Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
131~1 19
24439/SGEN-2
llUCLEIC ACID CXELAl`E CONJUGATE~
J~S THER~PEUTIC AND DIAGNOSTIC A(;ENTS
The field concerns the u~e of modi~ied oligo-
nucleotides as therapeutic agents, inhibiting matura-
tion or expression of transcrLption products.in vivoand in vitro.
The biological reYolution ha~ introduced a
15 ~arlety of new technique~ resulting ln the abilLty to
determine Yarious cellular and subcellular proces3es.
. As the understanding has increased a3 to how the cell
j maintains its viability and proli~erates, new opportu-
. nities have opened ~or utilizing novel therapeutic
20. approaches. One technique which has been u~ed in a
variety of ways in the laboratory and i~ being expanded
¦ from the laboratory into real life s1tuations, i9 the
¦ use of anti-~ense sequences to modulate the fate o~ a
transcription product in a host cell. ~.
For the mo~t part, the ap~roaches in employing
anti-sense ~eq,uences have been t~o~old. In one
appr~oach, cells in culture are modified by inJection o~
a large excess of a DNA sequence which i5 comple~entary
to the sequence o~ a mRNh present in the cell. A ~ub-
~tantial reduction in the expression product is
obser~ed. In another approach, one can lntroduce a
tranqcription caYsette comprising a promoter ~unctional
in the host and a DNA ~equence which result~ in the
production of a mRNA which is complementary to an endo-
:. 35 genous mRNA. Again, one obserYe~ a reduction in the
: expres3ion product to which the transcription cons~ruc~
',j ia d~rected.
;, ~ .
13191 19
If, however, anti-sense ~equences are to
become a useful therapeutic agent, there are many pro-
blems and difficulties to be overcome. As a therapeu-
tic agent, a method must be found which allows for the
transfer of the anti-sense sequence across the cellular
membrane. The anti-sense sequence must be designed so
as to be relatively stable to degradation, particularly
by nucleases. In addition, there remain concerns about
the specificity of the sequence, particularly where one
does not wish to kill the host cell. Also o~ concern
is how to reach the necessary concentration in the cell
to provide the desired level of inhibition of tran-
scription product maturation or expression. Finally,
any technique which is devised for ~ulfilling the above
objectives, must take into consideration such problems
as toxicity, immunogenicity, solubility, and the like.
There is, therefore, substantial interest in being able
to develop anti-sense sequences which will be effective
as therapeutic agents in the modulation of the forma-
tion of mRNA and its expression.
Relevant Literature
Anti-sense nucleic acid sequences have been
reported to selectively block translation of a number
25 of mRNAs (Izant and Weintraub, Cell (1984) 36:1007-
1015; Izant and Weintraub, Science (1985) 229:345-352;
Melton, ~roc.~Natl. Acad Sci. USA (1985) 82:144-148; ~-
; Mizuno et al. 9 Proc. Natl. Acad. S_i. USA (1984)
81:1966-1970). Short oligonucleotides have been
3 reported to provide for high selectivity. (Wallace et
al., Nucl. Acids Res. (1979) 6:3543-3557; Wallace et
al., Nucl. Acids Res. (1981) 9:879-894; Smith, et al.,
_
Proc. Natl._Acad. Sci. USA (1986) 83:2787-2791;
Szostak, et al., Methods Enzymol. (1979) 68:419~429; Wu
et al., Prog. Nucl. Acid Res. and Mol. Biol. (1978)
; 21:102). The short oligonucleotides are able to
rapidly and peci~ically bind to speci~ic target
3 ~3191 19
sequences. (Itakura and Rlggs, Science (1980) 209:
1401; Szostak et al., Methods Enzymol. (1979) 68:419-
429; Noyes et al., J. Biol. Chem. (1979) 254:7472-7475;
Noyes et al., Proc. Natl Acad. Sci. USA (1979) 76:1770-
5 1774; Agarwal et al., J. Biol. Chem. (1981) 256:1023-
1028; Tullis et al., Biochem. Biophys. Res. Comm.
(1980) 93:941). See also the use of oligonucleotide
probeq to detect point mutations. (Orkin et al., J.
Clin. Invest. (1983) 71:775; Conner et al., Proc Natl.
