest is expressed.
[0020] In one embodiment, the method is carried out by: (a)
obtaining cells from an individual and maintaining the cells under
appropriate conditions for cell growth and cell division; (b)
introducing into the cells a DNA construct of the invention; (c)
returning the cells produced in step (b) to the individual; and (d)
administering to the individual an agent which inhibits cleavage of
the self-cleaving schistosome RNA mutant motif. In a particular
embodiment, the DNA construct of the invention comprises: (a) a
promoter; (b) nucleic acid encoding a nucleic acid product to be
expressed, operably linked to the promoter; and (c) nucleic acid
encoding a self-cleaving schistosome RNA mutant motif of the
invention. The nucleic acid encoding the nucleic acid product to be
expressed and the nucleic acid encoding the self-cleaving
schistosome RNA mutant motif are downstream of the promoter.
Transcription of the nucleic acid encoding the nucleic acid product
and the nucleic acid encoding the self-cleaving schistosome RNA
mutant motif produces a mRNA comprising the self-cleaving
schistosome RNA mutant motif and mRNA encoding the nucleic acid
product. In this particular embodiment of the method of expressing
a nucleic acid product in an individual, the agent is, for example,
an antibiotic.
[0021] In one embodiment, the present invention relates to a method
of regulating expression of an endogenous gene (a gene resident in
a cell as the cell was obtained) to produce a nucleic acid product
and compositions useful in the method. The endogenous gene can be
one which is expressed ("on") in the cell or one which is normally
not expressed ("off") in the cell but whose expression is or has
been turned on or activated. In this embodiment, a DNA construct
encoding a self-cleaving schistosome RNA mutant motif of the
invention is introduced into genomic DNA of cells in such a
position that, in mRNA produced by the cells, the self-cleaving
schistosome RNA mutant motif is in a location which results in
control of expression of the encoded nucleic acid product. In the
absence of an agent which inhibits expression of the self-cleaving
schistosome RNA mutant motif, cleavage occurs and the nucleic acid
product is not expressed. In the presence of such an agent,
cleaving activity is inhibited and the nucleic acid product is
expressed. In one embodiment, DNA encoding a self-cleaving
schistosome RNA mutant motif of the invention can be introduced
alone or in a vector, into genomic DNA between the promoter
operably linked to (controlling expression of) the endogenous gene
encoding the nucleic acid product, in such a manner that the
endogenous gene remains operably linked to the promoter. In an
alternative embodiment, DNA encoding a self-cleaving schistosome
RNA mutant motif of the invention can be introduced alone or in a
vector, into genomic DNA 3' of the endogenous gene encoding the
nucleic acid product. The promoter which is operably linked to the
endogenous gene to be expressed can be the naturally occurring
(endogenous) promoter for the gene or can be an exogenous promoter
introduced into genomic DNA. The resulting cells can be used, as
described herein, to modulate production of the nucleic acid
product in an individual.
[0022] In certain embodiments, expression of a nucleic acid product
is effected by administering an antisense oligonucleotide of a
self-cleaving schistosome RNA mutant motif to a cell or an
individual. Some of those cells contain a DNA construct of the
present invention, wherein the DNA construct comprises: (a) a
promoter; (b) nucleic acid encoding the nucleic acid product
operably linked to the promoter; and (c) nucleic acid encoding a
self-cleaving schistosome RNA mutant motif of the invention.
Transcription of the nucleic acid of (b) and the nucleic acid of
(c) produces mRNA comprising the self-cleaving schistosome RNA
mutant motif and mRNA encoding the nucleic acid product. As a
result, activity of the encoded self-cleaving schistosome RNA
mutant motif is inhibited (partially or totally) by the antisense
oligonucleotide, with the result that the nucleic acid product of
interest is expressed in the cell or individual. Preferably, the
antisense oligonucleotide base pairs with a region of the
self-cleaving schistosome RNA mutant motif as depicted in SEQ ID
NO: 67. For example, the antisense oligonucleotide is a modified
oligonucleotide selected from the group consisting of: morpholino,
phosphorothioate RNA, 2'-O-methyl RNA, and phosphorothioate
2'-O-methoxyethyl RNA.
[0023] In a particular embodiment, the present invention relates to
a method of specifically inducing expression of a target gene in a
cell, comprising contacting the cell with an antisense
oligonucleotide which specifically inhibits a self-cleaving
schistosome RNA mutant motif, wherein the self-cleaving schistosome
RNA mutant motif is present in a RNA molecule encoding the target
gene product. The cell comprising the RNA molecule is cultured
under conditions appropriate for the antisense oligonucleotide to
inhibit cleaving the RNA molecule by the self-cleaving schistosome
RNA mutant motif. The method is based in part on the ability of
specific oligonucleotides to discriminate different self-cleaving
schistosome RNA mutant motifs. Optionally, the method of the
invention may be used for the generation of multiple independent
systems for gene regulation.
[0024] Similarly, the present invention relates to a method of
specifically modulating (inducing or inhibiting) expression of a
target gene in a cell, comprising contacting the cell with an
effector which specifically binds to an aptamer, wherein the
aptamer is engineered to be present in a RNA molecule encoding the
target gene product and a self-cleaving schistosome RNA mutant
motif. The cell comprising the RNA molecule is cultured under
conditions appropriate for the interaction between the effector and
the aptamer such that the interaction modulates cleaving the RNA
molecule by the self-cleaving schistosome RNA mutant motif as
described above. This method is based in part on the ability of
specific effectors to discriminate different aptamers. Optionally,
the method of the invention may be used for the generation of
multiple independent systems for gene regulation.
[0025] The present invention also provides modified antisense
oligonucleotides of a self-cleaving schistosome RNA mutant motif.
Preferably, the antisense oligonucleotides of the invention base
pair with a target region of the self-cleaving schistosome RNA
mutant motif as depicted in SEQ ID NO: 67. Examples of the modified
antisense oligonucleotides include, but are not limited to,
morpholino, phosphorothioate RNA, 2'-O-methyl RNA, and
phosphorothioate 2'-O-methoxyethyl RNA.
[0026] The present invention relates to a method of screening for
an agent which inhibits the catalytic activity of a self-cleaving
schistosome RNA mutant motif of the present invention comprising:
(a) introducing into host cells a DNA construct of the invention
which comprises: (1) a promoter, (2) nucleic acid encoding a
reporter which is operably linked to the promoter and (3) nucleic
acid encoding a self-cleaving schistosome RNA mutant motif of the
invention, wherein the nucleic acid of (2) and the nucleic acid of
(3) are downstream of the promoter, wherein the DNA construct is
introduced into the host cells under appropriate conditions for
expression of the nucleic acid encoding the reporter and the
nucleic acid encoding the self cleaving schistosome RNA mutant
motif; (b) contacting host cells with an agent to be assessed for
its ability to inhibit the catalytic activity of the self-cleaving
schistosome RNA mutant motif under conditions which result in
introduction of the agent into the cells; and (c) assaying reporter
activity in the host cells. If reporter activity detected in the
presence of the agent is greater than the reporter activity
detected in the absence of the agent, the agent is identified as
one which inhibits the catalytic activity of a self-cleaving
schistosome RNA mutant motif.
[0027] The present invention also relates to a method of screening
for an effector which binds to an aptamer (or RNA sequence) and
inhibits the catalytic activity of a self-cleaving schistosome RNA
mutant motif of the invention. In this method, host cells are
introduced a DNA construct of the invention which comprises: (1) a
promoter, (2) nucleic acid encoding a reporter operably linked to
the promoter and (3) nucleic acid encoding a self-cleaving
schistosome RNA mutant motif of the invention which includes an
aptamer located at a position such that the cleaving activity of
the self-cleaving schistosome RNA mutant motif is regulatable by
binding of an effector to the aptamer, wherein the nucleic acid of
(2) and the nucleic acid of (3) are downstream of the promoter, and
transcription of the nucleic acid of (2) and the nucleic acid of
(3) produces a mRNA comprising the aptamer-self-cleaving
schistosome RNA motif (or RNA sequence-self-cleaving schistosome
RNA motif) and mRNA encoding the reporter. The host cells are
contacted with an agent to be assessed for its ability to bind the
aptamer (or RNA sequence) under conditions appropriate for
expression of the reporter and the self-cleaving schistosome RNA
mutant motif, and reporter activity is assayed in the host cells.
If reporter activity detected in the presence of the agent is
greater than the reporter activity detected in the absence of the
agent, the agent is identified as an effector which can bind to the
aptamer (or RNA sequence) and inhibit the catalytic activity of a
self-cleaving schistosome RNA mutant motif.
[0028] The present invention also relates to a method for producing
a transgenic nonhuman animal using the self-cleaving schistosome
RNA motif. In one embodiment, the transgenic animal is produced by
introducing a DNA construct of the invention into a germ cell of a
nonhuman animal or the germ cell of its ancestor, wherein the DNA
construct comprises: (a) a promoter, (b) nucleic acid encoding a
nucleic acid product operably linked to the promoter and (c)
nucleic acid encoding a self-cleaving schistosome RNA mutant motif
of the invention. The nucleic acid of (b) and the nucleic acid of
(c) are downstream of the promoter, and transcription of the
nucleic acid of (b) and the nucleic acid of (c) produces an mRNA
comprising the self-cleaving schistosome RNA mutant motif and mRNA
encoding the nucleic acid product.
[0029] In certain embodiments, the present invention provides a kit
for regulating gene expression. For example, the kit comprises a
nucleic acid comprising: (a) a schistosome ribozyme mutant
sequence; and (b) a cloning site for introduction of a target
nucleotide sequence to be transcribed operatively linked to the
schistosome ribozyme mutant sequence. Optionally, the kit may
further comprise an inhibitor of the schistosome ribozyme mutant.
For example, the schistosome ribozyme mutant comprises a nucleotide
sequence selected from SEQ ID NOs: 1-63. The inhibitor of the kit
includes, but is not limited to, toyocamycin, 8-azaadenosine,
sangivamycin, tubercidin, tubercidin-cyclic monophosphate,
tubercidin-monophosphate, tubercidin-triphosphate, nebularine,
tricyclic nucleoside, 5-fluorouridine, 5-bromouridine,
5-fluorouracil, Syto-83, homidium bromide, and acridine orange.
[0030] In certain embodiments, the present invention provides a
method for determining the level of an inhibitor of a schistosome
ribozyme mutant in a cell. In this method, a cell is introduced a
DNA construct which comprises: (1) a promoter; (2) a nucleic acid
encoding a reporter; and (3) a nucleic acid encoding a schistosome
ribozyme mutant, wherein the nucleic acid of (2) and the nucleic
acid of (3) are downstream of the promoter and operably linked to
said promoter, under conditions which result in inhibition of the
ribozyme mutant and expression of the reporter. The reporter
activity is assayed, wherein the level of said inhibitor in the
cell is identified by comparing the reporter activity with an
appropriate control. Preferably, the inhibitor is 5-fluorouracil or
5-fluorouridine. In certain cases, the reporter is selected from
.beta.-galactosidase, .beta.-glucoronidase, .beta.-glucosidase,
chloramphenicol acetyl transferase (CAT), green flourescent
protein, and luciferase. Optionally, the cell is a cancer cell.
[0031] In certain embodiments, the present invention provides a
method for determining the level of an inhibitor of a schistosome
ribozyme mutant in a biological sample. In this method, a cell is
contacted with a biological sample, wherein the cell is engineered
to express a DNA construct which comprises: (1) a promoter; (2) a
nucleic acid encoding a reporter; and (3) a nucleic acid encoding a
schistosome ribozyme mutant, wherein the nucleic acid of (2) and
the nucleic acid of (3) are downstream of the promoter and operably
linked to said promoter, under conditions which result in
inhibition of the ribozyme mutant and expression of the reporter.
The reporter activity is assayed, wherein the level of said
inhibitor in the biological sample is identified by comparing the
reporter activity with an appropriate control.
[0032] In certain embodiments, the present invention provides a
method of inhibiting activity of a catalytic RNA in a cell,
comprising contacting a cell with an inhibitor of a schistosome
ribozyme mutant. Preferably, the cell has been infected or is at
risk of having infection with a virus or a pathogenic
microorganism. For example, the inhibitor is selected from
toyocamycin, 8-azaadenosine, sangivamycin, tubercidin,
tubercidin-cyclic monophosphate, tubercidin-monophosphate,
tubercidin-triphosphate, nebularine, tricyclic nucleoside,
5-fluorouridine, 5-bromouridine, 5-fluorouracil, Syto-83, homidium
bromide, and acridine orange. In certain cases, the inhibitor is an
antisense oligonucleotide, including a modified antisense
oligonucleotide (e.g., morpholino, phosphorothioate RNA,
2'-O-methyl RNA, or phosphorothioate 2'-O-methoxyethyl RNA).
[0033] In certain embodiments, the present invention provides a
method of inhibiting infection by a virus or a pathogenic
microorganism in a cell, comprising contacting a cell with an
inhibitor of a schistosome ribozyme mutant. The infection may be
caused by a virus (e.g., a human immunodeficiency virus, a herpes
virus, a hepatitis virus, or a human papillomavirus) or a
pathogenic microorganism (e.g., Notophthalmus viddescens, Ambystoma
talpoideum, Amphiuma tridactylum, and Schistosoma mansoni). The
cell can be an animal cell (e.g., a mammalian cell) or a plant cell
(e.g., tobacco).
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIGS. 1a-1g show the strategy for controlling gene
expression via the modulation of RNA self-cleavage and optimization
of Schistosome Sm1 rz self-cleavage activity. (a) When a cis-acting
hammerhead rz is embedded in the mRNA, self-cleavage leads to the
destruction of the mRNA and the absence of gene expression.
However, an inactive mutant or the administration of specific
inhibitors of the rz leads to the generation of intact mRNA's and
protein expression. (b) The reporter gene expression vector pMD
used for transfection assay, and the positions where rz were
placed. Cap site at the very beginning of the mRNA; A site upstream
of the intron; B, C, and D site in the intron; E site immediately
upstream of the translation start; F and G sites in the
3'-untranslated region. (c) to (g): optimization of Schistosome Sm1
activity (SEQ ID NOs: 1-12). (c) and (d), rz inserted at position A
of pMD vector; (e) to (g) at position E of vector. Corresponding
inactive mutants contained an A14 to G substitution. Name of rz is
shown at left; cleavage activity at right. (c) Cricket Pst3 motif.
Nucleotide numbering follows nomenclature of Hertel et al., 1992.
(d) Schistosome Sm1 motif. The original Sm1 lacked loop III and
exhibited no activity in cells. Nucleotides depicted in color
reflect changes made to Sm1. (e) Changes in stem I of N79 near the
core or near the restriction insertion site further enhanced
activity. Black line identifies the sequence targeted by antisense
morpholino oligonucleotides (SEQ ID NO: 67). (f) Shortening of stem
I vastly reduced rz cleavage activity. (g) Single nucleotide
changes in loop I of the N107 rz decreased its activity
dramatically. The N107 rz is a variant of N79 in which two `AUG`
were replaced by GUG and ACG to eliminate the potential start
codons. The .+-. signs indicate standard deviation from mean of at
least four independent measurements.
[0035] FIGS. 2a-2b show that efficient self-cleavage can occur in
different cells, with different vectors, and with rz sequences
positioned in different locations. (a) N79 functioned efficiently
in a variety of cell types. Numbers are the measurements of
.beta.-gal expressed. (b) N79 functioned efficiently at some but
not all positions within the vector. Numbers are `fold decrease` in
.beta.-gal expression (functional vs. inactive rz). The .+-. signs
indicate standard deviation from mean of at least four independent
measurements.
[0036] FIGS. 3a-3c show induction of gene expression in cultured
cells via inhibition of rz self-cleavage. (a) Effect of morpholino
oligonucleotide on N79 rz in transient transfection assay.
Induction was measured by `fold increase` in .beta.-gal expression
with vs. without morpholino application. The level of induction is
also shown as a percentage relative to the expression level of
inactive rz. The target for the oligonucleotide is shown in FIG.
1e. (b) Induction of luciferase expression by toyocamycin in a
stable cell line carrying an expression construct containing a
double N79 rz. Cells were treated with toyocamycin for 24 hours at
dosage of 0, 0.5, 1, and 1.5 .mu.M (toxic effects were observed at
concentrations higher than 1.5 .mu.M). Quantitative measurements of
luciferase activity revealed emission of 1,555, 66,774, 242, 546,
377,655 photons per second per 1000 cells respectively, as compared
to a background emission of 121 from cells carrying no luciferase
gene. Error bars indicate standard deviation from mean of at least
four independent measurements. (c) Induction by toyocamycin at the
RNA level as revealed by northern blot analyses. Experimental
conditions were similar to that of (b). RNA was purified from the
nucleus `N` or the cytoplasm `C` after 24 hours of treatment.
[0037] FIG. 4 show effective control of gene expression in vivo
using rz-based gene regulation system. Upper panel shows the animal
in which retina was injected with AAV carrying double inactive N79
and treated with toyocamycin. Middle panel shows the animal in
which retina was injected with AAV carrying double functional N79
and treated with adenosine. Lower panel shows the same as middle
panel but treated with toyocamycin. A strong induction in
luciferase expression at day 2 was observed in lower panel. Animals
were also injected in the hamstring muscles of the hind limb with
AAV carrying double inactive N79 as internal control. AAV injection
was done 3 weeks prior to the first imaging day (day 0).
