U.S. patent application number 10/209761 was filed with the patent office on 2002-12-19 for in situ binary synthesis of biologically effective molecules.
Invention is credited to Ecker, David J..
Application Number | 20020192700 10/209761 |
Document ID | / |
Family ID | 22740364 |
Filed Date | 2002-12-19 |
United States Patent
Application |
20020192700 |
Kind Code |
A1 |
Ecker, David J. |
December 19, 2002 |
In situ binary synthesis of biologically effective molecules
Abstract
A plurality of oligonucleotides are caused to bind specifically
to target nucleic acid which is predictive of a disease state or a
biological condition within cells containing the target nucleic
acid. The respective oligonucleotides, which may be functionilized
or may be present in the form of oligonucleotide analogs, carry
with them a plurality of synthons. Such synthons, which may be
identiphores, toxiphores, or other precursors to biologically
effective molecules, interact when specific binding of the
respective oligonucleotides occurs at sites adjacent to each other
on the target nucleic acid. The resulting interaction gives rise to
the synthesis, generation or release of highly active biological
molecules in situ in the cell in which the specific binding takes
place. This permits the use of extraordinarily toxic molecules for
use in killing cells containing the target nucleic acids. Imaging
and other uses are also provided by the present invention.
Inventors: |
Ecker, David J.; (Encinitas,
CA) |
Correspondence
Address: |
WOODCOCK WASHBURN LLP
ONE LIBERTY PLACE, 46TH FLOOR
1650 MARKET STREET
PHILADELPHIA
PA
19103
US
|
Family ID: |
22740364 |
Appl. No.: |
10/209761 |
Filed: |
August 1, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10209761 |
Aug 1, 2002 |
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09200107 |
Nov 25, 1998 |
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Current U.S.
Class: |
435/6.14 ;
435/6.16 |
Current CPC
Class: |
A61K 38/00 20130101;
C12Q 2565/101 20130101; C12Q 1/6841 20130101; Y02A 50/469 20180101;
C12N 2310/351 20130101; C12N 15/113 20130101; C12Q 1/6841
20130101 |
Class at
Publication: |
435/6 |
International
Class: |
C12Q 001/68 |
Claims
What is claimed is:
1. A method for identifying cells containing a preselected nucleic
acid sequence comprising: a. contacting said cells with a first
oligonucleotide specifically bindable with said preselected nucleic
acid, said first oligonucleotide comprising a first identiphore; b.
contacting said cells with a second oligonucleotide specifically
bindable with said preselected nucleic acid, said second
oligonucleotide comprising a second identiphore; c. said first and
second identiphores being detectable when in proximity with each
other.
2. The method of claim 1 wherein said oligonucleotides orient said
identiphores into spatial proximity when bound to said nucleic
acid.
3. The method of claim 1 wherein said oligonucleotides orient said
identiphores into a preselected geometric configuration when bound
to said nucleic acid.
4. The method of claim 1 wherein said identiphores form a charge
transfer complex when in proximity with each other.
5. The method of claim 1 wherein said identiphores become
covalently bonded upon being placed into proximity.
6. The method of claim 1 wherein said detection comprises X-ray,
magnetic resonance, proton magnetic resonance, electron spin
resonance, fluorescence, or electromagnetic spectroscopy.
7. The method of claim 1 wherein the presence of said preselected
nucleic acid is diagnostic for a cellular biological state.
8. The method of claim 7 wherein said cellular biological state is
a disease state.
9. The method of claim 7 wherein said cellular biological state is
a hyperproliferative state.
10. The method of claim 7 wherein said cellular biological state is
a cancerous state.
11. The method of claim 1 wherein said identiphores form a new
molecular species upon coming into proximity with each other.
12. A method of killing cells containing a preselected nucleic acid
sequence comprising: contacting said cells with a first
oligonucleotide specifically bindable with said preselected nucleic
acid, said first oligonucleotide comprising a first toxiphore; and
a second oligonucleotide specifically bindable with said
preselected nucleic acid, said second oligonucleotide comprising a
second toxiphore; said first and second toxiphores forming a toxin
upon coming into proximity with each other; said first and second
oligonucleotides being specifically bindable to adjacent sites on
the preselected nucleic acid.
13. The method of claim 12 wherein said oligonucleotides orient
said toxiphores into spatial proximity when bound to said nucleic
acid.
14. The method of claim 12 wherein said oligonucleotides orient
said toxiphores into a preselected geometric configuration when
bound to said nucleic acid.
15. The method of claim 12 wherein said toxiphores become
covalently bonded upon being placed into proximity.
16. The method of claim 12 wherein the presence of said preselected
nucleic acid is predictive of a cellular biological state.
17. The method of claim 16 wherein said cellular biological state
is a disease state.
18. The method of claim 16 wherein said cellular biological state
is a hyperproliferative state.
19. The method of claim 16 wherein said cellular biological state
is a cancerous state.
20. The method of claim 12 wherein said toxiphores form a new
molecular species upon coming into proximity with each other.
21. The method of claim 20 wherein said toxin is the functional
domain of a peptide.