10 Acad. Sci. USA (1983) 80:278; Piratsu et al., New Eng.
J. Med. (l983) 309:284-287; Wallace et al., Nucl.
Acids. Res. (1981) 9:879-894). The rapidity with which
-
synthetic DNAs hybridize is related to the complexity
of the~probe. (Wetmur and Davidson, J. Mol. Biol.
(1967) 31:349; Wallace et al., Nucl. Acids Res. (1979)
6:3543-3557; Tullis et al., Bioche_. Bio~y~
Comm. (1980) 93:941; Meinkoth and Wahl, Anal. Biochem.
(1984) 138:267-284~.
Both normal and phosphorous modified oligonu-
cleoides have been reported to selectively block the
expression of specific RNAs. Zamecnik and Stephenson,
Proc. Natl. Acad. Sci. USA (1978) 75:280-284; Tullis et
-
al., J. Cellular Biochem. Suppl. (1984) 8A:58
(Abstract); Kawasaki, Nucl. Acids Res. (1985) 13:4991;
Haeptule et al., Nucl. Acids R_s. (1986) 14:1427-1448;
~alder et al., Science August 1, 1986; Stephenson and
Zamecni~, Proc. Natl. Acad. Sci. USA (1978) 75:285-288
Cornelissen et al., Nucl. Acids Res._ ~1986) 14:5605-
5614; Minshall and Hunt, Nucl. Acids Res. (1986)
3 14:6433-6451).
Protection from nuclease degradation can be
achieved by employing phosphotriesters and methylphos-
phonates. (Barrett et al., Biochemistry (1974) 13:
4897-4906; Miller et al., Biochemiqtry (1977) 16:1988-
- 35 1997; Miller et al., Biochemistry (1981) 20:1873-1880;
; Jayaraman et al., Proc. Natl. Acad~ Sci. USA (1981)
77:1537-1541; Blake et al., Biochemistry (1985)
4 13191 19
24:6132-6138; 81ake et al., Biochemiqtr~ (1985) 24:
6139-6145; Smith et al., Proc. Natl. Acad._Sci. USA
(1986) 83:2787-2791; Agrls et al., Biochemistry (1986)
25:6268-6275; Miller st al., Nuci. Acids. Res. (1983)
5 11 :6225-6242) .
A number of group~ have tried to enhance the
binding affinity by modifying the nucleotide ~equence.
(Summerton, J. Theor. Biol. (1978) 78:77-99; Knorre et
al., Adv. Enz. Reg. (1984) pp . 277 -300; ~la~ov et al.,
0 Adv. Enz. Reg. (1986) pp. 301-320).
Oligonucleotide con~ugates are provided, where
a specific sequence of at least eight nucleotide3 i~
15 covalently linked to an ion chelating group and option-
ally to other groups to enhance tran~port across the
cell membrane. The resulting composition~ are found to
effectively block the function o~ a sequence comple-
mentary to the oligonucleotide. The compo~ition3 find
use a~ agentq in vivo and in vitro for modulating
intracellular tranqcription product maturation or
expression.
Method3 and compositions are provided ~or
modulating transcriptional maturation or expression by
employing oligonucleotide conjugate~. The oligonucleo-
; tide con~ugate~ have at least two components: an oli-
gonucleotide qequence Or at least eight nucleotide~;
3 and a chelating agent. In addition~ other groups may
be preqent which include a linker ~oining the chelating
agent to the oligonucleotide sequence, a hydrophobic
group ror enhancing the transport acro~ the membrane,
or other moietieq to enhance binding af~inity, reduce
~ 35 toxicity, enhance solubilityg or other characteristias
:. Or intere~tO
1 ~1 ql 1 9
The sub~ect CompositiOns will generally have a
hybridizing sequence, namely a polynucleotide unit from
about 8 to 30, more usually from about 8 to 20, prefer-
ably from about 12 to 18 members. The molecular weight
will normally be under about 10 kD, usually under about
6 kD, although high molecular weight moleCUles may be
used in special circumstances. The chelating agent may
be any one of a large number of chelating agents which
are able to chelate metal ions capable of acting as
scis~ile and/or free radical initiating agents, by
themselVes or in conjunction with other compounds which
may be present in the host cell or introduced in the
host cell. The chelating agent will be able to chelate
one of a variety of metals, such as iron, cobalt,
nickel, molybdenum, vanadium, or other metal Lon whiCh
may be encountered in the cytoplasm of the cell and may
serve to initiate the formation of free radicals,
resulting in the scission or other modification of the
transcription product, preventing its normal function
in the host cell.