Drug-releasing pellets were implanted subcutaneously in the dorsal
neck immediately after the first imaging, and released the drug
over 7 days. The toyocamycin pellet contains 10 .mu.g of drug;
adenosine pellet 50 .mu.g.
[0038] FIGS. 5A-5F show sequences of some self-cleaving schistosome
RNA mutant motifs (SEQ ID NOs: 13-46), locations of these
nucleotide modifications, and their self-cleaving activities in
mammalian cells. Changes in the resultant ribozyme activity were
measured by monitoring the fold of difference in the reporter
beta-galactosidase activity.
[0039] FIG. 6 is a diagram illustrating the partial nucleotide
sequence of a self-cleaving schistosome RNA motif and sites for
loop III and core modification; SEQ ID NO: 64.
[0040] FIG. 7 is a diagram illustrating the partial nucleotide
sequence of a self-cleaving schistosome RNA motif with a
four-nucleotide loop III and a site for core modification; SEQ ID
NO: 65.
[0041] FIG. 8 shows the nucleotide sequence of an exemplary vector
named HDM-nLacZ; SEQ ID NO: 66.
[0042] FIGS. 9A-9B show that 5'-FUridine (A) and 5'-FUracil (B)
induced gene expression via inhibition of rz self-cleavage in a
dose-dependent manner. FIG. 2 shows structures of four small
ribozymes: (a) the hammerhead; (b) hairpin; (c) hepatitis delta
virus; and (d) Neurospora VS ribozymes. Hammerhead ribozymes (a)
are composed of three stem helices designated I, II, and III. The
core region comprises unpaired nucleotides. Loop structures can be
present branching off stems I, II, and III.
[0043] FIG. 10 shows structures of four small ribozymes: (a) the
hammerhead; (b) hairpin; (c) hepatitis delta virus; and (d)
Neurospora VS ribozymes. Hammerhead ribozymes (a) are composed of
three stem helices designated I, II, and III. The core region
comprises unpaired nucleotides. Loop structures can be present
branching off stems I, II, and III.
[0044] FIG. 11 shows the secondary structure and nucleotide
sequence of Cricket ribozyme.
[0045] FIG. 12 shows the secondary structure and nucleotide
sequence of TRSV ribozyme.
[0046] FIG. 13 shows the secondary structure and nucleotide
sequence of the naturally-occurring (wildtype) schistosome ribozyme
and the location of the initial modifications. Stems I, II, and III
are noted.
DETAILED DESCRIPTION OF THE INVENTION
[0047] In certain embodiments, the present invention provides
compositions and methods for controlling gene expression with a
ribozyme-based system. The term "ribozyme" as used herein, includes
naturally-occurring (wildtype) ribozymes and modified ribozymes
(also referred to as mutants or variants). In particular, the
invention relates to novel self-cleaving ribozyme motifs (e.g.,
schistosome ribozymes), nucleic acids encoding at least one
self-cleaving ribozyme motif, regulators (e.g., inhibitors) of the
self-cleaving ribozyme motifs, and methods involving uses of the
ribozyme motifs and its regulators for diagnostic and therapeutic
applications. Although the application mostly discusses
compositions and methods derived from a particular ribozyme
(schistosome ribozyme), one of ordinary skill in the art will
readily recognize that similar compositions and methods can be
derived from any other ribozyme which functions in mammalian
cells.
[0048] In certain embodiments, the present invention relates to
self-cleaving ribozymes that are functional in mammalian cells. The
term "functional self-cleaving ribozyme" refers to a self-cleaving
ribozyme that efficiently cleaves an RNA molecule in which the
ribozyme is embedded and leads to at least 90% (preferably 95%,
98%, 99% or 100%) reduction in the RNA molecule relative to an
inactive ribozyme. For example, the activity of the self-cleaving
ribozyme can be assayed by the methods described in the working
examples below.
[0049] As one example, the ribozyme of the invention is a
hammerhead ribozyme (wildtype or mutants) selected from cherry
small circular RNA+(Scc+), cherry small circular RNA (Scc-),
Lucerne transient streak virusoid+ (sLTSV+), Lucerne transient
streak virusoid- (sLTSV-), Tobacco ringspot virus satellite RNA+
(sTRSV+), Arabis mosaic virus (sArMV), Chicory yellow mottle virus
satellite RNA (sCYMV), Barley yellow dwarf virus satellite RNA-
(sBYDV-), Barley yellow dwarf virus satellite RNA+ (sBYDV+), Peach
latent mosaic virus RNA+ (PLMVd+), Peach latent mosaic virus RNA-
(PLMVd-), Chrysanthemum chlorotic mottle viroid+ (CChMVd+),
Chrysanthemum chlorotic mottle viroid- (CChMVd-), Subterraneurn
clover mottle virusoid (vSCMoV), and velvet tobacco mottle virusoid
(vVTMoV).
[0050] As another example, the ribozyme of the invention is a
hammerhead ribozyme (wildtype or mutants) selected from
Notophthalmus viddescens satellite RNA (newt), Ambystoma talpoideum
(Am. ta.), Amphiuma tridactylum (Am. tr.), Schistosoma mansoni
hammerhead ribozyme (Schistozyme), D. bacceffli cricket hammerhead
ribozyme (cricketzyme A), D. schiavazzii cricket hammerhead
ribozyme (cricketzyme B), and Avocado sunblotch viroid+ (ASBV+). A
specific example of the ribozyme sequence is the Dolichopoda cave
cricket as illustrated in FIG. 1C (SEQ ID NO: 1.
[0051] As a further example, the ribozyme of the invention can be
other self-cleaving ribozymes, such as a hepatitis delta virus
(HDV) ribozyme, a hairpin ribozyme, and a Neurospora Varkud
satellite (VS) ribozyme (see, e.g., FIG. 10). Like hammerhead
ribozymes, these three self-cleaving ribozymes are found in viral,
virusoid, or satellite RNA genomes, and process the products of
rolling circle replication into genome-length strands (Doherty et
al., 2001, Annu Rev Biophys Biomol Struct. 30:457-75; Branch et
al., 1984, Science 223:45055).
[0052] In particular, the invention relates to novel self-cleaving
schistosome RNA mutant motifs, nucleic acids encoding at least one
self-cleaving schistosome RNA mutant motif, regulators (e.g.,
inhibitors) of the self-cleaving schistosome RNA mutant motifs, and
methods involving uses of the schistosome RNA mutant motif and its
regulators for diagnostic and therapeutic applications.
[0053] Self-Cleaving RNA Mutant Motifs
[0054] The self-cleaving RNA mutant motifs (e.g., schistosome
mutant motifs) are also referred to as ribozyme mutants or modified
ribozyme motifs). These modified ribozymes can be used to modulate
expression of a desired nucleic acid product in cells. Expression
in cells in accordance with the invention is modulated through
control of the activity of a self-cleaving RNA mutant motif of the
invention. In particular, expression of a nucleic acid product is
modulated by the activity of a self-cleaving RNA mutant motif which
is located in the mRNA at a position such that the desired nucleic
acid product is not expressed when the mutant motif is active.
Under conditions which are appropriate for expression of the
self-cleaving RNA mutant motif, the mRNA is cleaved and as a
result, the desired nucleic acid product encoded by the mRNA is not
produced (FIG. 1A). Administration to cells of an agent such as a
drug or other molecule or composition, which inhibits or reduces
the cleaving activity of the self-cleaving RNA mutant motif,
prevents cleavage of the mRNA and, therefore, the nucleic acid
product is expressed (FIG. 1A).
[0055] Ribozymes are RNA structural motifs of about 40-60
nucleotides that can self-cleave in a sequence-specific manner. The
term "ribozyme" refers to an RNA sequence that hybridizes to a
complementary sequence in a substrate RNA and cleaves the substrate
RNA in a sequence specific manner at a substrate cleavage site.
Typically, a ribozyme contains a catalytic region flanked by two
binding regions. The ribozyme binding regions hybridize to the
substrate RNA, while the catalytic region cleaves the substrate RNA
at a substrate cleavage site to yield a cleaved RNA product. The
nucleotide sequence of the ribozyme binding regions may be
completely complementary or partially complementary to the
substrate RNA sequence with which the ribozyme binding regions.
Generally, ribozymes are embedded in highly repetitive satellite
DNA and have been identified in plant viruses, newts, cave
crickets, and schistosomes.
[0056] Schistosomes are a family of parasitic blood flukes which
infect humans. Recently, several members of the Schistosome family
were found to encode hammerhead ribozymes (Ferbeyre et al., Mol.
Cell Bio. 18:3880-3888 (1998)). Hammerhead ribozymes are one of the
four known classes of self-cleaving RNA motifs and the term refers
to the secondary structure of this class of ribozymes. The
hammerhead secondary structure is composed of three helices,
referred to as stem I, stem II and stem III, joined at a central
core of 11-12 single stranded nucleotides that are necessary and
sufficient for the self-cleavage reaction (Uhlenbeck, Nature,
328:596-600 (1987); and Foster and Symons, Cell, 50:9-16 (1987)).
Systematic site-directed mutagenesis studies have determined that
most nucleotides in the conserved core cannot undergo mutation
without significant loss of catalytic activity (Ruffner et al.,
Biochemistry, 29:10695-10702 (1990)). This observation was used to
generate inactive self cleaving RNA motifs used herein as negative
controls in ribozyme activity assays.
[0057] Ribozymes can have additional structural elements such as
loops, which are designated by the stem from which they branch. For
example, schistosome ribozymes have a six nucleotide loop on stem
II (loop II), as illustrated in FIG. 1c. Naturally occurring
(wildtype) schistosome ribozymes include stems I-III, loops I-II,
but not loop III. By loop II is meant a loop on stem II. By loop
III is meant a loop on stem III. Other loops are identified
similarly herein by this convention (e.g., loop I). Standard
nomenclature for nucleotide position in hammerhead ribozymes is
used herein (Clouet-d'Orval and Uhlenbeck, Biochemistry,
36:9087-9092 (1997)). In certain embodiments, the present invention
contemplates sequences that are outside the conserved or consensus
region of the ribozymes. Optionally, sequences outside of the
canonical hammerhead ribozyme structure can be incorporated into
the ribozyme structure. The resulting compositions are examples of
self-cleaving schistosome RNA mutant motifs of the invention.
[0058] As described herein, modification or alteration of the
nucleotide sequence of naturally occurring ribozymes (e.g.,
schistosome ribozyme) can increase or decrease their catalytic
activity. As used herein, catalytic activity refers to the ability
of ribozyme to autocatalytically cleave its RNA.
[0059] Self-cleaving RNA mutant motifs (e.g., schistosome ribozyme
mutants) and methods disclosed in the present invention are
particularly useful in exogenous control of gene expression. The
present invention makes it possible to modulate expression of a
nucleic acid product without the need to use special
transcriptional control elements or chimeric transactivators. Thus,
the present invention has a number of distinct advantages over
previously-available methodologies and has broad applications in
such fields as protein production, gene therapy, and developmental
biology. In addition, the essential genetic element for gene
regulation is very small in size and does not encode any gene
product. Accordingly, it is unlikely that the introduction of the
element into cells will result in any toxicity, and it should be
possible to incorporate the necessary sequences for obtaining
regulated expression into many different types of vectors.
[0060] An additional benefit of the methods described herein for
modulating expression of a nucleic acid product is that gene
regulation is not sensitive to chromosomal position, since
modulation does not depend upon control of the initiation of
transcription. Furthermore, in contrast to existing methods for
controlling expression of a nucleic acid product, which require
that specific hybrid promoters be used, it is possible to modulate
expression within the context of the normal cell type specific or
developmental stage specific transcriptional elements of any gene
or vector. In fact, by incorporation of the essential genetic
element for gene regulation within a transcriptional unit, it is
even possible to provide gene regulation in the context of the
normal mRNA structure used for gene expression (e.g., a structure
devoid of any exogenous regulatory elements). These features may
prove to be particularly important for transgenic and knockout
experiments in animals designed to assess the role of a specific
gene product at different stages of development, where the
essential role of a gene product in embryonal development may
preclude the ability to determine the role of the gene product at a
later stage of development.
[0061] In certain aspects, the present invention relates to
self-cleaving RNA mutant motifs (e.g., schistosome ribozyme
mutants) comprising modifications that modulate or alter their
catalytic activity. The modifications can either positively
(increase) or negatively (decrease) modulate the catalytic activity
of the self-cleaving RNA motif. In a particular embodiment, the
modifications can modulate, either positively or negatively, the
ability and/or rate of the self-cleaving RNA motif to self-cleave.
Modifications can also have no measurable effect on the activity of
the self-cleaving RNA motif. By "modification" is meant a
modification (e.g., alternation or change) of the naturally
occurring ribozyme structural elements or the addition of other
structural elements not found in the naturally occurring ribozyme.
The term "modification" is meant to include nucleotide additions,
deletions or substitutions to the self-cleaving RNA motif sequence
or adjacent sequences comprising the self-cleaving RNA motif.
Modifications include the addition (e.g., by grafting) of stem
and/or loop structures to a naturally occurring ribozyme and the
modification of stem and/or loop structures of a naturally
occurring ribozyme. Modifications also include the addition of one
or more (multiple) aptamers to a self-cleaving RNA motif and the
addition of other structural features to a self-cleaving RNA motif,
such as hairpins. These modifications can be used separately or in
combination with one or more other modifications and are not meant
to be limiting in any way.
[0062] In one embodiment, the present invention relates to a
self-cleaving schistosome RNA motif modified to include a loop on
stem III. A "loop," as used herein, refers to a secondary structure
in an RNA sequence in which a single-stranded RNA sequence is
flanked by RNA sequences which are capable of pairing with each
other to form a "stem" structure. A loop comprises at least three
nucleotides, and preferably from about 3-40 nucleotides. A "loop"
can include nucleotides which can base pair and result in non-loop
structures. For example, a loop can include nucleotides which can
base pair and optionally elongate the stem from which it branches.
By "branches" is meant that the nucleotide sequence of the
modification starts where the stem originally ended. By "base pair"
is meant the formation of hydrogen bond(s) between two nucleic acid
sequences by either traditional Watson-Crick or other
non-traditional types (for example, Hoogsteen type) of
interactions. A self-cleaving schistosome RNA motif including a
loop on stem III is an example of a self-cleaving schistosome RNA
mutant motif of the invention. Preferably, the catalytic activity
of such a self-cleaving schistosome RNA mutant motif comprising a
loop on stem III is greater than the catalytic activity of the
corresponding naturally occurring self-cleaving schistosome RNA
motif.
[0063] In a particular embodiment, a self-cleaving schistosome RNA
mutant motif of the invention comprises a four-nucleotide loop III.
In a particular embodiment, the loop comprises 5'-UUCG-3'. An
exemplary self-cleaving schistosome RNA mutant motif of the
invention (N99) is illustrated in FIG. 1e. The present invention
also provides other self-cleaving schistosome RNA mutant motifs and
their sequences in FIG. 5 (SEQ ID NOs: 13-63).
[0064] In another embodiment, a self-cleaving schistosome RNA
mutant motif of the invention comprises a six- or eight-nucleotide
loop III that also elongates stem III. Examples of self-cleaving
schistosome RNA mutant motifs comprising a six nucleotide loop III
are illustrated in FIG. 5 (e.g., N27 and N53). Examples of
self-cleaving schistosome RNA mutant motifs comprising an eight
nucleotide loop III are also illustrated in FIG. 5 (e.g., N73, N79,
N99, and N117).
[0065] Self-cleaving schistosome RNA mutant motifs of the invention
can also include substitutions at position 5, 7 and/or 14 (e.g.,
C.fwdarw.U of the core, as illustrated in FIGS. 1 and 5). In a
particular embodiment, a self-cleaving schistosome RNA mutant motif
of the invention comprises a loop III and a substitution in the
conserved core. In a preferred embodiment, loop III comprises
5'-UUCG-3' and a U at position 7 of the core, as illustrated in
FIGS. 1 and 5 (e.g., N53, N73, N79, N99, and N117). In another
embodiment, a self-cleaving schistosome RNA mutant motif of the
invention comprises an addition of six nucleotides on the end of
stem III and a U at position 7 of the core, as illustrated in FIGS.
1 and 5.
[0066] In certain embodiments, the present invention also relates
to other self-cleaving schistosome RNA motifs illustrated in FIG. 5
(SEQ ID NOs: 13-63).
[0067] The term "substitution," as used herein, refers to one or
more than one (one or multiple) nucleotide changes in the naturally
occurring self-cleaving RNA motif. The term "self-cleaving
schistosome RNA mutant motif" refers to a self-cleaving schistosome
RNA motif, comprising at least one nucleotide alteration compared
to a naturally occurring (wildtype) schistosome ribozyme (e.g.,
sm1) as depicted in FIG. 1d (Ferbeyre et al., Mol. Cell Bio.,
18:3880-3888 (1998)). Methods described herein can be used to
identify any self-cleaving ribozyme mutant motifs (e.g.,
self-cleaving schistosome RNA mutant motifs with altered catalytic
properties). Methods of assaying catalytic activity are well known
in the art. For example, labeled self-cleaving schistosome RNA
mutant motifs can be incubated under appropriate conditions for
cleavage, fractionated by gel electrophoresis and the extent of
cleavage quantitated by autoradiography. Methods of quantitating
catalytic activity of the self-cleaving schistosome RNA mutant
motifs of the invention are known in the art and include the use of
a reporter gene, such as, for example, as described herein in
Example 1.