22. The method of claim 21 wherein said peptide comprises the
functional domain of ricin, sarcin, diphtheria toxin, or botulism
toxin.
23. The method of claim 20 wherein said toxin comprises the
functional domain of a bacterial toxin.
24. The method of claim 20 wherein said toxin is an invertebrate
toxin.
25. The method of claim 24 wherein said toxin is an arachnid
toxin.
26. The method of claim 20 wherein said toxin is an amphibian
toxin.
27. The method of claim 12 wherein fewer than ten molecules of said
toxin is sufficient to kill each of said cells.
28. The method of claim 12 wherein said contacting is under nucleic
acid heteroduplex forming conditions.
29. The method of claim 12 wherein said contacting is of a
mammalian tissue.
30. A method for the in situ synthesis of a chemical species
comprising specifically binding first and second oligonucleotides
to a preselected nucleic acid at adjacent sites thereof; said first
and second oligonucleotides comprising first and second
synthons.
31. The method of claim 30 wherein said first and second synthons
form said chemical species upon coming into proximity with each
other.
32. The method of claim 31 wherein said proximity comprises spatial
proximity and geometric proximity.
33. The method of claim 30 wherein said chemical species is toxic
to cells containing the nucleic acid.
34. The method of claim 30 wherein said chemical species is
detectible in cells containing the nucleic acid.
35. The method of claim 30 wherein said preselected nucleic acid is
predictive of a biological state in cells containing said nucleic
acid.
36. The method of claim 35 wherein said biological state is a
disease state.
37. The method of claim 35 wherein said biological state is
hyperproliferation.
38. The method of claim 35 wherein said biological state is a
neoplastic state.
39. The method of claim 30 wherein the first and second synthons
are brought into spatial and geometric proximity upon the specific
binding of the oligonucleotides to the preselected nucleic
acid.
40. A pair of oligonucleotides: each of said oligonucleotides being
specifically bindable with a preselected nucleic acid at adjacent
binding sites; each of said oligonucleotides comprising a portion
of a molecular species, which species is formed when said adjacent,
specific binding of the oligonucleotides to the nucleic acid
occurs.
41. The pair of claim 40 wherein said molecular species is a
toxin.
42. The pair of claim 40 wherein said molecular species is a charge
transfer complex.
43. The pair of claim 40 wherein said molecular species is a
detectable molecule.
44. The pair of claim 40 wherein said molecular species is a cell
regulatory moiety.
45. A pharmaceutical comprising the pair of oligonucleotides of
claim 40 in a pharmaceutically acceptable carrier or diluent.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to the synthesis,
generation or release of molecules within cells and tissues, which
molecules have significant, biological effects. Such molecules are
synthesized which permit the identification of the cells or
tissues, which destroy the cells or tissues, which moderate,
modulate, or enhance cellular or tissue function, or which
otherwise have a diagnostic, therapeutic, nutritional or other
biological effect.
[0002] The in situ generation of such molecules is specific to
particular cells and tissues such that the molecules thus formed or
released are delivered specifically to the cells or tissues in
question. Such high specificity of delivery overcomes many
therapeutic impediments and can provide high "leverage" with
concomitant low side effects.
BACKGROUND OF THE INVENTION
[0003] It has long been known to employ nucleic acid structures
present in cells to serve as "targets" for diagnostic and
therapeutic regimes. A classical example of such employment is
evidenced by the field of antisense therapeutics. According to the
antisense paradigm, a nucleic acid sequence or structure is
identified as being associated with the production of a gene
product--usually a peptide--which has deleterious effects upon
cells or tissues. The target nucleic acid may be an oncogene, an
mRNA associated with the genesis or development of a disease state
or a hyperproliferative gene structures which proliferation is to
be reduced or eliminated.
[0004] Under the antisense paradigm, oligonucleotides are designed
which are specifically bindable to a nucleic acids sequence or
structure, usually mRNA, the down regulation of which is desired.
The oligonucleotide, usually in the form of a chemically modified
analog or construct, is administered to the cells containing the
targeted nucleic acid. The specific binding of the oligonucleotide
interferes with expression of the mRNA thus interfering with its
function and expression. Protein normally expressed by the mRNA is
either not expressed at all or is expressed in much lower quantity
with a concomitant, beneficial therapeutic effect. The paradigm may
also be used for diagnosis and research in ways which are well
known to persons of ordinary skill in the art.
[0005] The antisense paradigm is well understood per se to persons
of ordinary skill in the art and there are numerous review articles
which describe in detail varying approaches to this practice.
[0006] Nucleic acids which can be used to target in accordance with
some embodiments of this invention include mRNA molecules, which
preferably have secondary structures such as stem-loop structures,
or unique secondary structural sites, such as the molecular
interaction sites taught in U.S. application Ser. Nos. 09/076,440,
09/076,447, and 09/076,404, each of which is incorporated herein by
reference in its entirety.
[0007] Potential target nucleic acids include, but are not limited
to, mammalian, bacterial, fungal and viral mRNA. Oligonucleotides
have been shown to bind to numerous targets including, for example,
Candida (U.S. Pat. No. 5,691,461), protein kinase C (U.S. Pat. Nos.