[0068] Expression Vectors and Cell Lines
[0069] In certain embodiments, the present invention encompasses
nucleic acids (e.g., DNA vectors) which encode a self-cleaving
ribozyme (naturally occurring or mutants such as schistosome
ribozyme mutants) and their use in modulating expression of a
nucleic acid product. The present invention relates to methods of
inserting the self-cleaving RNA mutant motif sequence into an
endogenous gene in a cell, or into an exogenous gene to be
introduced into a cell by a vector. In either case, necessary
elements (e.g., promoters) are present for the transcription of the
inserted sequence. For example, a self-cleaving ribozyme is
inserted into an appropriate expression vector which contains the
necessary elements for the transcription of the inserted sequence.
The expression vector is then transfected into a host cell in order
to effectuate expression of the ribozyme-encoding sequence and to
determine its effect on gene function in the transfected cell
and/or its progeny.
[0070] In one embodiment, the present invention relates to a
nucleic acid (e.g., a DNA construct) which comprises a promoter,
DNA encoding a nucleic acid product operably linked to the
promoter, and DNA encoding a self-cleaving ribozyme (naturally
occurring or mutants such as schistosome ribozyme mutants) which is
downstream of the promoter. Transcription of the two DNA components
in the construct produces a RNA molecule comprising the
self-cleaving ribozyme and mRNA encoding the nucleic acid product,
and the self-cleaving ribozyme can cleave the RNA
intramolecularly.
[0071] In another embodiment, the present invention relates to a
viral vector comprising a promoter, a nucleic acid encoding a
nucleic acid product operably linked to the promoter and a nucleic
acid encoding a self-cleaving ribozyme (naturally occurring or
mutants such as schistosome ribozyme mutants) which is downstream
of the promoter. Transcription of the two nucleic acid components
in the viral vector produces a RNA molecule comprising a
self-cleaving ribozyme and mRNA encoding the nucleic acid product,
and the self-cleaving ribozyme can cleave the RNA intramolecularly.
In another embodiment, the present invention relates to a virus
comprising a promoter, a nucleotide sequence encoding a nucleic
acid product operably linked to the promoter and a nucleotide
sequence encoding a self-cleaving ribozyme which is downstream of
the promoter. Transcription of the two nucleotide sequences in the
virus produces a RNA molecule comprising a self-cleaving ribozyme
and mRNA encoding the nucleic acid product, and the self-cleaving
ribozyme can cleave RNA intramolecularly.
[0072] DNA constructs encoding a self-cleaving ribozyme (naturally
occurring or mutants such as schistosome ribozyme mutants) of the
present invention can be manufactured according to methods
generally known in the art. For example, nucleic acids encoding a
self-cleaving ribozyme can be manufactured by chemical synthesis or
recombinant DNA/RNA technology (see, e.g., Sambrook et al., Eds.,
Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring
Harbor University Press, New York (1989); and Ausubel et al., Eds.,
Current Protocols In Molecular Biology, John Wiley & Sons, New
York (1997)).
[0073] In certain embodiments, the present invention provides a kit
for regulating gene expression. The subject kit comprises a nucleic
acid comprising: (a) a self-cleaving ribozyme (naturally occurring
or mutants such as schistosome ribozyme mutants); and (b) a cloning
site for introduction of a target nucleotide sequence to be
transcribed operatively linked to the self-cleaving ribozyme.
Optionally, the kit may further comprise an inhibitor of the
self-cleaving ribozyme. Transcription of the target nucleotide
sequence is inhibited in the absence of the inhibitor, while
transcription of the target nucleotide sequence is induced in the
presence of the inhibitor. For example, a schistosome ribozyme
mutant comprises a nucleotide sequence selected from SEQ ID NOs:
1-63. The inhibitor of the kit as described below includes, but is
not limited to, toyocamycin, 8-azaadenosine, sangivamycin,
tubercidin, tubercidin-cyclic monophosphate,
tubercidin-monophosphate, tubercidin-triphosphate, nebularine,
tricyclic nucleoside, 5-fluorouridine, 5-bromouridine,
5-fluorouracil, Syto-83, homidium bromide, and acridine orange.
[0074] Another aspect of the present invention relates to a method
of modulating expression of a nucleic acid product comprising
producing a nucleic acid encoding a self-cleaving ribozyme
(naturally occurring or mutants such as schistosome ribozyme
mutants), as described herein, and a nucleic acid product, wherein
the self-cleaving ribozyme modulates expression of the nucleic acid
product. The nucleic acid product can be a polypeptide, DNA or RNA
other than self-cleaving RNA and can be expressed in cells as a
component of a DNA construct. The nucleic acid product to be
expressed can be a therapeutic protein.
[0075] It is desirable that the subject ribozyme (naturally
occurring or mutants such as schistosome ribozyme mutants)
sufficiently self cleaves such that the target nucleic acid product
is not expressed. Such substantial self cleavage would facilitate
the observation of the effect of depletion of gene function in the
organism. While desirable, complete self cleavage of the ribozyme
is not required by the methods of the invention, so long as the
ribozyme results in a reduced level of a target nucleic acid
product relative to a control. The term "reduced level of a target
nucleic acid product relative to a control" refers to a quantity of
a target nucleic acid product which is less than, preferably at
least 20% less than, more preferably at least 50% less than, yet
more preferably at least 90% less than the quantity of a target
nucleic acid product in a control (e.g., a corresponding sample in
the absence of the ribozyme, or in the presence of a ribozyme which
is incapable of self-cleaving), and most preferably is at the
background level of, or is undetectable by, Northern blot
hybridization as described herein. The invention does not require,
and is not limited to, methods in which a target nucleic acid
product is 99% or 100% ablated.
[0076] In another embodiment, the present invention relates to a
method of modulating expression of a nucleic acid product in a cell
comprising introducing into a cell a DNA construct which comprises:
(a) promoter, (b) a nucleic acid encoding a nucleic acid product
which is operably linked to the promoter and (c) a nucleic acid
encoding a self-cleaving ribozyme (naturally occurring or mutants
such as schistosome ribozyme mutants) downstream of the promoter.
Transcription of the nucleic acid components produces a RNA
molecule (mRNA) comprising the self-cleaving ribozyme and mRNA
encoding the nucleic acid product such that the self-cleaving
ribozyme cleaves the RNA intramolecularly, thus modulating
expression of the nucleic acid product.
[0077] If the DNA construct is present in cells under conditions
which permit expression of the two nucleotide components, the mRNA
molecule comprising the self-cleaving ribozyme and mRNA encoding
the nucleic acid product is produced, the encoded self-cleaving
ribozyme is spontaneously cleaved and, as a result, the nucleic
acid product is not produced. On the other hand, if the DNA
construct is present in cells in the presence of an agent, such as
a drug (e.g., an antibiotic), which inhibits (totally or partially)
cleaving activity of the encoded self-cleaving ribozyme, the
desired nucleic acid product is produced.
[0078] The present invention also encompasses other structural
elements that can affect the stability and/or the activity of the
self-cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants), either positively or negatively. For
example, it has been determined that RNA sequences adjacent to the
catalytic site of the self-cleaving ribozyme affect its cleaving
activity. The cleaving activity of the self-cleaving ribozyme can
be modulated by binding of an effector to an aptamer which is
grafted onto the self-cleaving ribozyme at a location such that the
cleaving activity can be controlled by binding of the effector to
the aptamer.
[0079] Also the subject of the present invention is a DNA construct
useful in the present method of controlling expression of a desired
nucleic acid product in a cell. In one embodiment, the DNA
construct comprises: (a) DNA encoding a nucleic acid product to be
expressed in the cell; and (b) DNA encoding a self-cleaving
ribozyme (naturally occurring or mutants such as schistosome
ribozyme mutants). Transcription of the two DNA components in the
construct yields an mRNA comprising the self-cleaving ribozyme and
mRNA encoding the nucleic acid product to be expressed. The
construct components can be separated by intervening DNA, such as a
linker, provided that the intervening DNA does not interfere with
the ability of the cleaving activity of the encoded self-cleaving
ribozyme to disrupt (cleave) the mRNA coding for the desired
nucleic acid product, thereby inhibiting/blocking expression of the
desired nucleic acid product. This embodiment of the DNA construct
can be introduced into appropriate recipient/host cells in such a
manner that the construct integrates into host cell genomic DNA at
a location which results in its being operably linked to a host
cell promoter (DNA sufficient to initiate transcription) and, as a
result, expressed under the control of the host cell machinery. If
the host cell is maintained under conditions appropriate for
expression of DNA in the host cell (including expression of the DNA
of the introduced and now integrated DNA construct), the encoded
desired nucleic acid product is not expressed because the
self-cleaving ribozyme is produced and its activity results in
disruption of the resulting transcript (mRNA), which cannot
subsequently be translated. As a result, the encoded nucleic acid
product is not expressed. If the host cell which contains the DNA
construct of this embodiment is maintained under conditions
appropriate for expression of DNA in the host cell and in the
presence of an agent such as an antibiotic (which prevents activity
of the encoded self-cleaving ribozyme), disruption of the resulting
transcript does not occur and the encoded desired nucleic acid
product is expressed. In this embodiment, in which the DNA
construct integrates into host cell genomic DNA, the construct can
comprise additional DNA which increases the extent to which the DNA
construct integrates into host cell genomic DNA and/or targets or
directs introduction of the construct to a specific genomic
location. The construct of this embodiment can also include
additional components, such as an enhancer and transcriptional
binding sites.
[0080] In an alternative embodiment, the DNA construct further
comprises DNA sufficient for initiation of transcription (such as a
promoter) operably linked to the DNA encoding the desired nucleic
acid product. In a particular embodiment, the DNA encoding the
self-cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants) is 5' of the DNA encoding the desired
nucleic acid product. Thus, the order of the components in the
construct (from 5' to 3') is: promoter--DNA encoding self-cleaving
ribozyme--DNA encoding the desired nucleic acid product. In a
second embodiment, the DNA encoding the self-cleaving ribozyme is
3' of the DNA encoding the desired nucleic acid product. Thus, the
order of the components in the construct (from 5' to 3') is:
promoter--DNA encoding the desired nucleic acid product--DNA
encoding self-cleaving ribozyme.
[0081] In certain aspects, the DNA construct of the invention
comprises a promoter which includes, but is not limited to, tRNA
promoter, 5S rRNA promoters, histone gene promoters, CMV promoter,
RSV promoter, SV40 promoter, PEPCK promoter, MT promoter, SR.alpha.
promoter, P450 family promoters, GAL7 promoter, T.sub.7 promoter,
T.sub.3 promoter, SP6 promoter, and K11 promoter. The T7 promoter,
T.sub.3 promoter, SP6 promoter, and K.sub.11 promoter have been
described in U.S. Pat. No. 5,591,601, the entire contents of which
are incorporated by reference.
[0082] The invention relates to packaging cell lines useful for
generating recombinant viral vectors and viruses comprising a
recombinant genome which includes a nucleotide sequence (RNA or
DNA) which represents a DNA construct of the present invention;
construction of such cell lines; and methods of using the
recombinant viral vectors to modulate production of a desired
nucleic acid product in vitro, in vivo and ex vivo. In a particular
embodiment, the recombinant viral vectors and viruses comprise a
recombinant genome which includes a nucleotide sequence encoding a
self-cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants), a nucleotide sequence encoding a
desired nucleic acid product and a promoter operably linked to the
nucleotide sequence encoding the desired nucleic acid product, as
described herein.
[0083] Cell lines useful for generating recombinant viral vectors
and viruses comprising a recombinant genome which includes a
nucleotide sequence which represents a DNA construct of the present
invention are produced by transfecting host cells, such as
mammalian host cells, with a viral vector including the DNA
construct integrated into the genome of the virus, as described
herein. Viral stocks are harvested according to methods generally
known in the art. See, e.g., Ausubel et al., Eds., Current
Protocols In Molecular Biology, John Wiley & Sons, New York
(1998); Sambrook et al., Eds., Molecular Cloning: A Laboratory
Manual, 2nd edition, Cold Spring Harbor University Press, New York
(1989); Danos and Mulligan, U.S. Pat. No. 5,449,614; and Mulligan
and Wilson, U.S. Pat. No. 5,460,959, the teachings of which are
incorporated herein by reference. The recombinant viral vectors
produced by the packaging cell lines of the present invention are
also referred to herein as viral vectors which represent the DNA
construct.
[0084] Methods of Delivering Nucleic Acids
[0085] DNA constructs encoding self-cleaving ribozyme (naturally
occurring or mutants such as schistosome ribozyme mutants) can be
introduced into a cell by a variety of methods (e.g.,
transformation, transfection, direct uptake, projectile
bombardment, using liposomes). The present invention contemplates
any methods generally known in the art which are appropriate for
the particular agent or effector and cell type. For example, agents
and effectors can be introduced into a cell by direct uptake,
DEAE-dextran, calcium phosphate precipitation, lipofection, cell
fusion, electroporation, biolistics, microinjection, infection
(e.g., by DNA viruses and RNA viruses) and retrovirus-mediated
transduction. Such methods are described in more detail, for
example, in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Second Edition, Cold Spring Harbor University Press, New
York (1989); and Ausubel, et al., Current Protocols in Molecular
Biology, John Wiley & Sons, New York (1998). Other suitable
methods are also described in the art.
[0086] A vector comprising a DNA construct can also be introduced
into a cell by targeting the vector to cell membrane phospholipids.
For example, targeting of a vector of the present invention can be
accomplished by linking the vector molecule to a VSV-G protein, a
viral protein with affinity for all cell membrane phospholipids.
Such a construct can be produced using methods well known to those
practiced in the art.
[0087] In a particular embodiment, a DNA construct encoding a
self-cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants) is inserted into a nucleic acid
vector, e.g., a DNA plasmid, virus or other suitable replicon
(e.g., viral vector). Viral vectors include retrovirus, adenovirus,
parvovirus (e.g., adeno-associated viruses), coronavirus, negative
strand RNA viruses such as orthomyxovirus (e.g., influenza virus),
rhabdovirus (e.g., rabies and vesicular stomatitis virus),
paramyxovirus (e.g. measles and Sendai), positive strand RNA
viruses such as picornavirus and alphavirus, and double stranded
DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex
virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and
poxvirus (e.g., vaccinia, fowlpox and canarypox). Other viruses
include Norwalk virus, togavirus, flavivirus, reoviruses,
papovavirus, hepadnavirus, and hepatitis virus, for example.
Examples of retroviruses include: avian leukosis-sarcoma, mammalian
C-type, B-type viruses, D-type viruses, HTLV-BLV group, lentivirus,
spumavirus (Coffin, J. M., Retroviridae: The viruses and their
replication, In Fundamental Virology, Third Edition, B. N. Fields,
et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).
Other examples include murine leukemia viruses, murine sarcoma
viruses, mouse mammary tumor virus, bovine leukemia virus, feline
leukemia virus, feline sarcoma virus, avian leukemia virus, human
T-cell leukemia virus, baboon endogenous virus, Gibbon ape leukemia
virus, Mason Pfizer monkey virus, simian immunodeficiency virus,
simian sarcoma virus, Rous sarcoma virus and lentiviruses. Other
examples of vectors are described, for example, in McVey et al.,
U.S. Pat. No. 5,801,030, the teachings of which are incorporated
herein by reference.
[0088] As a particular example of the above approach, a DNA
construct of the invention can be integrated into the genome of a
virus that enters the cell. By infection of the cell, the
components of a system which permit expression of the DNA encoding
the desired nucleic acid product and the spontaneous cleavage of
the corresponding mRNA, are introduced into the cell. Under
appropriate conditions, spontaneous cleavage of the corresponding
mRNA occurs and expression of the encoded product is inhibited.
[0089] Virus stocks consisting of recombinant viral vectors
comprising a recombinant genome which includes a nucleotide (DNA or
RNA) sequence which represents a DNA construct of the present
invention, are produced by maintaining the transfected cells under
conditions suitable for virus production. Such conditions, which
are not critical to the invention, are generally known in the art.
See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual,
Second Edition, Cold Spring Harbor University Press, New York
(1989); Ausubel et al., Current Protocols in Molecular Biology,
John Wiley & Sons, New York (1998); U.S. Pat. No. 5,449,614;
and U.S. Pat. No. 5,460,959, the teachings of which are
incorporated herein by reference. The resulting recombinant viral
vectors can be used, as described herein, to modulate production of
a desired nucleic acid product in vitro, in vivo and ex vivo.
[0090] Thus, the invention also relates to recombinant viral
vectors and viruses comprising a recombinant genome which includes
a nucleotide (DNA or RNA) sequence which represents a DNA construct
of the present invention. Viral vectors and viruses which comprise
the DNA constructs or the encoded (reverse transcribed) RNA are
also the subject of the present invention.
[0091] In certain embodiments, the present invention contemplates a
method of inhibiting expression of a nucleic acid product in host
cells, comprising introducing a self-cleaving ribozyme alone
(naturally occurring or mutants such as schistosome ribozyme
mutants) into cells. Cells comprising the RNA mutant motif are
cultured under conditions appropriate for the RNA mutant motif to
pair with and cleave the mRNA encoding the nucleic acid product.
The self-cleaving ribozyme sequence can be produced by chemical
synthetic methods or by recombinant nucleic acid techniques. For
example, cloned RNA polymerase can be used for transcription in
vitro. The produced self-cleaving ribozyme sequence may be made to
include modifications to either the phosphate-sugar backbone or the
nucleoside, e.g., to reduce susceptibility to cellular nucleases,
improve bioavailability, improve formulation characteristics,
and/or change other pharmacokinetic properties. The self-cleaving
ribozyme sequence may be produced enzymatically or by partial/total
organic synthesis, any modified ribonucleotide can be introduced by
in vitro enzymatic or organic synthesis. The self-cleaving ribozyme
sequence can then be introduced into cells by the conventional
methods described above which are routinely used to deliver nucleic
acids.
[0092] Methods of Identifying Ribozyme Regulators
[0093] The present invention relates to a method of screening for
an agent which is capable of inhibiting the catalytic activity of a
self-cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants). In one embodiment of this method,
host cells are introduced a DNA construct which comprises: (1) DNA
encoding a reporter, (2) a promoter operably linked to the DNA
encoding the reporter, and (3) DNA encoding a self-cleaving
ribozyme, wherein the DNA of (1) and the DNA of (3) are downstream
of the promoter, and transcription of the DNA of (1) and the DNA of
(2) yields a mRNA comprising the self-cleaving ribozyme and mRNA
encoding the reporter. Host cells are contacted with an agent to be
assessed for its ability to inhibit the catalytic activity of the
self-cleaving ribozyme under conditions appropriate for expression
of the reporter, and reporter activity is assayed in the host
cells. If reporter activity is detected, the mRNA coding for the
reporter is not cleaved, indicating that the catalytic activity of
the self-cleaving ribozyme is inhibited by the agent.
[0094] The term "reporter" refers to a protein or polypeptide whose
activity can be readily and easily assayed using standard
techniques. Examples of reporters include enzymes, such as
.beta.-galactosidase, .beta.-glucoronidase, .beta.-glucosidase,
bacterial chloramphenicol acetyl transferase (CAT), luminescent
molecules, such as green flourescent protein and firefly
luciferase, and auxotrophic markers such as His3p and Ura3p. See,
e.g., Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Chapter 9, John Wiley & Sons, Inc. (1998).
[0095] The present invention also relates to a method of screening
for an effector which binds to a desired aptamer (or RNA sequence).
In one embodiment of this method, host cells are introduced a DNA
construct representing the DNA construct, wherein the DNA construct
comprises: (1) DNA encoding a reporter, (2) a promoter operably
linked to the DNA encoding the reporter and (3) DNA encoding a
self-cleaving ribozyme which comprises a desired aptamer (or RNA
sequence) grafted at a position such that the cleaving activity of
the self-cleaving ribozyme is regulatable by the binding of an
effector to the aptamer (or RNA sequence), wherein the DNA of (1)
and the DNA of (3) are downstream of the promoter, and
transcription of the DNA of (1) and the DNA of (3) produces a mRNA
comprising the aptamer-self-cleaving ribozyme (or RNA
sequence-self-cleaving ribozyme) and mRNA encoding the reporter.
Host cells are contacted with an agent to be assessed for its
ability to bind the aptamer (or RNA sequence) under conditions
appropriate for expression of the reporter, and reporter activity
is assayed in the host cells. If the agent binds to the aptamer (or
RNA sequence), the cleaving activity of the self-cleaving RNA motif
is inhibited and, as a result, the mRNA coding for the reporter is
not cleaved and the reporter is produced. Therefore, if reporter
activity is detected, the agent is identified as an effector which
binds to the desired aptamer (or RNA sequence).
[0096] The invention also relates to a method of screening for an
agent which is capable of inhibiting the catalytic activity of a
self-cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants) including a random sequence at a
position in the self-cleaving ribozyme capable of modulating the
cleaving activity of the self-cleaving ribozyme comprising: (a)
introducing into host cells a DNA construct which represents the
DNA construct, wherein the DNA construct comprises: (1) a promoter;
(2) DNA encoding a reporter operably linked to the promoter; and
(3) DNA encoding a self-cleaving ribozyme modified to include a
random sequence at a position in the self-cleaving ribozyme capable
of modulating the cleaving activity of the self-cleaving
schistosome RNA mutant motif, wherein the DNA of (2) and the DNA of
(3) are downstream of the promoter, and transcription of the DNA of
(2) and the DNA of (3) yields a mRNA comprising the self-cleaving
ribozyme including the random sequence and mRNA encoding the
reporter; (b) contacting the host cells with an agent to be
assessed for its ability to inhibit the catalytic activity of the
self-cleaving ribozyme including the random sequence under
conditions appropriate for expression of the reporter; and (c)
assaying reporter activity in the host cells. If reporter activity
is detected, the mRNA coding for the reporter is not cleaved,
indicating that the catalytic activity of the self-cleaving
ribozyme including the random sequence is inhibited by the
agent.
[0097] Agents, such as drugs, chemical compounds, ionic compounds,
organic compounds, organic ligands, including cofactors,
saccharides, recombinant and synthetic peptides, proteins,
peptoids, and other molecules and compositions, can be individually
screened or one or more agents can be tested simultaneously for the
ability to modulate the cleaving activity of a self-cleaving
ribozyme or for the ability to bind to a aptamer moiety in
accordance with the methods described herein. Where a mixture of
agents is tested, the agents selected by the methods described can
be separated and identified by suitable methods (e.g., PCR,
sequencing, chromatography). One or more agents in a test sample
which modulate the cleaving activity of a self-cleaving ribozyme
can be determined according to these methods. Similarly, agents in
a test sample which bind an aptamer moiety can also be
determined.
[0098] Large combinatorial libraries of agents (e.g., organic
compounds, recombinant or synthetic peptides, peptoids, nucleic
acids) produced by combinatorial chemical synthesis or other
methods can be tested (see e.g., Zuckerman, R. N. et al., J. Med.
Chem., 37:2678-2685 (1994) and references cited therein; see also,
Ohlmeyer, M. H. J. et al., Proc. Natl. Acad. Sci. USA
90:10922-10926 (1993) and DeWitt, S. H. et al., Proc. Natl. Acad.
Sci. USA 90:6909-6913 (1993), relating to tagged compounds; Rutter,
W. J. et al. U.S. Pat. No. 5,010,175; Huebner, V. D. et al., U.S.
Pat. No. 5,182,366; and Geysen, H. M., U.S. Pat. No. 4,833,092).
The teachings of these references are incorporated herein by
reference. Where agents selected from a combinatorial library carry
unique tags, identification of individual agents by chromatographic
methods is possible. In addition, chemical libraries, microbial
broths and phage display libraries can be tested (screened) in
accordance with the methods herein.
[0099] In a further embodiment, the cleaving activity of a self
cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants) can be inhibited (partially or
totally) using an agent such as a drug (e.g., an antibiotic) or
other molecule or composition, which inhibits (partially or
totally) the cleaving activity of the self-cleaving ribozyme.
Inhibition of spontaneous cleavage of the corresponding mRNA
results in the efficient induction of expression of the nucleic
acid product of interest. Antibiotics that can be used to inhibit
the cleaving activity of a self cleaving ribozyme include
aminoglycoside antibiotics, such as, but not limited to, neomycin
B, neomycin sulfate, adriamycin RDF, doxorubicin, Bisbenzimide,
chelocardin, diminazene aceturate, ribostamycin, paromomycin,
neamine, gentamicin, gentamicin C complex, gentamicin C1A sulfate,
gentamicin sulfate, gramicidin S HCL, lincomycin, kanamycin,
tobramycin, tuberactinomycin A, tuberactinomycin B,
6'-amino-6'-deoxykanamycin and 5'-epi-sisomicin; tetracyclines and
their derivatives and analogs, such as, but not limited to,
tetracycline, chlortetracycline, demeclocycline, chelocardin and
4-epi-anhydrochlortetracycline; peptide antibiotics, such as, but
not limited to, viomycin, di-.beta.-lysyl capreomycin IIA and
tuberactinomycin A; and pseudodisaccharide antibiotics, such as,
but not limited to, 2'-de-N-1-.beta.-lysyllysinomicin,
3-epi-6'-de-C-methylfortim- icin B and
3-epi-2'-N-1-.beta.-lysyl-6'-de-C-methylfortimicin B. Other
antibiotics that can be used to inhibit the cleaving activity of a
self cleaving ribozyme are known and described in the art. See, for
example, Stage et al., RNA, 1:95-101 (1995); Clouet-d'Orval et al.,
Biochem., 34:11186-11190 (1995); Murray and Arnold, Biochem. J.,
317:855-860 (1996); Hermann and Westhof, J. Mol. Biol., 276:903-912
(1998); and Rogers et al., J. Mol. Biol., 259:916-925 (1996), the
teachings of which are entirely incorporated herein by
reference.
[0100] In certain specific embodiments, inhibitors of a self
cleaving ribozyme (naturally occurring or mutants such as
schistosome ribozyme mutants) include, but are not limited to,
antisense oligonucleotides of the ribozyme and any modified form of
the antisense oligonucleotides. For example, an antisense
oligonucleotide bases pair with a region of the self-cleaving
schistosome RNA mutant motif as depicted in FIG. 1e. As a result,
activity of the self-cleaving ribozyme is inhibited (partially or
totally) by the antisense oligonucleotide. In certain cases, the
antisense oligonucleotide is a modified oligonucleotide selected
from the group consisting of: morpholino, phosphorothioate RNA,
2'-O-methyl RNA, and phosphorothioate 2'-O-methoxyethyl RNA.
[0101] In other specific embodiments, inhibitors of a self cleaving
ribozyme (naturally occurring or mutants such as schistosome
ribozyme mutants) include, but are not limited to, the compounds
listed in Table 2, such as toyocamycin, 5-fluorouridine, and
5-fluorouracil.
[0102] In certain embodiments, the present invention contemplates a
method for determining the level of an inhibitor of a ribozyme
(naturally occurring or mutants such as schistosome ribozyme
mutants) in a biological sample. The term "determining" is used
herein to refer to any process of observing an inhibitor in a
biological sample, whether or not the inhibitor is actually
detected. Determining the level of an inhibitor may be a
quantitative, semi-quantitative or non-quantitative observation.
Such methods can be used for determining an inhibitor which is
present in a cell or in a biological sample. Exemplary inhibitors
include 5-fluorouracil and 5-fluorouridine. As used herein, the
5-FU compounds include 5-fluorouracil and its metabolite
derivatives such as 5-fluorouridine.
[0103] 5-fluorouracil is widely used in the treatment of a large
range of tumors and according to various schedules. Some studies
have proved a relationship between 5-fluorouracil plasma
concentrations and the toxic and therapeutic effects of the
treatment in different types of tumors (Beerblock, et al., 1997,
Cancer 79:1100; Trump, et al., 1991, J. Clin. Oncol. 11:2027;
Gamelin, et al., 1996, Cancer 77:441; Gamelin, et al., 1998, J.
Clin. Oncol. 16:1470; Milano, et al., 1994, J. Clin. Oncol.
12:1291). These findings were the basis for the determination of a
therapeutic range for the 5-FU compound, which is essential for
individual adjustment the dosage of compound. For example, by means
of individual dosage based on 5-FU concentrations, Gamelin et al.
reached a percentage of objective responses of 56% Gamelin, et al.,
1998, J. Clin. Oncol. 16:1470), while this value was approximately
15% for 5-FU in monotherapy.
[0104] In one specific embodiment, the invention provides a method
for determining the level of 5-FU compound in a cell,
comprising:
[0105] (a) introducing into a cell a DNA construct which comprises:
(1) a promoter; (2) nucleic acid encoding a reporter; and (3)
nucleic acid encoding a ribozyme (naturally occurring or mutants
such as schistosome ribozyme mutants), wherein the nucleic acid of
(2) and the nucleic acid of (3) are downstream of the promoter and
operably linked to said promoter, under conditions which result in
inhibition of the ribozyme and expression of the reporter; and
[0106] (b) assaying reporter activity in the cell produced by (a),
wherein the level of said 5-FU compound in the cell is identified
by comparing the reporter activity with an appropriate control.
[0107] In another specific embodiment, the invention provides a
method for determining the level of a 5-FU compound in a biological
sample comprises:
[0108] (a) contacting a cell with a biological sample, wherein the
cell expresses a DNA construct which comprises: (1) a promoter; (2)
a nucleic acid encoding a reporter; and (3) a nucleic acid encoding
a ribozyme (naturally occurring or mutants such as schistosome
ribozyme mutants), wherein the nucleic acid of (2) and the nucleic
acid of (3) are downstream of the promoter and operably linked to
said promoter, under conditions which result in inhibition of the
ribozyme and expression of the reporter; and
[0109] (b) assaying the reporter activity in the presence of the
biological sample, wherein the level of the 5-FU compound in the
biological sample is identified by comparing the reporter activity
with an appropriate control.
[0110] As described in the working examples, Applicants have found
that 5-FU compounds inhibited activity of a ribozyme (e.g., a
self-cleaving schistosome ribozyme mutant) in a dose-dependent
manner. In certain cases, an appropriate control of the method may
be a reference panel including predetermined mean values which have
been developed by contacting the cell with various doses of a 5-FU
compound. Exemplary biological samples of the method include, but
are not limited to, living cells or tissues (in vivo or in vitro),
lysates or extracts of cells or tissues, and bodily fluids (e.g.,
blood, serum, plasma, a blood-derived fraction, stool, colonic
effluent or urine).
[0111] In other embodiments, the present invention provides methods
of inhibiting activity of a catalytic RNA (e.g., a ribozyme or
mutants thereof) in a cell and methods of inhibiting infection by a
virus or a pathogenic microorganism in a cell. Traditionally,
pharmaceutical discovery has been focused on the compounds that
target the protein products of genes, while RNAs as drug targets
have remained largely unexplored. Recently, interests in RNAs are
increasing (Zaman G J R 2003; Hermann T, 1998; Pearson N D, 1997).
There have been efforts to target catalytic RNAs in viruses and
pathogenic microorganisms with small molecules (Rogers J, 1996,
supra). In the methods of the present invention, cells which have
been infected or are at risk of having infection by a virus or a
pathogenic microorganism (e.g., a parasite) are contacted with any
of the inhibitors as described above (e.g., Table 2). These
inhibitors interfere with RNA catalytic activity preferably through
RNA incorporation and can be used to target any RNA catalytic
activities involved in function (e.g., genome replication) of the
viruses or pathogenic microorganisms.
[0112] In certain aspects, viruses of the methods include, but are
not limited to, hepatitis virus (e.g., C, B, and delta), human
immunodeficiency virus (HIV), herpes virus, and human
papillomavirus (HPV). In other aspects, pathogenic microorganisms
of the methods include, but are not limited to, Notophthalmus
viddescens, Ambystoma talpoideum, Amphiuma tridactylum, and
Schistosoma mansoni. Cells of the methods can be animal cells
(e.g., mammalian cells such as human cells) or plant cells (e.g.,
tobacco).
[0113] Methods of Treatment and Administration
[0114] In certain embodiments, agents and effectors of the present
invention can be introduced into a cell for therapeutic
applications. As used herein, a cell includes, but is not limited
to, a prokaryotic cell, such as a bacterial cell, and eukaryotic
cell, such as an animal, plant or yeast cell. A cell which is of
animal or plant origin can be a stem cell or somatic cell. Suitable
animal cells can be of, for example, mammalian or avian origin.
Examples of mammalian cells include human (such as HeLa cells),
bovine, ovine, porcine, murine (such as embryonic stem cells),
rabbit and monkey (such as COS1 cells) cells. The cell may be an
embryonic cell, bone marrow stem cell or other progenitor cell.
Where the cell is a somatic cell, the cell can be, for example, an
epithelial cell, fibroblast, smooth muscle cell, blood cell
(including a hematopoietic cell, red blood cell, T-cell, B-cell,
etc.), tumor cell, cardiac muscle cell, macrophage, dendritic cell,
neuronal cell (e.g., a glial cell or astrocyte), or
pathogen-infected cell (e.g., those infected by bacteria, viruses,
virusoids, parasites, or prions).
[0115] The cells can be obtained commercially or from a depository
or obtained directly from an individual, such as by biopsy. The
cells used can be obtained from an individual to whom they will be
returned or from another/different individual of the same or
different species. For example, nonhuman cells, such as pig cells,
can be modified to include a DNA construct and then introduced into
a human. Alternatively, the cell need not be isolated from the
individual where, for example, it is desirable to deliver the
vector to the individual in gene therapy.