5,703,054, 5,681,944, and 5,620,963), papillomavirus (U.S. Pat.
Nos. 5,681,944 and 5,756,282), herpesvirus (U.S. Pat. No.
5,658,891), cytomegalovirus (U.S. Pat. Nos. 5,607,923 and
5,595,978), human immunodeficiency virus (U.S. Pat. No. 5,523,389),
ras (U.S. Pat. Nos. 5,661,134, 5,582,986, and 5,576,208),
Epstein-Barr virus (U.S. Pat. No. 5,242,906), cell adhesion
molecules (U.S. Pat. Nos. 5,599,797 and 5,514,788), hepatitis virus
(U.S. Pat. No. 5,576,302), Raf kinase (U.S. Pat. Nos. 5,654,284,
5,563,255, 5,656,612, and 5,744,361), p120 (U.S. Pat. No.
5,814,629), cell growth (U.S. Pat. No. 5,656,743), and multi-drug
resistance associated protein (U.S. Pat. No. 5,510,239). Each of
the above-identified patents is incorporated herein by reference in
its entirety.
[0008] In addition to traditional antisense mechanisms,
oligonucleotides have also been shown to act through mechanisms,
for example, involving pseudo-half-knot formulations (U.S. Pat. No.
5,512,438), 5'-cap inhibition (U.S. Pat. No. 5,643,780), and triple
helix formation (U.S. Pat. No. 5,834,185), each of which is
incorporated herein by reference in its entirety. All such targets
and many more may be used herein.
[0009] It has also been proposed to employ a pair of
oligonucleotides to bind specifically with a mRNA in an antisense
fashion. (see Viassov et al., "Binary Systems of Oligonucleotide
Conjugates for Sequence Specific Energy--Transfer Sensitized
Photomodification of Nucleic Acids" NATO ASI Ser., Ser. C, 479 (DNA
and RNA Cleavers and Chemotherapy of Cancer and Viral Diseases)
195-207, (1996). Viassov discloses that, along with one
oligonucleotide there is a flourescent sensitizer, while the other
oligonucleotide carries a structure which, when irradiated with
ultraviolet radiation, is known to react with nucleic acids.
Viassov demonstrates a rapid modification of the DNA to which the
oligonucleotides were targeted when the two oligonucleotides are
allowed to bind to adjacent sites on the DNA and be subsequently
irradiated. The proximity of the photosensitizer to the group which
transfers energy to the DNA was viewed to be important to the
reaction with the DNA.
[0010] The technique of fluorescence resonance energy transfer
(FRET) has been used, inter alia to identify point mutations in
nucleic acids. A pair of oligonucleotides, each of which carries a
fluorophores are caused to bind to nucleic acids. Through study of
their fluorescence behavior, determination of the proximity of the
fluorophores and, hence, the presence or absence of intervening
base units, can give rise to the desired mutational information.
FRET can also monitor ribozime interactions and seems to be useful
generally in nucleic acid research.
[0011] Viassov et al in, "Sequence Specific Cleavage of Yeast
tRNA-Phe with Oligonucelotides Conjugated to Diimidazole
Construction", Antisense Nucleic Drug Development, 7(1): 39-42,
(1997), prepares oligonucleotides conjugated to a chemical
construction having two histidine residues. Yurchenko et al,
"Cleavage of Leishmania Mini--Exon Sequence by Oligonucleotides
Conjugated to a Diimidazole Construction", Nucleosides and
Nucleosides, 16 (7-9): 1721-1725, (1996), is directed to similar
subject matter. While it has been known to conjugate a plurality of
oligonucleotides to a single nucleic acid in a sequence specific
fashion, all such techniques are believe to have been directed to
the incapacitation or destruction of the nucleic acid target. While
this can certainly have some useful applications, it is limited in
its applicability.
[0012] It has been greatly desired to provide for the targeting of
nucleic acids in a way different from the traditional, antisense
approach. Thus, improvements over antisense methodology and over
the methodologies for nucleic acid cleavage of, e.g. Viassov and
Yurchenko are greatly desired. In particular, a methodology that
does not necessarily involve the incapacitation of the target
nucleic acid but which has a vastly more significant therapeutic
potential have long been desired. The present invention is directed
to this new paradigm of therapeutics and diagnostics.
SUMMARY OF THE INVENTION
[0013] The present invention provides a dramatic divergence in
oligonucleotide therapeutics, diagnostics and related paradigms. In
accordance with the invention, a nucleic acid target which has been
identified as being present in the cell or tissue of interest is
caused to be the object of specific binding by a plurality of
oligonucleotides. By virtue of the specific binding of the two or
more oligonucleotides, which binding is caused to take place at
adjacent sites on the target nucleic acid, a molecular species is
either synthesized, formed or released. The molecular species has
great biological effect within the cell or tissue which, of course,
carries the target nucleic acid. By virtue of the formation,
synthesis or release of such biologically effective molecules,
cells having the target nucleic acid may either be identified,
imaged, killed, benefitted or modified in some significant way.