[0116] For example, the present invention relates to a method of
regulating expression of an endogenous gene (a gene resident in a
cell as the cell was obtained) to produce a desired nucleic acid
product and compositions useful in the method. The endogenous gene
can be one which is expressed ("on") in the cell or one which is
normally not expressed ("off") in the cell but whose expression is
or has been turned on or activated. DNA encoding a self-cleaving
ribozyme (naturally occurring or mutants such as schistosome
ribozyme mutants), or a virus or viral vector comprising a
recombinant genome which includes a nucleotide (RNA or DNA)
sequence which represents DNA encoding a self-cleaving ribozyme,
can be introduced into genomic DNA of cells in such a position that
in mRNA produced by the cells, the self-cleaving ribozyme is in a
location which results in control of expression of the encoded
product. In the absence of an agent which inhibits expression of
the self-cleaving ribozyme, cleavage occurs and the desired nucleic
acid product is not expressed. In the presence of such an agent,
cleaving activity is inhibited and the desired nucleic acid product
is expressed. In one embodiment, DNA encoding a self-cleaving
ribozyme, or a virus or viral vector comprising a recombinant
genome which includes a nucleotide (RNA or DNA) sequence which
represents DNA encoding a self-cleaving ribozyme, is introduced
into genomic DNA between the promoter operably linked to
(controlling expression of) the endogenous gene encoding the
desired nucleic acid product, in such a manner that the endogenous
gene remains operably linked to the promoter. In an alternative
embodiment, the DNA encoding a self-cleaving ribozyme, or the virus
or viral vector, is introduced into genomic DNA 3' of the
endogenous gene encoding the desired nucleic acid product. The
promoter which is operably linked to the endogenous gene to be
expressed can be the naturally occurring (endogenous) promoter for
the gene or can be an exogenous promoter introduced into genomic
DNA. The resulting cells can be used, as described herein, to
modulate production of the desired nucleic acid product in an
individual.
[0117] The present invention also relates to cells (host cells)
which comprise a DNA construct of the invention. Particular cells
which comprise a DNA construct of the invention are discussed
above.
[0118] In a particular embodiment, a ribozyme (naturally occurring
or mutants such as schistosome ribozyme mutants) of the invention
and DNA construct encoding the ribozyme can be used to produce
transgenic animals whose cells contain and express the ribozyme.
There is a variety of techniques for producing transgenic animals
of the present invention. For example, foreign nucleic acid can be
introduced into the germline of an animal by, for example,
introducing the additional foreign genetic material into a gamete,
such as an egg. Alternatively, transgenic animals can be produced
by breeding animals which transfer the foreign DNA to their
progeny. It is also possible to produce transgenic animals by
introducing foreign DNA into somatic cells from which an animal is
produced. As used herein, the term "transgenic animal" includes
animals produced from cells modified to contain foreign DNA or by
breeding; that is, it includes the progeny of animals (ancestors)
which were produced from such modified cells. As used herein, the
term "foreign nucleic acid" refers to genetic material obtained
from a source other than the parental germplasm. Preferably, the
transgenic animals are derived from mammalian embryos.
[0119] In certain aspects, the invention provides a homologous
recombinant non-human animal expressing the ribozyme (naturally
occurring or mutants such as schistosome ribozyme mutants). The
term "homologous recombinant animal" as used herein is intended to
describe an animal containing a gene which has been modified by
homologous recombination between the gene and a DNA molecule
introduced into a cell of the animal, e.g., an embryonic cell of
the animal. An animal can be created in which nucleic acid encoding
the ribozyme has been introduced into a specific site of the
genome.
[0120] To create such a homologous recombinant animal, a vector is
prepared which contains DNA encoding the ribozyme flanked at its 5'
and 3' ends by additional nucleic acid of a eukaryotic gene at
which homologous recombination is to occur. The additional nucleic
acid flanking that encoding the ribozyme is of sufficient length
for successful homologous recombination with the eukaryotic gene.
Typically, several kilobases of flanking DNA (both at the 5' and 3'
ends) are included in the vector (see e.g., Thomas, K. R. and
Capecchi, M. R. (1987) Cell 51:503 for a description of homologous
recombination vectors). The vector is introduced into an embryonic
stem cell line (e.g., by electroporation) and cells in which the
introduced DNA has homologously recombined with the endogenous DNA
are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
[0121] In addition to the homologous recombination approaches
described above, enzyme-assisted site-specific integration systems
are known in the art and can be applied to the components of the
regulatory system of the invention to integrate a DNA molecule at a
predetermined location in a second target DNA molecule. Examples of
such enzyme-assisted integration systems include the Cre
recombinase-lox target system (e.g., as described in Baubonis, W.
and Sauer, B. (1993) Nucl. Acids Res. 21:2025-2029; and Fukushige,
S. and Sauer, B. (1992) Proc. Natl. Acad. Sci. USA 89:7905-7909)
and the FLP recombinase-FRT target system (e.g., as described in
Dang, D. T. and Perrimon, N. (1992) Dev. Genet. 13:367-375; and
Fiering, S. et al. (1993) Proc. Natl. Acad. Sci. USA
90:8469-8473).
[0122] Methods for acquiring, culturing, maintaining and
introducing foreign nucleic acid sequences into recipient eggs for
transgenic animal production are well known in the art. See, for
example, Manipulating the Mouse Embryo: A Laboratory Manual, Hogan
et al., Cold Spring Harbor Laboratory, New York (1986). Preferably,
a DNA construct will be delivered into the embryo at a very early
stage in development so that only a small frequency of the embryos
are mosaic (e.g., an embryo in which integration of the foreign
nucleic acid occurs after the one cell stage of development).
[0123] The DNA constructs of the present invention can be used in
methods of inducing expression of a desired nucleic acid product in
an individual (e.g., a human or other mammal or vertebrate). In
these methods, a DNA construct of the present invention can be
introduced into cells obtained from the individual. The cells can
be migratory, such as a hematopoietic cell, or non-migratory, such
as a solid tumor cell or fibroblast. After treatment in this
manner, the resulting cells can be administered to (introduced
into) the individual according to methods known to those practiced
in the art. To induce expression of the nucleic acid product, an
agent (such as a drug) which is capable of inhibiting cleavage of
the encoded self-cleaving ribozyme (naturally occurring or mutants
such as schistosome ribozyme mutants), can be administered to the
individual according to methods known to those practiced in the
art. Such a treating procedure is sometimes referred to as ex vivo
treatment. Ex vivo therapy has been described, for example, in
Kasid et al., Proc. Natl. Acad. Sci. USA, 87:473 (1990); Rosenberg
et al., N. Engl. J. Med., 323:570 (1990); Williams et al., Nature,
310:476 (1984); Dick et al., Cell, 42: 71 (1985); Keller et al.,
Nature, 318:149 (1985); and Anderson et al., U.S. Pat. No.
5,399,346.
[0124] In a particular embodiment, the DNA constructs of the
present invention can be used in a method of expressing a desired
nucleic acid product in an individual. In this method, cells which
comprise a DNA construct of the present invention are introduced
into an individual. An agent (such as a drug) which is capable of
inhibiting cleavage of the encoded self-cleaving ribozyme
(naturally occurring or mutants such as schistosome ribozyme
mutants), is then administered to the individual, in whom the DNA
encoding the desired nucleic acid product is expressed, resulting
in production of the product. In a particular embodiment of this
method, the DNA construct which comprises: (a) DNA encoding the
desired nucleic acid product; (b) a promoter operably linked to the
DNA encoding the desired nucleic acid product; and (c) DNA encoding
a self-cleaving ribozyme. The DNA encoding the desired nucleic acid
product and the DNA encoding the self-cleaving ribozyme are
downstream of the promoter. The DNA encoding the self-cleaving
ribozyme can be 5' or 3' of the DNA encoding the desired nucleic
acid product. Transcription of the two DNA components in the
construct produces a mRNA comprising the self-cleaving ribozyme and
mRNA encoding the desired nucleic acid product.
[0125] Alternatively, in a method for expressing a desired nucleic
acid product in an individual, a DNA construct of the present
invention can be administered directly to the individual. The mode
of administration is preferably at the location of the target
cells. The administration can be nasally or by injection. Other
modes of administration (parenteral, mucosal, systemic, implant,
intraperitoneal, oral, intradermal, transdermal, intramuscular,
intravenous including infusion and/or bolus injection,
subcutaneous, topical, epidural, buccal, rectal, vaginal, etc.) are
generally known in the art. The DNA construct can, preferably, be
administered in a pharmaceutically acceptable carrier, such as
saline, sterile water, Ringer's solution, or isotonic sodium
chloride solution. An agent (such as a drug) which is capable of
inhibiting cleavage of the encoded self-cleaving ribozyme
(naturally occurring or mutants such as schistosome ribozyme
mutants), is then administered to the individual, in whom the DNA
encoding the desired nucleic acid product is expressed, resulting
in production of the product.
[0126] In another embodiment, the DNA constructs of the present
invention can be used in a method of modulating expression of a
desired nucleic acid product in an individual. In this method,
cells which comprise a DNA construct of the present invention are
introduced into an individual. An effector which is capable of
binding to the aptamer moiety of the aptamer-self-cleaving ribozyme
complex is then administered to the individual, whereupon
expression of the DNA encoding the desired nucleic acid product can
be induced, enhanced, reduced, inhibited or regulated, depending
upon the design of the complex as discussed above. In a particular
embodiment of this method, the DNA construct which comprises: (a)
DNA encoding the desired nucleic acid product; (b) a promoter
operably linked to the DNA encoding the desired nucleic acid
product; and (c) DNA encoding an aptamer-self-cleaving-ribozyme
complex (e.g., a self-cleaving schistosome RNA mutant motif which
comprises an aptamer grafted onto to the self-cleaving schistosome
RNA mutant motif at a location such that the cleaving activity of
the self-cleaving schistosome RNA mutant motif can be controlled by
binding of an effector to the aptamer). Alternatively, in a method
for modulating expression of a desired nucleic acid product in an
individual, a DNA construct of the present invention can be
administered directly to the individual. The modes of
administration include those described above. The DNA construct
can, preferably, be administered in a pharmaceutically acceptable
carrier, such as saline, sterile water, Ringer's solution, or
isotonic sodium chloride solution. An effector which is capable of
binding to the aptamer moiety of the aptamer-self-cleaving-ribozyme
complex can then be administered to the individual, whereupon
expression of the DNA encoding the desired nucleic acid product can
be induced, enhanced, reduced, inhibited or regulated, depending
upon the design of the complex as discussed above.
[0127] Agents and effectors can be administered to an individual in
a variety of ways. The route of administration depends upon the
particular agent or effector. Routes of administration are
generally known in the art and include oral, intradermal,
transdermal (e.g., in slow release polymers), intramuscular,
intraperitoneal, intravenous including infusion and/or bolus
injection, subcutaneous, topical, epidural, buccal, rectal, vaginal
and intranasal routes. Other suitable routes of administration can
also be used, for example, to achieve absorption through epithelial
or mucocutaneous linings.
[0128] The dosage of agent, effector, DNA construct of the present
invention administered to an individual, including frequency of
administration, will vary depending upon a variety of factors,
including mode and route of administration; size, age, sex, health,
body weight and diet of the recipient; nature and extent of
symptoms of the disease or disorder being treated; kind of
concurrent treatment, frequency of treatment, and the effect
desired.
[0129] Pharmaceutical Compositions
[0130] In certain embodiments, the agent, effector, and DNA
construct (collectively referred to herein as therapeutic agents)
of the present disclosure are formulated with a pharmaceutically
acceptable carrier. Such therapeutic agents can be administered
alone or as a component of a pharmaceutical formulation
(composition). Recombinant nucleic acid sequences (e.g., expression
constructs) encoding a ribozyme (naturally occurring or mutants
such as schistosome ribozyme mutants) can be used in therapeutic
(or pharmaceutical) compositions for regulating expression of a
target nucleic acid product. The therapeutic compositions of the
invention can be used alone or in admixture, or in chemical
combination, with one or more materials, including other
recombinant vectors, materials that increase the biological
stability of the recombinant vectors, or materials that increase
the ability of the therapeutic compositions to specifically
penetrate the relevant cell type. The therapeutic compositions of
the invention are administered in pharmaceutically acceptable
carriers (e.g., physiological saline), which are selected on the
basis of the mode and route of administration, and standard
pharmaceutical practice. Suitable pharmaceutical carriers, as well
as pharmaceutical necessities for use in pharmaceutical
formulations, are described in Remington's Pharmaceutical Sciences,
a standard reference text in this field.
[0131] The therapeutic compositions of the invention are
administered in dosages determined to be appropriate by one skilled
in the art. An appropriate dosage is one that effects a desired
result, e.g., a reduction in a symptom of a disease sought to be
treated. It is expected that the dosages will vary, depending upon
the pharmacokinetic and pharmacodynamic characteristics of the
particular agent, and its mode and route of administration, as well
as the age, weight, and health of the recipient; the nature and
extent of any relevant disease; the frequency and duration of the
treatment; the type of, if any, concurrent therapy; and the desired
effect.
[0132] The therapeutic compositions of the invention can be
administered to a patient by any appropriate mode, e.g.,
parenterally, intraperitoneally, orally, topically (e.g., with
dimethyl sulfoxide), or intravenously, as determined by one skilled
in the art. Alternatively, it may by necessary to administer the
compositions surgically to the target tissue. The treatments of the
invention can be repeated as needed, as determined by one skilled
in the art.
[0133] Any method that accomplishes in vivo transfer of nucleic
acids into eukaryotic cells can be used. For example, expression
constructs thereof can be packaged into liposomes, non-viral
nucleic acid-based vectors, erythrocyte ghosts, or microspheres
(e.g., microparticles; see, e.g., U.S. Pat. Nos. 4,789,734; and
4,925,673; 3,625,214; and Gregoriadis, Drug Carriers in Biology and
Medicine, pp. 287-341 (Academic Press, 1979)). Further, delivery of
expression constructs encoding a ribozyme mutant motif can be
accomplished by direct injection into target tissues, for example,
in a calcium phosphate precipitate or coupled with lipids.
[0134] Exogenously provided ribozyme (naturally occurring or
mutants such as schistosome ribozyme mutants) can contain modified
nucleotides, e.g., modified nucleotides that enhance stability. For
example, the ribozyme mutant motifs can contain inter-nucleotide
linkages other than phosphodiester bonds, such as phosphorothioate,
methylphosphonate, methylphosphodiester, phosphorodithioate,
phosphoramidate, phosphotriester, or phosphate ester linkages
(Uhlman et al., Chem. Rev. 90(4):544-584, 1990; Tidd et al.,
Anticancer Research 10:1169, 1990). Ribozymes' stability can also
be increased by incorporating 3'-deoxythymidine or 2'-substituted
nucleotides (substituted with, e.g., alkyl groups) into the
ribozymes during synthesis, by providing the ribozymes as
phenylisourea derivatives, or by having other molecules, such as
aminoacridine or poly-lysine, linked to the 3' ends of the snoRNAs
(see, e.g., Tidd et al, Anticancer Research 10:1169-1182, 1990).
Modifications of the RNA nucleotides of the ribozyme motifs of the
invention may be present throughout the ribozymes, or in selected
regions. The DNA vectors encoding ribozyme motifs can be modified
to increase their ability to penetrate the target tissue by, e.g.,
coupling them to lipophilic compounds. In addition, DNA vectors can
be targeted to particular cells by coupling them to ligands
specific for receptors on the cell surface of a target cell. DNA
vectors can also be targeted to specific cell types by being
conjugated to monoclonal antibodies that specifically bind to
cell-type-specific receptors.
[0135] For topical administration, a therapeutically effective
amount of one or more of the therapeutic agents is applied to the
desired site on the skin, preferably in combination with a
pharmaceutically acceptable carrier, e.g., a spreadable cream, gel,
lotion, or ointment, or a liquid such as saline. For use on the
skin, the penetration of the nucleic acids into the tissue may be
accomplished by a variety of methods known to those of ordinary
skill in this field. For example, the expression constructs may be
incorporated into a transdermal patch that is applied to the skin.
Preferably, the penetration resulting from these methods is
enhanced with a chemical transdermal delivery agent such as
dimethyl sulfoxide (DMSO) or the nonionic surfactant, n-decylmethyl
sulfoxide (NDMS), as described in Choi et al., Pharmaceutical Res.,
7(11):1099, 1990. Dosages for a therapeutically effective amount
for topical application would be in the range of 100 ng to 10 mg
per treated surface area per day.
[0136] Exemplification
[0137] The invention now being generally described, it will be more
readily understood by reference to the following examples, which
are included merely for purposes of illustration of certain
embodiments and embodiments of the present invention, and are not
intended to limit the invention.
[0138] Recent studies of the control of specific metabolic pathways
in bacteria have documented the existence of entirely `RNA-based`
mechanisms for controlling gene expression which involve the
modulation of translation, transcription termination, or RNA
self-cleavage through the direct interaction of specific
intracellular metabolites and RNA sequences (Winkler, et al., 2002,
Nature 419, 952-6; Winkler, et al., 2004, Nature 428, 281-6;
Mandal, et al., 2004, Nat Struct Mol Biol 11, 29-35; Cech, et al.,
2004, Nature 428, 263-4). Here, Applicants show that an analogous
"RNA-based" gene regulation system can effectively be `designed`
for mammalian cells via the incorporation of sequences encoding
self-cleaving RNA motifs (Cech, et al., 1990, Biosci Rep 10,
239-61) into the transcriptional unit of a gene or vector. When
properly positioned, the sequences lead to potent inhibition of
gene or vector expression, due to the spontaneous cleavage of the
RNA transcript. Administration of either oligonucleotides
complementary to regions of the self-cleaving motif, or a specific
small molecule results in the efficient induction of gene
expression, due to inhibition of self-cleavage of the mRNA.
Efficient regulation of transgene expression is shown to be
possible in a variety of mammalian cell lines, and in live animals.
In conjunction with other emerging technologies (Silverman, et al.,
2003, RNA 9, 377-83), the general methodology may be particularly
applicable to the development of gene regulation systems tailored
to any small inducer molecule, and provide for a novel means of
biological sensing in vivo that may have an important application
in the regulated delivery of protein therapeutics.
[0139] The general strategy for controlling gene expression via
modulation of RNA processing is shown in FIG. 1a. The approach is
critically dependent upon both the ability of a specific
self-cleaving ribozyme (rz) to effect the highly efficient cleavage
(>99%) of an mRNA molecule into which it is embedded, and the
availability of a small molecule capable of efficiently inhibiting
self-cleavage of the cis-acting rz within an intracellular milieu.
A first step in our studies was to identify candidate rz sequences
capable of efficient cleavage in mammalian cells in the context of
an `expression` vector. For this purpose, Applicants made use of a
transient transfection assay involving a standard mammalian
expression vector (Ory, et al., 1996, Proc Natl Acad Sci USA 93,
11400-6) which encodes a LacZ reporter (FIG. 1b). Candidate rz
sequences were introduced into one of a number of different
locations within the vector transcriptional unit, and in parallel,
the corresponding inactive mutant rzs were introduced into the same
sites to provide a means of measuring the efficiency of reduction
of reporter gene expression. After transfection of 293 cells with
these vectors, protein extracts were prepared from cells and the
.beta.-gal level quantified. Cleavage activity of the functional rz
in cells was measured as `fold` suppression in reporter gene
expression relative to the vector with inactive rz.
[0140] A large number of different rz encoding motifs were chosen
for analysis, including unmanipulated natural rz sequences, rzs
shown to function in mammalian cells, and rzs engineered by others
to possess specific biochemical or catalytic properties in vitro
(e.g., high K.sub.cat, low Mg++ requirement, etc.). See Table 1
below. While the vast majority of rzs tested did not appreciably
affect reporter expression, as reflected by near equal expression
of LacZ by vectors encoding functional and inactive rzs (defined as
`fold`=1), two rz motifs were identified which did appear to
function effectively: the hammerhead Pst-3 rz derived from the
Dolichopoda cave cricket (Rojas, et al., 2000, Nucleic Acids Res
28, 4037-43) (FIG. 1c; 13-fold difference between functional and
inactive rz) and the hammerhead 5 ml rz derived from the trematode
Schistosoma mansoni (Ferbeyre, et al., 1998, Mol Cell Biol 18,
3880-8) (FIG. 1d) in which the tetraloop 5'UUCG3' was grafted onto
an extended stem III (19-fold difference). Interestingly, both rzs
possess unique structures relative to the other hammerhead rzs
tested, in that they contain an extended `stem-I` with an internal
loop (see FIGS. 1c and 1d).
1TABLE 1 Survey of ability of different ribozymes to function in
mammalian cells. Ribozyme Fold Commands Zillmen's hammerhead.sup.1
1 short stem II, worked at low Mg++ Lockett's hammerhead.sup.2 1
short stem II, worked at low Mg++ Szostak's hammerhead.sup.3 1
short stem II, higher catalytic rate Taira's hammerhead.sup.4 1
worked in cells Taira's embedded in tRNA.sup.4 1 tRNA helped to
stablize the ribozyme McSwiggen's hammerhead.sup.5 1 worked in
cells Uhlenbeck's hammerhead.sup.6 1 short stem I, higher catalytic
rate ABSV hammerhead.sup.7 1 Naturally occurring ribozyme TRSV
hairpin.sup.8 1 Naturally occurring ribozyme TRSV hammerhead.sup.8
1 Naturally occurring ribozyme Neurospora ribozyme.sup.9 1
Naturally occurring ribozyme Hepatitis Delta Virus 1 Naturally
occurring ribozyme ribozyme.sup.10 Newt hammerhead.sup.11 1
Naturally occurring ribozyme Cricket hammerhead.sup.12 13 Naturally
occurring ribozyme Schisto hammerhead.sup.13 1 Naturally occurring
ribozyme Schisto with new loop-III 19 Naturally occurring ribozyme
The above ribozymes can be found in the following references:
.sup.1Zillmann, et al., RNA 3, 734-47 (1997); .sup.2Conaty, et al.,
Nucleic Acids Res 27, 2400-7 (1999); .sup.3Salehi-Ashtiani, et al.,
Nature 414, 82-4 (2001); .sup.4Yuyama, et al., Nucleic Acids Res
22, 5060-7 (1994); .sup.5Chowrira, et al., # J Biol Chem 269,
25856-64 (1994); .sup.6Clouet-d'Orval, et al., Biochemistry 36,
9087-92 (1997); .sup.7Hutchins, et al., Nucleic Acids Res 14,
3627-40 (1986); .sup.8Buzayan, et al., Virology 151, 186-199
(1986); .sup.9Rastogi, et al., Embo J 15, 2820-5 (1996);
.sup.10Been, et al., Eur J Biochem 247, # 741-53 (1997);
.sup.11Zhang, et al., Gene 172, 183-90 (1996); .sup.12Rojas, et
al., Nucleic Acids Res 28, 4037-43 (2000); .sup.13Ferbeyre, et al.,
Mol Cell Biol 18, 3880-8 (1998).
[0141] Based on its apparent higher level of self-cleavage
activity, the Sm1 rz was chosen for further study and manipulation.
In an effort to improve the efficiency of the Sm1 rz self-cleavage
activity, a series of modifications of the Sm1 rz structure were
made and evaluated. As shown in FIG. 1d, specific modification at
nucleotide 7 (C to U) in the conserved catalytic core (Hertel, et
al., 1992, Nucleic Acids Res 20, 3252), and changes in distal stem
III led to a significant increase in the extent of self-cleavage
(the modified Sm1 rz was termed `N73`, up to 62-fold difference
between functional and inactive rz). Transfer of the N73 rz from
position A to E of the vector enhanced the activity to 225-fold
(this rz was termed `N79` and was used in the subsequent studies).
Additional modifications of stem I near the catalytic core and near
the restriction insertion site led to further increases in
activity, resulting ultimately in an overall 1401-fold difference
in expression levels between functional vs. inactive rz (FIG.
1e).
[0142] In addition to modifications that led to improved
self-cleavage activity, several modifications, notably those
involving the shortening of stem I (FIG. 1f), and alteration of
nucleotides within the internal loop of stem I (FIG. 1g),
dramatically reduced the level of self-cleavage. Interestingly,
neither of those modifications affect conserved core sequences
known to be required for rz cleavage in vitro (Ruffner, et al.,
1990, Biochemistry 29, 10695-702). Under standard in vitro
conditions of 10 mM Mg.sup.++, measurement of the catalytic
activity of the N79 rz and the N107 (U to G) rz, which carries a
single base U to G change in the loop I and is inactive in
mammalian cells (FIG. 1g), indicated that both rzs were equally
functional in vitro (K.sub.obs values of 0.84.sup.-min and,
1.06.sup.-min, respectively, see Supplementary Methods).
Intriguingly, however, determination of the cleavage rate at 0.5 mM
Mg.sup.++ indicated that only the N79 enzyme possessed significant
activity under low Mg.sup.++ conditions (N79 rz
K.sub.obs=0.84.sup.-min vs. N107 (U to G) rz
K.sub.obs=0.014.sup.-min). These results suggest that sequences
within the unique loop structure of stem-I of the Sm1 rz may enable
efficient self-cleavage in mammalian cells in part because they
facilitate self-cleavage at physiological Mg.sup.++ concentrations.
Consistent with this idea, measurement of the K.sub.obs of another
well-characterized hammerhead rz ("McSwiggen's hammerhead", Table
1) previously shown by others to function in mammalian cells, yet
shown in our transfection assay to possess no appreciable activity,
indicated that significant in vitro cleavage activity occurred only
under high Mg.sup.++ conditions (0.32.sup.-min at 10 mM Mg.sup.++
vs. 0.015 min at 0.5 mM Mg.sup.++). To what extent the ability to
function at low Mg.sup.++ concentration per se contributes to the
ability of a rz to function efficiently in mammalian cells remains
unclear, however, since several rzs engineered by others to
efficiently function in vitro under low Mg.sup.++
conditions.sup.13-14 were amongst the many rzs that were found to
be non-functional in our transient transfection assay.
[0143] Applicants also found that the efficiency of schistosoma
ribozymes was independent of the amount of plasmid DNA transfected
in mammalian cells. A wide range of plasmid DNA concentration was
tested in the transfection assay. It was found that the efficiency
of the schistosoma ribozymes remained unchanged with increasing
amounts of transfected DNA. This suggests that the function of
schistosoma ribozymes is largely independent of the cellular
resource and can be coupled to a strong promoter to produce high
copy numbers of regulatable mRNA. Applicants tested four kinds of
promoters (CMV promoter, EF1-alfa promoter, Ubiquitin C promoter,
and Lenti viral promoter) and found that they all worked well with
the Schistosoma ribozymes. Since the ribozyme mechanism should be
independent of the promoter systems, Applicants believe that the
schistosoma ribozymes should work with all different promoters.
Applicants also tested four kinds of reporter genes (Lac-Z, GFP,
dsRED, and Luciferase) and they all worked well with the
Schistosoma ribozymes. Applicants believe that schistosoma
ribozymes should work with other reporter genes. Applicants further
found that schistosoma ribozymes worked not only in transiently
transfected cells, but also in stably transfected cells. Several
stable HEK-293 cell lines containing schistosoma ribozymes were
generated. These cell lines were later used to screen libraries of
small compounds that eventually led to the discovery of drugs for
ribozyme inhibition and thus the regulation of gene expression (see
below).
[0144] For example, in addition to functioning in 293 cells within
the context of the original CMV-based pMD vector tested, the N79 rz
also dramatically reduced .beta.-gal expression in a variety of
other commonly used cell lines after transfection (FIG. 2a). In
addition, the N79 rz was able to function efficiently when placed
within other transcriptional units which made use of different
promoters and a different reporter gene (eGFP) (FIG. 2b). As
Applicants had observed in the primary screen of different rz
motifs for activity in mammalian cells, N79 was able to function
when placed in several, but not all locations of the pMD
transcriptional unit, albeit at different efficiencies (FIG. 2c).
Importantly, placement of two rz sequences in tandem, in some cases
(e.g., E position) led to a dramatic suppression of reporter gene
expression (FIG. 2c).
[0145] An essential requirement for the development of a gene
regulation system based on modulation of self-cleavage activity is
the availability of small inducer molecules capable of efficient
inhibition of rz activity in mammalian cells. In an effort to
identify such molecules, Applicants first surveyed a large number
of common antibiotics that had been shown to inhibit rz cleavage in
vitro (Table 1 above) (Hermann, et al., 1998, J Mol Biol 276,
903-12; Jenne, et al., 2001, Nat Biotechnol 19, 56-61; Murray, et
al., 1996, Biochem J 317 (Pt 3), 855-60; Stage, et al., 1995, RNA
1, 95-101; Tor, et al., 1998, Chem Biol 5, R277-83; von Ahsen, et
al., 1991, Nature 353, 368-70). In no case was significant
inhibition of self-cleavage by these antibiotics observed in our
transient transfection assay.
[0146] Applicants next surveyed the ability of different types of
antisense oligonucleotides (Braasch, et al., 2002, Biochemistry 41,
4503-10) to inhibit rz cleavage (see FIG. 1e for targeted sequence,
SEQ ID NO: 67). While transfection with PNAs, LNAs, and "grip" NAs
had no measurable effects on self-cleavage activity, and
phosphorothioate, 2'-O-methyl, and phosphorothioate 2'-O-methyl
derived RNAs led to modest inhibition of self-cleavage (<10-fold
induction), transfection of a morpholino oligonucleotide (Morcos,
et al., 2001, Genesis 30, 94-102) led to a strong inhibition of rz
self-cleavage, as revealed by a dramatic increase in reporter gene
expression (FIG. 3a). While the `fold` induction afforded by this
method was somewhat variable from experiment to experiment
(110-2000-fold in the case of double N79 construct) most likely due
to the variability of efficiency of oligo delivery and toxicity
associated with transfection of the oligo, the extent of induction
of gene expression was nonetheless comparable to that achieved with
other gene regulation systems, and clearly represents a range that
would be useful for a variety of experimental and clinical
settings. In some cases, the absolute levels of gene expression
achieved after morpholino administration approached 50% of the
theoretical maximum induction possible (i.e., the level of gene
expression produced by the inactive rz), suggesting that rz
cleavage can be very efficiently inhibited in mammalian cells.
[0147] Applicants generated stable cell lines carrying an
integrated expression construct in which a luciferase reporter was
placed under the control of two copies of the N79 rz, and made use
of the cells in high-throughput screening studies to identify small
molecule compounds capable of inhibiting rz self-cleavage. Of the
compounds identified (Table 1 below), toyocamycin (Aszalos, et al.,
1966, J Antibiot (Tokyo) 19, 285), a nucleoside analogue, was found
to be one of the most potent inhibitors of rz function. As shown in
FIG. 3b, administration of 1.5 .mu.m toyocamycin to the same cells
led to a dramatic increase in luciferase protein expression. A
parallel analysis of luciferase mRNA expression demonstrated that
in the absence of drug, little if any luciferase mRNA was produced
in the nucleus or cytoplasm, while in drug-treated cells, the
amount of luciferase mRNA was increased to a level comparable to
that of cells carrying inactive rz (FIG. 3c). The lack of
detectable mRNA in untreated cells suggests that cleaved mRNAs are
rapidly degraded, presumably because the cleaved fragments lack
conventional sequences at the ends of mRNAs.
2TABLE 2 Compounds found to inhibit ribozyme activity in cells
EC.sub.50 Compound name (.mu.M) Fold.sub.max Class Toyocamycin 0.4
365 ribo-nucleoside 8-Azaadenosine 4 45 ribo-nucleoside
Sangivamycin 1 38 ribo-nucleoside Tubercidin 2.5 58 ribo-nucleoside
Tubercidin-cyclic monophosphate 34 35 ribo-nucleoside
Tubercidin-monophosphate 2 39 ribo-nucleoside
Tubercidin-triphosphate 2 35 ribo-nucleoside Nebularine 10 8
ribo-nucleoside Tricyclic Nucleoside 40 12 ribo-nucleoside
5-FluoroUridine 5.9 120 ribo-nucleoside 5-BromoUridine 267 16
ribo-nucleoside 5-FluoroUracil 200 377 pyrimidine Syto-83 7 26
RNA-binding dye Homidium bromide 2 8 RNA-binding dye Acridine
orange 7 4 RNA-binding dye
[0148] In addition to toyocamycin, 5'-FU compounds (such as
5'-FluoroUridine and 5'-FluoroUracil) are two other potent
inhibitors of rz function (Table 2). These two 5'-FU compounds are
also nucleoside analogues. FIGS. 9A-9B show that 5'-FUridine (A)
and 5'-FUracil (B) induced gene expression via inhibition of rz
self-cleavage in a dose-dependent manner. As shown in FIGS. 9A and
9B, administration of the two 5'-FU compounds to the cells led to a
dramatic increase in luciferase protein expression in a
dose-dependent manner.
[0149] In contrast to toyocamycin, adenosine, a related compound,
possessed no ability to inhibit rz self-cleavage. While the above
assays indicate that self-cleavage is highly efficient in the
stable cell line, sensitive measurement of luciferase activity by
photon emission indicates that there is, nevertheless, an extremely
low but detectable amount of luciferase expression above the
background emission level of control cells that carry no luciferase
gene (see legend to FIG. 3).
[0150] Having documented the ability of the rz-based regulation
system to function in mammalian cell culture, a final important
issue to be addressed was whether the intracellular machinery and
`environment` necessary for self-cleavage was operable in primary
cells in vivo. To address this issue, Applicants generated
recombinant adeno-associated virus (AAV) genomes carrying
transcription units derived from pMD rz-luciferase vectors
possessing two copies of functional or inactive N79 rz at the E
position and prepared high titer virus possessing the host range of
AAV serotype 5. The two viruses were then used to inject nude mice
subretinally as described under "Methods." To provide a means of
normalization for differences in the capacity to measure luciferase
activity on different days (such as variations due to the delivery
of the luciferin substrate), all animals were also injected in the
hamstring muscles of the hind limb with AAV viruses carrying the
inactive N79 rzs. After 21 days, the injected animals were imaged
for luciferase gene expression using the Xenogen IVIS imager, which
provides a quantitative measure of luciferase expression based on
single-photon detection (Contag, et al., 1998, Nat Med 4, 245-7).
Immediately after imaging, cohorts of mice injected with virus
carrying the functional rz sequences were implanted under the
dorsal skin with seven day `time-release` pellets of either
toyocamycin or adenosine (Innovative Research of America, Inc),
while another cohort of mice injected with virus carrying the
inactive rz sequences were implanted with toyocamycin pellets. Two
days later, all animals were then imaged for luciferase expression.
Representative images of mice in each treatment group, taken before
and after drug treatment, are shown in FIG. 4. The images
demonstrate that, as expected, mice injected with virus carrying
inactive rzs showed robust luciferase expression in the retina, and
the expression was independent of the administration of toyocamycin
(FIG. 4, upper panel). Mice injected with virus carrying two
functional rzs and implanted with adenosine pellets showed little
if any gene expression before or after adenosine treatment (FIG. 4,
middle panel), consistent with the inability of adenosine to
inhibit rz self-cleavage. Importantly, mice injected with virus
carrying the functional rzs showed readily detectable expression
only after toyocamycin treatment (FIG. 4, lower panel).