Overall, this paradigm is denominated "in situ binary synthesis,"
since a molecule having biological activity is created upon the
specific binding of oligonucleotides to the target nucleic acid.
The method is in situ since the formation, synthesis or release of
the molecule takes place in situ--within the cell or tissue.
[0014] It is apparent that the generation of these biologically
active molecules, occurring as it does within the cell or tissue,
which has been targeted, is extraordinarily specific. Moreover, the
generation of only a few molecules in this way can have an
extraordinary and profound effect upon the cells or tissues. Thus,
the molecules to be synthesized, generated or released are not
spread out over an entire organism, but, rather, only within the
cells or tissues which have been targeted. Extraordinary "leverage"
can thus be obtained, such that a very small total amount of
biologically active material need be formed in order to obtain a
very large biological effect. Side effects are concomitantly
diminished. Indeed, molecules may be selected in this way for
generation, synthesis or release which could never be considered
for diagnostic or therapeutic use under any other paradigm. As will
be seen, even extraordinarily toxic molecules can find use in
therapeutics so long as they are confined though the use of the
present methodology to the particular cellular locus where they are
needed.
[0015] The methodologies of the present invention do not
necessarily involve modification of nucleic acid, or at least not
the nucleic acid which serves as a targeting nucleic acid. Thus,
unlike prior oligonucleotide diagnostic and therapeutic regimes.
the objective of a specific binding of an oligonucleotide is not
necessarily destruction or inhibition of the nucleic acid, usually
mRNA, which has been targeted. Rather, the nucleic acid simply
serves as the "locator beacon" and as a place for the docking of
oligonucleotides by virtue of their specific binding. As will be
seen, the ability of a plurality of oligonucleotides to
specifically bind to a target nucleic acid at adjacent sites upon
the nucleic acid permits the generation, synthesis or release of
the biologically effective molecules in a very highly controlled
fashion. Thus, wherever such specific binding at adjacent sites
does not occur, generation, synthesis or release of the biological
molecule does not take place. This high degree of control
contributes to the utility of the present invention in that
extraordinary toxic or potent molecules may be used without the
worry of accidental generation or release of such molecules at
locations other than the ones desired.
[0016] The present invention, to some extent, builds upon previous
methodologies of oligonucleotide interaction with cellular nucleic
acids, especially mRNA in the following way. It is well known to
identify nucleic acids structures or sequences which are related to
disease states. It is also known how to prepare oligonucleotides
which have antisense sequences to the nucleic acids whose function
or activity is to be moderated or ended. Moreover, a wide variety
of chemical modifications of nucleic acids, and, indeed, a host of
oligonucleotide analogs are known for this purpose. It is similarly
well understood how to contact cells or tissues with an
oligonucleotide or oligonucleotide analog in such a way as to cause
formation of a heteroduplex between the oligonucleotide and the
target nucleic acid. Such heteroduplex formation traditionally
leads to the destruction of the nucleic acid or to a failure in its
translation into protein. A number of intermediate results may
attend this interaction. Thus, the binding of the oligonucleotide
may activate RNAse H to cleave the nucleic acid. Alternatively,
specific binding may simply make it impossible for the nucleic acid
to be translated into protein. A number of other methodologies have
been proposed for oligonucleotide interaction with nucleic acids,
however these are generally attended by interference with the
structure or activity of the target nucleic acid.
[0017] It follows from the foregoing that it is essential that the
target nucleic acid be one which is important to disease states. It
is not enough, under these prime paradigms, for the nucleic acid
merely to be associated with a cell or tissue which is in a disease
state. It may be immediately seen that interaction with a
non-critical nucleic acid, such as an mRNA, even if very specific
and entirely efficacious, will not lead to any beneficial results
since the expression of the mRNA is not vital to survival of the
cell or tissue. Moreover, antisense interaction with nucleic acids,
while much more efficient than small molecule interaction with
peptides, still requires the individual, specific binding of
oligonucleotide to individual nucleic acids, such as mRNAs.
[0018] Contrarily, the present invention does not require the
identification of a target nucleic acid which is vital to the
function of a cell on which is causative of a disease state. Indeed
the nucleic acid thus identified need not actually be translated
into protein in large quantities within the cell or tissue in
question. Rather, it is only necessary that the target nucleic acid
be associated with or predictive of the existence of the disease
state or other state of interest in cells or tissues to be treated.
This is so because it is not the inactivation of the nucleic which
is sought, but rather the use of that nucleic acid to target
oligonucleotides to the cells or tissues which contain the target
nucleic acid.
[0019] In accordance with preferred embodiments of this invention,
a plurality of oligonucleotides are formulated to be specifically
bindable to the target nucleic acid. Moreover, such
oligonucleotides are formulated to have sequences which cause them
to specifically bind to the target nucleic acid at adjacent sites.