Quantification of the photon output indicated gene expression was
`induced` 39, 185, and 191-fold in the three mice infected with
virus carrying the functional rzs and treated with toyocamycin. In
the last case (animal showing 191-fold induction) the induced gene
expression reached a level within 40% of the gene expression of
virus carrying inactive rzs. These results indicate that
significant rz-mediated gene regulation can be accomplished in the
in vivo setting. While the retina may be particularly accessible to
`inducer` due to its extensive vascularization, Applicants have
shown in preliminary experiments that gene regulation can be
accomplished at a number of other anatomical sites in vivo (e.g.,
muscle and ear).
[0151] Overall, the studies reported here provide an important
`proof-of-principle` for gene regulation strategies based on the
modulation of RNA processing. Specifically, the fact that efficient
rz `self-cleavage` can be made to occur in a variety of different
mammalian cell lines, and in primary cells in vivo suggests that
mammalian cells may in general be `permissive` for efficient
ribozyme self-cleavage and therefore that rz-based regulation
systems may be generally applicable to the manipulation of gene
expression in cells and animals. In addition to implications for
the development of gene regulation strategies, the studies also
provide a compelling rationale for determining whether `naturally
occurring` RNA-only mechanisms for gene regulation exist in
mammalian cells exist.
[0152] The most commonly used systems for controlling gene
expression, which rely on the regulation of transcription (Gossen,
et al., 1992, Proc Natl Acad Sci USA 89, 5547-51; Rivera, et al.,
1996, Nat Med 2, 1028-32; Suhr, et al., 1998, Proc Natl Acad Sci
USA 95, 7999-8004; Wang, et al., 1994, Proc Natl Acad Sci USA 91,
8180-4) have proved to be extremely powerful experimental tools.
However, despite their utility, such systems possess at least some
practical and theoretical limitations due to their reliance on
chimeric transcriptional transactivators and specialized promoter
elements. These limitations include the need to co-introduce
expression constructs for both the transactivator and the transgene
to be regulated, the potential toxicities due to expression of a
chimeric transactivator, difficulties in application of such
systems to the regulation of endogenous cellular genes due to the
requirement of a specialized promotor, and the limited number of
small inducer molecules available for experimental and therapeutic
applications. In contrast to systems based on the regulation of
transcription, the rz-based system Applicants have described does
not require the expression of any protein transactivator products
and is not dependent upon the use of any specialized promoter
elements, and therefore, in theory, represents a `portable`
regulation system that could be `embedded` into any endogenous gene
or engineered vector transcription unit. Although the two
inhibitors of rz self-cleavage Applicants have described may not be
ideal for many experimental applications, it is likely that
additional inducers with more desirable pharmacokinetic properties
and toxicity profiles can be identified via high-throughput
screening, further evaluation of specific antisense
oligonucleotides and methods for their in vivo delivery to cells,
or through the application of several emerging technologies. In
this latter regard, recent studies have shown that it is possible
to generate rzs whose in vitro self-cleavage activity is controlled
by a specific ligand, either by the `judicious` linkage of RNA
aptamer sequences to specific regions of hammerhead rz (Breaker, et
al., 2002, Curr Opin Biotechnol 13, 31-9), or through the use of in
vitro evolution technologies (Wilson, et al., 1999, Annu Rev
Biochem 68, 611-47). Application of these technologies to the
strategy for controlling gene expression described here should make
it possible in the future to `tailor` specific rz-based gene
regulation systems to any small molecule ligand. Such an approach
would provide a general methodology for developing gene regulation
systems which rely on ligands with desirable and/or specific
pharmacokinetic properties. In addition, the combined technologies
should provide the means to independently and simultaneously
control the expression of multiple gene products, and to express
gene products in response to the concentration of any intracellular
molecule or combinations of molecules. Such a form of `biological
sensing` could have broad experimental and therapeutic
applications.
[0153] Methods:
[0154] 1) Transfection Protocol:
[0155] 1.5 .mu.l Fugene-6 (Roche, Basel, Switzerland) was diluted
in 100 .mu.l OPTIMEM (Gibco, Carlsbad, Calif.) and incubated for 5
minutes at room temperature. This solution was added dropwise to
0.45 .mu.g plasmid DNA and incubated for 15 minutes. The plasmid
DNA contains either a wildtype or a mutant ribozyme and a LacZ
reporter gene. The DNA mixture was then added dropwise to
3.times.10.sup.5 HEK 293 cells (plated the previous day in a 35 mm
dish). Cells were harvested for .beta.-galactosidase assay 24 hours
following transfection.
[0156] 2) Transient Reporter Expression Assay:
[0157] Plasmids carrying different ribozymes were transfected into
HEK293T cells and lysed 24 h later. The cell extracts were
incubated with ONPG, and the amount of .beta.-galactosidase in cell
extracts was measured by quantifying the processed ONPG using a
luminometer.
[0158] 3) Assays for Ribozyme Activity:
[0159] Cleaving activity was determined by comparing the level of
.beta.-galactosidase measured in the test sample to a control
comprising a point mutation (A.fwdarw.G) at position 14 which
attenuates ribozyme activity. Briefly, transfected HEK293 cells
were lysed with a lysis solution and the extracts of cells were
separated from cell debris. The extracts were then incubated with
ONPG, a chromogenic substrate of .beta.-galactosidase. Cleavage of
ONPG by .beta.-galactosidase resulted in a yellow color. The
intensity of yellow light emission was measured with a luminometer
(Miller J., Experiments in Molecular Genetics, Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y. (1972)).
[0160] Most of Applicants' assays on ribozyme activity were
performed at protein level. The active ribozymes (the wildtype)
produced little or no proteins while the inactive ribozymes (the
mutant) enabled high protein production. Applicants also performed
Northern analyses to compare the mRNA level. It was found that the
cell line containing the active ribozymes had no detectable level
of mRNA in both the nucleus and cytoplasm, as compared to the high
level of mRNA found in the cells with inactive ribozymes. This is
consistent with the idea that the ribozymes acted at the
transcriptional level.
[0161] 4) Catalytic Rate Measurement:
[0162] Ribozymes were generated by in vitro transcription in the
presence of 50 uM blocking antisense oligos. Full length ribozymes
were purified and the cleavage rate determined in 50 mM Tris-HCl,
pH7.5 at 23.degree. C. K.sub.obs was calculated according to the
equation F.sub.t=F.sub.0+F.sub..infin.(1-e.sup.kt).
[0163] 5) Non-Invasive Bioluminescent Imaging:
[0164] Prior to imaging, the anesthetized mice were injected with
150 .mu.l of luciferin (30 mg/ml) and the pupils were moistened and
dilated with 1% tropicamide. A series of bioluminescent images were
taken for up to 30 minutes using the Xenogen IVIS imager. Photon
output was quantified at the plateau of the time course using the
LivingImage software. Induction in fold was calculated based on the
photon output in the retina before and after drug treatment, and
was normalized to the photon output from the leg muscles. Adenosine
was purchased from Innovative Research of America.
[0165] Incorporation by Reference
[0166] All publications and patents mentioned herein are hereby
incorporated by reference in their entirety as if each individual
publication or patent was specifically and individually indicated
to be incorporated by reference.
[0167] While specific embodiments of the subject invention have
been discussed, the above specification is illustrative and not
restrictive. Many variations of the invention will become apparent
to those skilled in the art upon review of this specification and
the claims below. The full scope of the invention should be
determined by reference to the claims, along with their full scope
of equivalents, and the specification, along with such variations.
Sequence CWU 1
1
71 1 76 RNA Unknown modified schistosome ribozyme partial sequence
1 auguguguuc ccucugcccc gcugaugagg ucggggagac cgaaaggguc aacucuacgg
60 ggcuauuaca ugcaau 76 2 75 RNA Unknown modified schistosome
ribozyme partial sequence 2 cugagaugca gguacaucca gcugacgagu
cccaaauagg acgaaacgcg cguccuggau 60 uccacugcua uccac 75 3 81 RNA
Unknown modified schistosome ribozyme partial sequence 3 cugagaugca
gguacaucca gcugacgagu cccaaauagg acgaaacgcc uucgggcguc 60
cuggauucca cugcuaucca c 81 4 81 RNA Unknown modified schistosome
ribozyme partial sequence 4 cugagaugca gguacaucca gcugaugagu
cccaaauagg acgaaacgcc uucgggcguc 60 cuggauucca cugcuaucca c 81 5 83
RNA Unknown modified schistosome ribozyme partial sequence 5
cugagaugca gguacaucca gcugaugagu cccaaauagg acgaaacgcg cuucggugcg
60 uccuggauuc cacugcuauc cac 83 6 83 RNA Unknown modified
schistosome ribozyme partial sequence 6 cugagaugca gguacaucca
gcugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 uccuggauuc
cacugcuauc cac 83 7 83 RNA Unknown modified schistosome ribozyme
partial sequence 7 cugagaugca gguacauccc acugaugagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucugggauuc cacugcuauc cac 83 8 83 RNA
Unknown modified schistosome ribozyme partial sequence 8 cugagaugca
gguacauccc acugaugagu cccaaauagg acgaaacgcg cuucggugcg 60
ucugggauuc cacugcuauc cac 83 9 83 RNA Unknown modified schistosome
ribozyme partial sequence 9 cugagaugca gguacaucca gcugaugagu
cccaaauagg acgaaacgcg cuucggugcg 60 uccuggauuc cacugcuauc cac 83 10
65 RNA Unknown modified schistosome ribozyme partial sequence 10
agguacaucc agcugaugag ucccaaauag gacgaaacgc gcuucggugc guccuggauu
60 ccacu 65 11 49 RNA Unknown modified schistosome ribozyme partial
sequence 11 ccagcugaug agucccaaau aggacgaaac gcgcuucggu gcguccugg
49 12 83 RNA Unknown modified schistosome ribozyme partial sequence
12 cugaggugca gguacaucca gcugacgagu cccaaauagg acgaaacgcg
cuucggugcg 60 uccuggauuc cacugcuauc cac 83 13 75 RNA Unknown
modified schistosome ribozyme partial sequence 13 cugagaugca
gguacaucca gcugacgagu cccaaauagg acgaaacgcg cguccuggau 60
uccacugcua uccac 75 14 81 RNA Unknown modified schistosome ribozyme
partial sequence 14 cugagaugca gguacaucca gcugacgagu cccaaauagg
acgaaacgcc uucgggcguc 60 cuggauucca cugcuaucca c 81 15 81 RNA
Unknown modified schistosome ribozyme partial sequence 15
cugagaugca gguacaucca gcugaugagu cccaaauagg acgaaacgcc uucgggcguc
60 cuggauucca cugcuaucca c 81 16 83 RNA Unknown modified
schistosome ribozyme partial sequence 16 cugagaugca gguacaucca
gcugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 uccuggauuc
cacugcuauc cac 83 17 83 RNA Unknown modified schistosome ribozyme
partial sequence 17 cugagaugca gguacaucca gcugaugagu cccaaauagg
acgaaacgcg cuucggugcg 60 uccuggauuc cacugcuauc cac 83 18 83 RNA
Unknown modified schistosome ribozyme partial sequence 18
cugagaugca gguacauccc acugaugagu cccaaauagg acgaaacgcg cuucggugcg
60 ucugggauuc cacugcuauc cac 83 19 83 RNA Unknown modified
schistosome ribozyme partial sequence 19 cugagaugca gguacauccc
acugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 ucugggauuc
cacugcuauc cac 83 20 81 RNA Unknown modified schistosome ribozyme
partial sequence 20 cugagaugca gguacaucca gcugaugagu cccaaauagg
acgaaacgcc uucgggcguc 60 cuggauucca cugcuaucca c 81 21 81 RNA
Unknown modified schistosome ribozyme partial sequence 21
cugagaugca gguacaucca gcugaugagu cccaaauagg acgaaacgcc uucgggcgug
60 cuggauucca cugcuaucca c 81 22 81 RNA Unknown modified
schistosome ribozyme partial sequence 22 cugagaugca gguacaucca
gcugaugagu cccaaauagg acgaaacgcc uucgggcguu 60 cuggauucca
cugcuaucca c 81 23 81 RNA Unknown modified schistosome ribozyme
partial sequence 23 cugagaugca gguacaucca gcugaugagu cccaaauagg
acgaaacgcc uucgggcgua 60 cuggauucca cugcuaucca c 81 24 80 RNA
Unknown modified schistosome ribozyme partial sequence 24
cugagaugca gguacaucca gcugaugagu cccaaauagg acgaaacgcu ucgggccucc
60 uggauuccac ugcuauccac 80 25 81 RNA Unknown modified schistosome
ribozyme partial sequence 25 cugagaugca gguacaucca gcugaugagu
cccaaauagg acgaaacgcc uucgggccug 60 cuggauucca cugcuaucca c 81 26
81 RNA Unknown modified schistosome ribozyme partial sequence 26
cugagaugca gguacaucca gcugaugagu cccaaauagg acgaaacgcc uucgggccuu
60 cuggauucca cugcuaucca c 81 27 81 RNA Unknown modified
schistosome ribozyme partial sequence 27 cugagaugca gguacaucca
gcugaugagu cccaaauagg acgaaacgcc uucgggccua 60 cuggauucca
cugcuaucca c 81 28 79 RNA Unknown modified schistosome ribozyme
partial sequence 28 cugagaugca gguacaucca gcugaugagu ccuucgggac
gaaacgccuu cgggcguccu 60 ggauuccacu gcuauccac 79 29 83 RNA Unknown
modified schistosome ribozyme partial sequence 29 cugagaugca
gguacaucca gcugaugagu cccaaauagg acgaaacgcg cuucggugcg 60
uccuggauuc cacugcuauc cac 83 30 126 RNA Unknown modified
schistosome ribozyme partial sequence 30 cugagaugca gguacaucca
gcugaugagu ccuaaaacau accagauuuc gaucuggaga 60 ggugaagaau
ucgaccaccu aggacgaaac gcgcuucggu gcguccugga uuccacugcu 120 auccac
126 31 82 RNA Unknown modified schistosome ribozyme partial
sequence 31 cugagaugca gguacaucca gcugaugagu cccaaauagg acgaaacgcc
cuucgggcgu 60 ccuggauucc acugcuaucc ac 82 32 82 RNA Unknown
modified schistosome ribozyme partial sequence 32 cugagaugca
gguacaucca gcugaugagu cccaaauagg acgaaacgcc uuucgggcgu 60
ccuggauucc acugcuaucc ac 82 33 82 RNA Unknown modified schistosome
ribozyme partial sequence 33 cugagaugca gguacaucca gcugaugagu
cccaaauagg acgaaacgcc auucgggcgu 60 ccuggauucc acugcuaucc ac 82 34
82 RNA Unknown modified schistosome ribozyme partial sequence 34
cugagaugca gguacaucca gcugaugagu cccaaauagg acgaaacgcc guucgggcgu
60 ccuggauucc acugcuaucc ac 82 35 83 RNA Unknown modified
schistosome ribozyme partial sequence 35 cugaggugca gguacauccc
acugacgagu cccaaauagg acgaaacgcg cuucggugcg 60 ucugggauuc
cacugcuauc cac 83 36 83 RNA Unknown modified schistosome ribozyme
partial sequence 36 cugaggugca gguacauccc acugacgagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucugggauac cacugcuauc cac 83 37 83 RNA
Unknown modified schistosome ribozyme partial sequence 37