While it is possible that the oligonucleotides could bind at sites
which are removed by one or two bases, it is greatly preferred that
the oligonucleotides bind immediately adjacent to each other. It
will, thus, be seen that the present methodology causes two
oligonucleotides to enter a cell, tissue, or preparation having
target nucleic acid and to bind to the target nucleic acid "right
next to" each other. This close, predicable, and regulatable
proximity of oligonucleotides to each other that contributes to the
present invention.
[0020] Reliable and precise, specific binding of two designed
oligonucleotides to each other within a cell containing a
preselected, target nucleic acid permits the specific synthesis,
formulation or release of a molecular species when--but only
when--such specific binding occurs. Thus, a first one of the
oligonucleotides carries with it a first portion of the
biologically effective molecule to be synthesized, formulated or
released. The second oligonucleotide carries with it a second
portion of the biologically effective molecule. It is only when the
first and second portions of the molecules are brought together in
physical proximity and, preferably, with careful geometric
alignment, that the biological molecule is either synthesized,
generated, or released. It is also possible to have an essentially
complete biologically effective molecule from part of one of the
oligonucleotides with a moiety on the second oligonucleotide being
capable of either altering, transforming, cleaving, or releasing
the first biologically effective molecule or molecule precursor. In
any event, whether the biologically effective molecule is actually
synthesized through the joining together with covalent bonds of two
or more fragments or synthons thereof, whether a precursor molecule
is transformed, modified, or cleaved during this process, or
whether a pre-formed, biologically effective molecule is cleaved or
released from the oligonucleotides through the present methodology,
the biologically effected molecule is not rendered effective unless
and until the two oligonucleotides specifically bind at adjacent
sites on the target nucleic acid within the cell, tissue, or in
vitro preparation.
[0021] It will be seen that the first oligonucleotide carries with
it a first synthon while the second oligonucleotide carries with or
on it, or otherwise comprises, a second synthon. Persons of
ordinary skill in the art will understand that the term "synthon"
is extraordinarily broad and means a chemical moiety which is
capable of interacting with another chemical moiety to give rise to
chemical alteration.
[0022] While the invention is amenable to the use of a pair of
oligonucleotides for interaction with the target nucleic acid,
other paradigms are also possible within the spirit of the
invention. Thus, an oligonucleotide may specifically bind to the
target nucleic acid followed by the interaction of the heteroduplex
with another species, which is not an oligonucleotide. Such other
species may be, for example, a peptide, a carbohydrate or complex
sugar, or a "small" molecule, e.g. a non-oligomer. The confluence
of two or more molecules in some specific relationship with target
nucleic acid followed by the synthesis, generation or release of a
molecular species having significant biological effect, especially
toxic effect.
[0023] Certain aspects of the invention provide for the employment
of two or more non-oligonucleotides in the practice hereof. Thus,
specific binding of two or more non-oligonucleotide oligomers,
carbohydrates or sugars, or small molecules may serve the purposes
described herein. Thus, two or more small molecules could bind
specifically and adjacently to the target nucleic acid, giving rise
to a biologically active molecular species for, e.g. destruction of
the cell containing the target nucleic acid or detection
thereof.
[0024] The present invention provides for the detection of cells or
nucleic acids found in the cells, with the killing of cells having
target nucleic acids, and with certain other beneficial results.
While, in all such cases, it may be seen that the respective
oligonucleotides carry with them synthons, in accordance with the
broad definition of that term, it may also be convenient to refer
to the chemical moieties carried by the respective oligonucleotides
in terms which are more nearly suited to their function. Thus, when
it is desired to achieve the identification of cells or tissues
containing target nucleic acid, the respective synthons carried by
the oligonucleotides may be referred to as "identiphores." In this
context, the identiphores are moieties which can interact with each
other to give rise to either an interaction or a molecular species
which can be detected in some way.
[0025] Detection may take place using any form of spectroscopy,
x-ray examination, biochemical or biological analyses or otherwise.
What is required is that the confluence of the two identiphores
carried, respectively, by the oligonucleotides which specifically
bind to the target nucleic acid, be detectable. The presence of the
detectable moiety formed, synthesized or released by the
conjunction in space of the two identiphores is, according to this
embodiment, probative of the presence of the target nucleic acid in
the cell, tissue or in vitro assay.
[0026] As persons of skill in the art will appreciate, x-ray
detection may be employed. This is particularly beneficial when the
identiphore contains a heavy metal such as a heavy metal contained
within an heterocyclic structure such as a porphyrin, heme or
similar species. The identiphores may form a charged transfer
complex when in proximity with each other which complex may be
detectable through spectroscopy. Such identiphores may also become
cobandedly bonded with each other upon coming into proximity giving
rise to a chemical moiety or molecule which can, itself be
detected. Other forms of detection including magnetic resonance
spectroscopy, proton magnetic resonance spectroscopy, electron
resonance spectroscopy, fluorescence spectroscopy or
electromagnetic spectroscopy may also be profitable employed in
connection with this embodiment of the invention.