cugaggugca gguacauccc acugacgagu cccaaauagg acgaaacgcg cuucggugcg
60 ucugggaucc cacugcuauc cac 83 38 83 RNA Unknown modified
schistosome ribozyme partial sequence 38 cugaggugca gguacauccc
acugacgagu cccaaauagg acgaaacgcg cuucggugcg 60 ucugggaugc
cacugcuauc cac 83 39 83 RNA Unknown modified schistosome ribozyme
partial sequence 39 cugaggugca gguacauccc acugacgagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucugggauua cacugcuauc cac 83 40 83 RNA
Unknown modified schistosome ribozyme partial sequence 40
cugaggugca gguacauccc acugacgagu cccaaauagg acgaaacgcg cuucggugcg
60 ucugggauug cacugcuauc cac 83 41 83 RNA Unknown modified
schistosome ribozyme partial sequence 41 cugaggugca gguacauccc
acugacgagu cccaaauagg acgaaacgcg cuucggugcg 60 ucugggauuu
cacugcuauc cac 83 42 83 RNA Unknown modified schistosome ribozyme
partial sequence 42 cugaggugca gguacauccc acugacgagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucugggauuc aacugcuauc cac 83 43 83 RNA
Unknown modified schistosome ribozyme partial sequence 43
cugaggugca gguacauccc acugacgagu cccaaauagg acgaaacgcg cuucggugcg
60 ucugggauuc gacugcuauc cac 83 44 83 RNA Unknown modified
schistosome ribozyme partial sequence 44 cugaggugca gguacauccc
acugacgagu cccaaauagg acgaaacgcg cuucggugcg 60 ucugggauuc
uacugcuauc cac 83 45 82 RNA Unknown modified schistosome ribozyme
partial sequence 45 cugaccagau gguacaucca gcugaugagu cccaaauagg
acgaaacgcg cuucggugcg 60 uccuggauuc cacaucuggc ac 82 46 82 RNA
Unknown modified schistosome ribozyme partial sequence 46
cugaccagau gguacaucca gcugaugagu cccaaauagg acgaaacgcg cuucggugcg
60 uccuggauac uacaucuggc ac 82 47 83 RNA Unknown modified
schistosome ribozyme partial sequence 47 cugagaugca gguacaucca
gcugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 uccuggauuc
cacugcuauc cac 83 48 65 RNA Unknown modified schistosome ribozyme
partial sequence 48 agguacaucc agcugaugag ucccaaauag gacgaaacgc
gcuucggugc guccuggauu 60 ccacu 65 49 49 RNA Unknown modified
schistosome ribozyme partial sequence 49 ccagcugaug agucccaaau
aggacgaaac gcgcuucggu gcguccugg 49 50 83 RNA Unknown modified
schistosome ribozyme partial sequence 50 cugagaugca gguacaucca
ucugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 ucauggauuc
cacugcuauc cac 83 51 83 RNA Unknown modified schistosome ribozyme
partial sequence 51 cugagaugca gguacauccc ucugaugagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucagggauuc cacugcuauc cac 83 52 83 RNA
Unknown modified schistosome ribozyme partial sequence 52
cugagaugca gguacauccu acugaugagu cccaaauagg acgaaacgcg cuucggugcg
60 ucuaggauuc cacugcuauc cac 83 53 83 RNA Unknown modified
schistosome ribozyme partial sequence 53 cugagaugca gguacauccc
acugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 ucugggauuc
cacugcuauc cac 83 54 83 RNA Unknown modified schistosome ribozyme
partial sequence 54 cugagaugca gguacauccg ucugaugagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucacggauuc cacugcuauc cac 83 55 82 RNA
Unknown modified schistosome ribozyme partial sequence 55
cugaccagau gguacaucca gcugaugagu cccaaauagg acgaaacgcg cuucggugcg
60 uccuggauuc cacaucuggc ac 82 56 82 RNA Unknown modified
schistosome ribozyme partial sequence 56 cugaccaggu gguacaucca
gcugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 uccuggauuc
cacaucuggc ac 82 57 82 RNA Unknown modified schistosome ribozyme
partial sequence 57 cugaccaggu gguacauccc ucugaugagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucagggauuc cacaucuggc ac 82 58 82 RNA
Unknown modified schistosome ribozyme partial sequence 58
cugaccaggu gguacauccc acugaugagu cccaaauagg acgaaacgcg cuucggugcg
60 ucugggauuc cacaucuggc ac 82 59 83 RNA Unknown modified
schistosome ribozyme partial sequence 59 cugaggugca gguacaucca
gcugaugagu cccaaauagg acgaaacgcg cuucggugcg 60 uccuggauuc
cacugcuauc cac 83 60 83 RNA Unknown modified schistosome ribozyme
partial sequence 60 cugaggugca gguacauccc acugaugagu cccaaauagg
acgaaacgcg cuucggugcg 60 ucugggauuc cacugcuauc cac 83 61 83 RNA
Unknown modified schistosome ribozyme partial sequence 61
cugaggugca gguacaucca gcuggugagu cccaaauagg acgaaacgcg cuucggugcg
60 uccuggauuc cacugcuauc cac 83 62 114 RNA Unknown modified
schistosome ribozyme partial sequence 62 gccuaaaaca uaccagaugg
uacauccagc ugaugagucc caaauaggac gaaacgcgcu 60 ucggugcguc
cuggauucca caucuggaga ggugaagaau ucgaccaccu aggc 114 63 78 RNA
Unknown modified schistosome ribozyme partial sequence 63
cugagaugcu uuacgcgucu gaugaguccc aaauaggacg aaacgcgcuu cggugcguca
60 cgcguuguug cuauccac 78 64 73 RNA Unknown modified schistosome
ribozyme partial sequence 64 cugagaugca gguacaucag cugangaguc
ccaaauagga cgaaacgcng gguccugauu 60 ccacugcauc cac 73 65 76 RNA
Unknown modified schistosome ribozyme partial sequence 65
cugagaugca gguacaucag cugangaguc ccaaauagga cgaaacgcuu cgggguccug
60 auuccacugc auccac 76 66 7684 DNA Unknown vector HDM-nLacZ
nucleotide sequence 66 agcttggccc attgcatacg ttgtatccat atcataatat
gtacatttat attggctcat 60 gtccaacatt accgccatgt tgacattgat
tattgactag ttattaatag taatcaatta 120 cggggtcatt agttcatagc
ccatatatgg agttccgcgt tacataactt acggtaaatg 180 gcccgcctgg
ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc 240
ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
300 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct
attgacgtca 360 atgacggtaa atggcccgcc tggcattatg cccagtacat
gaccttatgg gactttccta 420 cttggcagta catctacgta ttagtcatcg
ctattaccat ggtgatgcgg ttttggcagt 480 acatcaatgg gcgtggatag
cggtttgact cacggggatt tccaagtctc caccccattg 540 acgtcaatgg
gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca 600
actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
660 gagctcgttt agtgaaccgt cagatcgcct ggagacgcca tccacgctgt
tttgacctcc 720 atagaagaca ccgggaccga tccagcctcc cctcgaagct
gatcctgaga acttcagggt 780 gagtctatgg gacccttgat gttttctttc
cccttctttt ctatggttaa gttcatgtca 840 taggaagggg agaagtaaca
gggtacacat attgaccaaa tcagggtaat tttgcatttg 900 taattttaaa
aaatgctttc ttcttttaat atactttttt gtttatctta tttctaatac 960
tttccctaat ctctttcttt cagggcaata atgatacaat gtatcatgcc tctttgcacc
1020 attctaaaga ataacagtga taatttctgg gttaaggcaa tagcaatatt
tctgcatata 1080 aatatttctg catataaatt gtaactgatg taagaggttt
catattgcta atagcagcta 1140 caatccagct accattctgc ttttatttta
tggttgggat aaggctggat tattctgagt 1200 ccaagctagg cccttttgct
aatcatgttc atacctctta tcttcctccc acagctcctg 1260 ggcaacgtgc
tggtctgtgt gctggcccat cactttggca aagaattccg cgggcggccg 1320
ccatggcgcc aaaaaagaag agaaaggtaa agatccccgg gaattcactg gccgtcgttt
1380 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
gcagcacatc 1440 cccctttcgc cagctggcgt aatagcgaag aggcccgcac
cgatcgccct tcccaacagt 1500 tgcgcagcct gaatggcgaa tggcgctttg
cctggtttcc ggcaccagaa gcggtgccgg 1560 aaagctggct ggagtgcgat
cttcctgagg ccgatactgt cgtcgtcccc tcaaactggc 1620 agatgcacgg
ttacgatgcg cccatctaca ccaacgtgac ctatcccatt acggtcaatc 1680
cgccgtttgt tcccacggag aatccgacgg gttgttactc gctcacattt aatgttgatg
1740 aaagctggct acaggaaggc cagacgcgaa ttatttttga tggcgttaac
tcggcgtttc 1800 atctgtggtg caacgggcgc tgggtcggtt acggccagga
cagtcgtttg ccgtctgaat 1860 ttgacctgag cgcattttta cgcgccggag
aaaaccgcct cgcggtgatg gtgctgcgct 1920 ggagtgacgg cagttatctg
gaagatcagg atatgtggcg gatgagcggc attttccgtg 1980 acgtctcgtt
gctgcataaa ccgactacac aaatcagcga tttccatgtt gccactcgct 2040
ttaatgatga tttcagccgc gctgtactgg aggctgaagt tcagatgtgc ggcgagttgc
2100 gtgactacct acgggtaaca gtttctttat ggcagggtga aacgcaggtc
gccagcggca 2160 ccgcgccttt cggcggtgaa attatcgatg agcgtggtgg
ttatgccgat cgcgtcacac 2220 tacgtctgaa cgtcgaaaac ccgaaactgt
ggagcgccga aatcccgaat ctctatcgtg 2280 cggtggttga actgcacacc
gccgacggca cgctgattga agcagaagcc tgcgatgtcg 2340 gtttccgcga
ggtgcggatt gaaaatggtc tgctgctgct gaacggcaag ccgttgctga 2400
ttcgaggcgt taaccgtcac gagcatcatc ctctgcatgg tcaggtcatg gatgagcaga
2460 cgatggtgca ggatatcctg ctgatgaagc agaacaactt taacgccgtg
cgctgttcgc 2520 attatccgaa ccatccgctg tggtacacgc tgtgcgaccg
ctacggcctg tatgtggtgg 2580 atgaagccaa tattgaaacc cacggcatgg
tgccaatgaa tcgtctgacc gatgatccgc 2640 gctggctacc ggcgatgagc
gaacgcgtaa cgcgaatggt gcagcgcgat cgtaatcacc 2700 cgagtgtgat
catctggtcg ctggggaatg aatcaggcca cggcgctaat cacgacgcgc 2760
tgtatcgctg gatcaaatct gtcgatcctt cccgcccggt gcagtatgaa ggcggcggag
2820 ccgacaccac ggccaccgat attatttgcc cgatgtacgc gcgcgtggat
gaagaccagc 2880 ccttcccggc tgtgccgaaa tggtccatca aaaaatggct
ttcgctacct ggagagacgc 2940 gcccgctgat cctttgcgaa tacgcccacg
cgatgggtaa cagtcttggc ggtttcgcta 3000 aatactggca ggcgtttcgt
cagtatcccc gtttacaggg cggcttcgtc tgggactggg 3060 tggatcagtc
gctgattaaa tatgatgaaa acggcaaccc gtggtcggct tacggcggtg 3120
attttggcga tacgccgaac gatcgccagt tctgtatgaa cggtctggtc tttgccgacc
3180 gcacgccgca tccagcgctg acggaagcaa aacaccagca gcagtttttc
cagttccgtt 3240 tatccgggca aaccatcgaa gtgaccagcg aatacctgtt
ccgtcatagc gataacgagc 3300 tcctgcactg gatggtggcg ctggatggta
agccgctggc aagcggtgaa gtgcctctgg 3360 atgtcgctcc
acaaggtaaa cagttgattg aactgcctga actaccgcag ccggagagcg 3420
ccgggcaact ctggctcaca gtacgcgtag tgcaaccgaa cgcgaccgca tggtcagaag
3480 ccgggcacat cagcgcctgg cagcagtggc gtctggcgga aaacctcagt
gtgacgctcc 3540 ccgccgcgtc ccacgccatc ccgcatctga ccaccagcga
aatggatttt tgcatcgagc 3600 tgggtaataa gcgttggcaa tttaaccgcc
agtcaggctt tctttcacag atgtggattg 3660 gcgataaaaa acaactgctg
acgccgctgc gcgatcagtt cacccgtgca ccgctggata 3720 acgacattgg
cgtaagtgaa gcgacccgca ttgaccctaa cgcctgggtc gaacgctgga 3780
aggcggcggg ccattaccag gccgaagcag cgttgttgca gtgcacggca gatacacttg
3840 ctgatgcggt gctgattacg accgctcacg cgtggcagca tcaggggaaa
accttattta 3900 tcagccggaa aacctaccgg attgatggta gtggtcaaat
ggcgattacc gttgatgttg 3960 aagtggcgag cgatacaccg catccggcgc
ggattggcct gaactgccag ctggcgcagg 4020 tagcagagcg ggtaaactgg
ctcggattag ggccgcaaga aaactatccc gaccgcctta 4080 ctgccgcctg
ttttgaccgc tgggatctgc cattgtcaga catgtatacc ccgtacgtct 4140
tcccgagcga aaacggtctg cgctgcggga cgcgcgaatt gaattatggc ccacaccagt
4200 ggcgcggcga cttccagttc aacatcagcc gctacagtca acagcaactg
atggaaacca 4260 gccatcgcca tctgctgcac gcggaagaag gcacatggct
gaatatcgac ggtttccata 4320 tggggattgg tggcgacgac tcctggagcc
cgtcagtatc ggcggaattc cagctgagcg 4380 ccggtcgcta ccattaccag
ttggtctggt gtcaaaaata ataataaccg ggcagggggg 4440 atccaagctt
atcgataccg tcgacctcga gggcccagat ctaattcacc ccaccagtgc 4500
aggctgccta tcagaaagtg gtggctggtg tggctaatgc cctggcccac aagtatcact
4560 aagctcgctt tcttgctgtc caatttctat taaaggttcc tttgttccct
aagtccaact 4620 actaaactgg gggatattat gaagggcctt ccggagcatc
tggattctgc ctaataaaaa 4680 acatttattt tcattgcaat gatgtattta
aattatttct gaatatttta ctaaaaaggg 4740 aatgtgggag gtcagtgcat
ttaaaacata aagaaatgaa gagctagttc aaaccttggg 4800 aaaatacact
atatcttaaa ctccatgaaa gaaggtgagg ctgcaaacag ctaatgcaca 4860
ttggcaacag cccctgatgc ctatgcctta ttcatccctc agaaaaggat tcaagtagag
4920 gcttgatttg gaggttaaag ttttgctatg ctgtatttta cattacttat
tgttttagct 4980 gtcctcatga atgtcttttc actacccatt tgcttatcct
gcatctctca gccttgactc 5040 cactcagttc tcttgcttag agataccacc
tttcccctga agtgttcctt ccatgtttta 5100 cggcgagatg gtttctcctc
gcctggccac tcagccttag ttgtctctgt tgtcttatag 5160 aggtctactt
gaagaaggaa aaacaggggg catggtttga ctgtcctgtg agcccttctt 5220
ccctgcctcc cccactcaca gtgacccgga atccctcgac atggcagtct agatcattct
5280 tgaagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
gataataatg 5340 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc
gcggaacccc tatttgttta 5400 tttttctaaa tacattcaaa tatgtatccg
ctcatgagac aataaccctg ataaatgctt 5460 caataatatt gaaaaaggaa
gagtatgagt attcaacatt tccgtgtcgc ccttattccc 5520 ttttttgcgg
cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 5580
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
5640 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
ttttaaagtt 5700 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
aagagcaact cggtcgccgc 5760 atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa gcatcttacg 5820 gatggcatga cagtaagaga
attatgcagt gctgccataa ccatgagtga taacactgcg 5880 gccaacttac
ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 5940
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
6000 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
caaactatta 6060 actggcgaac tacttactct agcttcccgg caacaattaa
tagactggat ggaggcggat 6120 aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat tgctgataaa 6180 tctggagccg gtgagcgtgg
gtctcgcggt atcattgcag cactggggcc agatggtaag 6240 ccctcccgta
tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 6300
agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
6360 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
gatctaggtg 6420 aagatccttt ttgataatct catgaccaaa atcccttaac
gtgagttttc gttccactga 6480 gcgtcagacc ccgtagaaaa gatcaaagga
tcttcttgag atcctttttt tctgcgcgta 6540 atctgctgct tgcaaacaaa
aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 6600 gagctaccaa
ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 6660
gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
6720 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
gtcgtgtctt 6780 accgggttgg actcaagacg atagttaccg gataaggcgc
agcggtcggg ctgaacgggg 6840 ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag atacctacag 6900 cgtgagctat gagaaagcgc
cacgcttccc gaagggagaa aggcggacag gtatccggta 6960 agcggcaggg
tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 7020
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
7080 tcaggggggc ggagcctatg gaaaaacgcc agcaacggat gcgccgcgtg
cggctgctgg 7140 agatggcgga cgcgatggat atgttctgcc aagggttggt
ttgcgcattc acagttctcc 7200 gcaagaattg attggctcca attcttggag
tggtgaatcc gttagcgagg tgccgccggc 7260 ttccattcag gtcgaggtgg
cccggctcca tgcaccgcga cgcaacgcgg ggaggcagac 7320 aaggtatagg
gcggcgccta caatccatgc caacccgttc catgtgctcg ccgaggcggc 7380
ataaatcccc gtgacgatca gcggtccaat gatcgaagtt aggctggtaa gagccgcgag
7440 cgatccttga agctgtccct gatggtcgtc atctacctgc ctggacagca
tggcctgcaa 7500 cgcgggcatc ccgatgccgc cggaagcgag aagaatcata
atggggaagg ccatccagcc 7560 tcgcgtcggg gagctttttg caaaagccta
ggcctccaaa aaagcctcct cactacttct 7620 ggaatagctc agaggccgag
gcggcctcgg cctctgcata aataaaaaaa attagtcagc 7680 catg 7684 67 26
RNA Unknown ribozyme nucleotide sequence targeted by antisense
oligonucleotides 67 gugcguccug gauuccacug cuaucc 26 68 166 RNA
Neurospora 68 ugcgaagggc gucgucgccc cgagcgguag uaagcaggga
acucaccucc aauuugagua 60 cugaaauugu cguagcaguu gacuacuguu
augugauugg ugaggcuaag ugacgguauu 120 ggcguaaguc aguauugcag
cacagcacaa gcccgcuugc gagaau 166 69 86 RNA Cricket 69 caugaugugu
guucccucug ccccgcugau gaggucaggg aagaccgaaa gugucgacuc 60
uacggggcua uaacaugcaa uggugg 86 70 62 RNA Tobacco ringspot virus 70
auacccuguc accggaugug cuuuccgguc ugaugagucc gugaggacga aacaggacug
60 uc 62 71 75 RNA Shistosome 71 cugagaugca gguacaucca gcugacgagu
cccaaauagg acgaaacgcg cguccuggau 60 uccacugcua uccac 75
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