[0027] For example, the identiphores can comprise portions of a
charge transfer complex of TTF-TCNQ, (Tetrathiafulvalene and
Tetracyanoquinodimethane). Alternately,
Bis-(ethylenedithio)dithiapyrene (ETDTYP) and Dibenzo barreleno
tetracyano quino dimethane or Triphenylphosphine and Acrylonitrile
could form such complexes. Other charge transfer complexes are
known to persons of ordinary skill in the art. Thus, these
molecular moieties may be caused to comprise the oligonucleotides
which specifically bind to the target nucleic acid. Bringing the
two identiphores into proximity gives rise to the detectable
complex.
[0028] The use of identiphores for identifying the presence of
target nucleic acid in cells or tissues may be used to diagnose
certain cellular biological states such as disease states,
especially hyperproliferative states and cancers.
[0029] As with most aspects of the present invention, the
identiphores are best placed into spacial proximity with each other
and also to have a preselected geometric relationship one to the
other. This is especially true than intermolecular interactions are
required such as charge transfer complexes and the like.
[0030] This aspect of the invention provides methods for
identifying cells containing a preselected nucleic acid sequence.
These methods comprise contacting the cells with a first
oligonucleotide specifically bindable with the preselected nucleic
acid, the first oligonucleotide comprising a first identiphore. The
cells are also contacted with the second oligonucleotide
specifically bindable with the preselected nucleic acid, preferably
at a site immediately adjacent to the site where the first
oligonucleotide has bound to the nucleic acid. The second
oligonucleotide also comprises an identiphore. The first and second
identiphores are detectable when in proximity with each other.
Thus, they may either react with each other to form a new molecular
species, cause a species to be released from the oligonucleotides
for detection, or may give rise to an entirely new moiety which,
itself, may be detected in any of the ways known to persons of
ordinary skill in the art.
[0031] In accordance with other preferred embodiments of the
present invention, the synthons carried by the respective
oligonucleotides may give rise to toxic molecules. Such molecules
may either be synthesized, generated or released upon the specific
binding of the two oligonucleotides to adjacent sites on the target
nucleic acid. The generation of a toxic molecule as a result of
this specific binding gives rise to cell death and possibly even
destruction of tissue material in adjacent cells. It will be
appreciated that many highly toxic molecules exist in nature and
otherwise which can effectuate widespread destruction on a cellular
and tissue level. Prior therapeutic regimes have never been able to
exploit this phenomenon, since systemic or untargeted toxicity has
never been able to be avoided heretofore. The present invention,
however, permits this.
[0032] Thus, the present invention provides methods of killing
cells, which cells contain a preselected nucleic acid. The method
comprises contacting the cells with a first oligonucleotide
specifically bindable with the selected preselected nucleic acid.
The first oligonucleotide comprises a first toxiphore. It would be
understood that the term "toxiphore" is meant herein to mean a
chemical species which, when combined with one or more additional
toxiphores, gives rise to a toxic molecular species, interaction or
phenomenon. Such toxicity is to be expressed on a cellular or
tissue level; it is not necessary that any particular nucleic acid
be inactivated or "killed" thereby. The method further comprises
contacting these preselected nucleic acid sequence with a second
oligonucleotide specifically bindable with it. The second
oligonucleotide contains a second toxiphore. The first and second
toxiphores either synthesize, generate or release a toxin upon
coming into proximity with each other as is the case when the two
oligonucleotides, carrying their toxiphores, specifically bind to
adjacent sites on the target nucleic acid.
[0033] It is, of course, preferred that the toxiphores be placed
into spatial proximity when the oligonucleotides specifically bind
to the target nucleic acids. Additionally, it is greatly preferred
that the toxiphores be oriented geometrically so as to facilitate
the formation of the molecular species, its generation or
release.
[0034] In accordance with preferred embodiments, the toxiphores
become chemically bonded one to the other to give rise to the
toxic, molecular specie. Such specie is, of course, highly
biologically effective. It is also within the spirit of this
invention that one toxiphore modify the second toxiphore to give
rise to the biologically effective, toxic molecule. One toxiphore
may also cleave from the oligonucleotide, the heteroduplex, or
otherwise, a toxic species.
[0035] As will be appreciated, it is desired that the preselected
nucleic acid, to which the oligonucleotides and their related
toxiphores will specifically attach, be predictive of a cellular
biological state. Such state is usually a disease state, especially
a hyperproliferative state or one indicative of cancer. Other
cellular or tissues states may also be associated with the
preselected nucleic acid and, indeed, the present invention may be
used in the sense of a probe as well as in the sense of a
therapeutic or diagnostic. A host of research functions may also be
benefitted through use of the present invention.
[0036] The toxic molecule which, in accordance with preferred
embodiments of this invention, are synthesized, generated or
released may be anything which has effective. preferably powerful
cellular toxicity within related tissues or organs. For example,
the toxin may be a peptide such as ricin, sarcin, or diphtheria,
botulism, etc. toxins. In such cases, the toxin would comprise a
functional domain of the toxin. The toxin may be bacterial. The
toxin need not be a peptide. For example, the toxin could be an
amphibian toxin such as tetradotoxin or bufotoxin. The toxins may
also be from invertebrates, such as arachnids, or from reptiles or
amphibians. In short, any number of toxins may be employed in
conjunction with the present invention. It is preferred that the
toxin be very powerful such that only a few, perhaps as few as one
or two molecules of the toxin are necessary to effect cell death.
Numbers of toxin molecules fewer than ten are preferred in
accordance with certain embodiments to kill any given cell. As will
be appreciated, a preferred embodiment of the present aspect of the
invention is for the treatment of disease state in mammals. Thus, a
tissue of a mammal is contacted with the oligonucleotides having
toxiphores associated therewith under conditions such that
heteroduplexes can be formed. Alternately, in vitro embodiments,
heteroduplex formation is encouraged through appropriate selection
of reaction conditions. Upon the specific binding of the
oligonucleotides to the preselected nucleic acid, the toxiphores
interact with each other to give rise to a toxic molecule or other
toxic effect.
[0037] When the toxin is a peptide, it can be sufficient that a
carboxylic acid "end" of a toxiphore be brought into spatial
proximity with an amine "end" of another toxiphore, and the same be
carefully oriented in space and with geometric precision. In such
case, the activation energy required for the formation of the
peptide bond may be significantly lowered such that spontaneous
generation of the peptide bond occurs. The resulting, toxin is then
either active immediately or can be released or cleaved from the
complex for toxic activity. Organic molecules other than peptides
may also be prepared in this way, cleaved. modified, or
released.
[0038] The chemical species which effects the toxic activity may
also be other than an organic species; they may be organometallic
or even inorganic. Thus, one toxiphore might contain a complex
metal ion which, in uncomplexed form or in a different oxidation
state is highly toxic. The interaction with the second toxiphore
may give rise to the toxic form of the metal ion or to its complex.
A number of "heavy" metallic species and some which are not so
"heavy" are known to be highly toxic in one or another form. All of
these are contemplated by the present invention.
[0039] As is apparent, the present invention provides for the in
situ synthesis of chemical species. This is accomplished through
specifically binding first and second oligonucleotides to a
preselected nucleic acid at adjacent sites of the nucleic acid.
First and second sythons, related to or forming part of the
respective oligonucleotides, are then reacted with each other or
caused to interact to give rise to a new molecular species or an
altered form of an existing molecular species. Significant
biological effects may thus be generated upon the cell or tissues
comprising those cells.
[0040] As will be apparent, a wide variety of organic,
organometallic and other inorganic molecules can be formed through
the practice of the present invention. As discussed herein above,
peptides may be prepared by bringing together two synthons
comprising peptide residues, which residues, when carefully
oriented in space and oriented as to geometry may form the peptide
bond either spontaneously or through the intervention of a
catalyst. Indeed, a catalytic agent may also be included in or more
of the oligonucleotides. A host of other organic molecules may
similarly be formed as will be apparent to persons of ordinary
skill in the art. Organometallic materials may also be prepared
hereby and the oxidation state of organic, and organometallic
species may be changed through intermediation of oxidizing and
reducing agents forming part or all of one or both of the synthons.
The charged transfer complexes as discussed herein before may also
be placed into apposition and may be used in either a catalytic,
synthetic, biologically active or other functional applications. As
is apparent, the ability of the present invention to orient
synthons in precise proximity and in exact geometric relationship
one with the other permits the overcoming of activation energies
which would otherwise apply. Accordingly, even reactions which seem
extraordinarily slow under solution conditions, may run at sensible
rates when synthesized in accordance with the present
invention.
[0041] It is also within the spirit of the present invention to
alter, cleave, release or otherwise furnish chemical moieties
through the interaction with the plurality of synthons placed into
proximity in this way. Thus, active chemical species may be cleaved
from the parent oligonucleotides and from the heteroduplexes to
which they have been attracted through the action of the other
synthon or otherwise.
[0042] Accordingly, the present invention provides for the
preparation of such chemical species in amounts which are
detectable in cells containing the target nucleic acid. It is, of
course, preferred that such nucleic acid be predictive of a
biological condition or disease state such that the preparation and
delivery of the molecules thus formed or furnished is delivered in
situ to such cells. Diagnosis, therapy, or killing of the cell may
ensue.
[0043] The present invention also provides compositions of matter.
In accordance with one embodiment, the invention provides a pair of
oligonucleotides each of which is specifically bindable with a
preselected nucleic acid. The binding of the pair of
oligonucleotides is preferably at adjacent sites on the preselected
nucleic acid. Each of the oligonucleotides comprises a portion of a
molecular species, which species is formed when the adjacent,
specific binding of the oligonucleotides to the nucleic acid
occurs. Any of the foregoing methodologies may give rise to pairs
of oligonucleotides which are contemplated by the present
invention. Indeed, the present invention may also be applied to
additional oligonucleotides with additional synthons (toxiphores,
identiphores. etc.) whereupon binding of three or more
oligonucleotides to adjacent sites on the target nucleic acid is
accomplished with results analogous to those described herein
above. As will be appreciated, the oligonucleotides may give rise
to toxins, charge transfer complexes. detectable molecules for
detection and sensing, cell regulatory moieties, nutritional
species, and to a wide variety of other compounds having biological
activity. The present invention is also directed to compositions of
matter comprising a pair of oligonucleotides as herein above
together with a diluent or carrier, especially a pharmaceutically
acceptable diluent or carrier.
[0044] In the present description of the invention, the term
oligonucleotide has been used very broadly. Persons of ordinary
skill in art will appreciate that the term includes wild type
oligonucleotides, those which are unmodified in any way in terms of
their chemical construction or substituents. The term also includes
semisynthetic and modified oligonucleotides and molecules which are
analogous to oligonucleotides. In this context, oligonucleotide
includes peptide nucleic acids, "PNAs", molecules which are
analogous in function and spatial relationships to nucleic acids
and which may be used in oligonucleotide therapeutics such as
antisense and the like. These have been well-characterized in the
literature and are set forth in number of U.S. patents, many of
which are assigned to the assignee of the present application. Each
of these are incorporated herein by reference.
[0045] Thus, numerous other identophores have been shown to bind
mRNA including, for example, peptide nucleic acids (U.S. Pat. Nos.
5,641,625, 5,700,922, 5,719,262, 5,714,331, 5,766,855, 5,773,571,
5,786,461, and 5,736,336, each of which is incorporated herein by
reference in its entirety), ribozymes (U.S. Pat. Nos. 5,599,706,
5,801,158, 5,639,655, 5,635,385, 5,599,704, 5,610,052, 5,766,942,
and 5,747,335, each of which is incorporated herein by reference in
its entirety), and small molecules (U.S. Pat. Nos. 5,668,165,
5,641,486, and 5,683,873, each of which is incorporated herein by
reference in its entirety).
[0046] Chemically modified oligonucleotides include those which are
modified in the backbone, e.g. phosphorothioate, methylphosphonates
and a host of other backbone modifications as well as modifications
to the substituents on the sugar rings present in the oligomers.
Further modifications may be had on the base structure in a number
of ways. These are exemplified by a number of U.S. patents
including many owned by the assignee of the present application.
Each of these in incorporated in full by reference in order to
provide more explanation of the full scope of the oligonucleotides
which may be employed in connection with the present invention:
U.S. Pat. Nos. 5,618,704, 5,608,046, 5,792,844, 5,808,023,
5,834,607, 5,138,045, 5,378,825, 5,359,051, 5,212,295, 5,736,294,
5,614,617, 5,670,633, 5,681,941, 5,359,044, 5,210,264, 5,218,105,
5,610,289, 5,506,351, 5,391,667, 5,514,786, 5,489,677, 5,541,307,
5,386,023, 5,457,191, 5,578,718, 5,644,048, 5,623,065, 5,719,271,
5,637,684, 5,783,682, 5,539,082, 5,614,621, 5,459,255, 5,521,023,
5,539,083, 5,510,476, 5,629,152, 5,554,746, 5,571,902, 5,519,134,
5,543,507, 5,734,041, 5,506,212, 5,714,606, 5,717,083, 5,831,014,
5,677,437, 5,623,070, 5,688,941, 5,808,027, 5,750,692, 5,639,873,
5,731,438, 5,798,360, 5,646,265, 5,760,202, 5,587,361, 5,635,488,
5,587,469, 5,587,420, 5,770,713, 5,214,597, 5,817,489, 5,811,534
and 5,602,240.
[0047] While the present invention has been exemplified in terms of
the use of a plurality of oligonucleotides to bind specifically to
target nucleic acid and to effect the synthesis, generation or
release of biologically useful molecules thereby, certain other
materials may also be employed. Thus, it may be seen that a first
oligonucleotide carrying a synthon (identiphore, toxiphore, etc.)
may be caused to bind specifically to a site of a target nucleic
acid. Another biooligomer may then be caused to bind to the
heteroduplex thus formed. This latter oligomer need not necessarily
be an oligonucleotide. Thus, certain peptides are known to be able
to complex with heteroduplexes formed between a nucleic acids and
oligonucleotides. It is possible to include a synthon along with
such peptide and to cause the same to bind the heteroduplex thus
bringing the synthon of the peptide and the synthon carried by the
oligonucleotide into proximity with the results explained above.
Many other variations are possible in accordance with the full
spirit of the present invention.
[0048] It is also believed to be possible to use a single
oligonucleotide to accomplish the function presently exemplified
through a plurality of oligonucleotides. Thus, a single
oligonucleotide having two portions, each of the two portions
carrying a synthon (identiphore, toxiphore, etc.) may be employed.
If the two portions of the single oligonucleotide can bind at
adjacent locations on the target nucleic acid, thus bringing the
synthons into proximity, the spirit of the present invention may be
accomplished. It may also be that a single oligomer which first
binds with the target nucleic acid and then binds with the
heterodymer formed by the first part of the oligomer and the target
nucleic acid could be used in conjunction with certain embodiments
of the present invention.
[0049] While the present invention has been exemplified with
respect to certain of its preferred embodiments in considerable
detail, persons of ordinary skill will know that other embodiments
remain within the scope of the true spirit of the invention.
* * * * *