For the most part, the compo~itions of the
subject invention will have the following formula:
{ ¦ N ~ N a ! N jb }
. Wherein:
K represents the chelating agent capable of
chelating a metal ion, which ion is capable of catalyz-
ing a chemical reaction in the phy9iological medium of
the cytoplasm of a cell, which re9ult9 in a chemical
transformation of mRNA inhibiting expression, particu-
larly degradative modification;
L is a bond or linking unit derived from a
; 35 polyvalent functional group having at least one atom,
which functional sroup may be of from about 1 to 20
atoms other than hydrogen, compri~ing carbon, nitrogen,
6 1;~191 19
oxygen, sulfur, and pho~phorous, where the linking
group may be aliphatic, aromatic, alicyclic, hetero-
cyclic, or combinations thereof; substituted or unsub-
stituted; generally having-from 0 to 10 heteroatoms,
usually from 0 to 6 heteroatoms, where the cyclic com-
pounds will usually have from 1 to 2 ring~, usually 1
ring, and the aliphatic groups may be branched,
straight chained, heterosubstituted or unsubstituted;
desirably the linking unit will have a chain o~ 2 to
10 20, usually 4 to 16 atoms normally free of linkages
capable of enzymatic degradation; L may be ~oined
through Y to the terminal phosphorous or may be joined
at any convenient site of the oligonucleotide chain,
being linked to P, C, N, 0 or S of the base (N),
saccharide (Z), or group linked to phosphorous (X);
X is usually a pair of electrons, alkyl of
from 1 to 3 carbon atoms, chalcogen (oxygen or sulfur),
or amino, particularly NH;
Z is a monosaccharide, particularly of 5 to 6
carbon atom~, more particularly of 5 carbon atom.~,
which may have from 0 to 1 hydroxyl groups replaced by
hydrogen, and will usually be substituted by phospho-
rous at the 2, 3, 5, or 6 positions, particularly at
the 3 and 5 positions, and ~ubstituted at ~he one posi-
tion, by the purine or pyrimidine, where the sugars mayinclude such sugars as ribose, arabino~e, xylylose
glucose, galactose, deoxy, particularly 2-deoxy,
derivatives thereof, etc.;
L' is a linker group which is derived from a
polyvalent functional group having at least one atom,
and not more than about 60 atoms other than hydrogen,
usually not more than about 30 atoms other than hydro-
gen, having up to about 30 carbon atoms, usually not
more than about 20 carbon atoms, and up to about 10
35 heteroatoms, more usually up to about 6 heteroatoms,
.particularly chalcogen, nitrogen, phosphorous, etc.;
7 1 3 1 9 1 1 q
M is a moiety, particularly imparting amphi-
philic properties to the compound, a hydrophobic or
amphiphilic moiety which will have a ratio of carbon to
heteroatom of at least about 2:1, usually at lea~t
about 3:1, frequently up to greater than about 20:1,
may include hydrocarbons o~' at lea~t 6 carbon atoms and
not more than about 30 carbon atoms, polyoxy compounds
(alkyleneoxy), where the oxygen atoms are joined by
from about 2 to 10 carbon atoms, usually 2 to 6 carbon
atoms, preferably 2 to 3 carbon atoms, and there will
be at least about 6 units and usually not more than
about 200 alkyleneoxy units, more usually not more than
about 100 units, generally not more than about 60
units;
Y is a bond to L or a terminal group; Y' is a
bond to L', linking L' to a terminal phosphorous, or a
terminal group; when a terminal group, Y and Y' are
oxy, thio, amino or substituted functionalities
thereof, e.g., oxyether, alkylamino, etc. or alkyl of
up to about 20, usually of up to about 6 carbon atoms,
Y and Y' usually not being more than about 20 carbon
atoms, more usually being not more than about 10 carbon
atoms;
N is any natural or unnatural base (purine or
pyrimidine), capable of binding to and hybridizing with
a natural purine or pyrimidine, where N may be adenine,
cytidine, thymidine, guanidine, uracil, orotidine,
inosine, etc.;
a is at least 4, usually at least 5, and not
more than about 50, u~ually not more than about 35;
b and c are each 0 or 1.
The functional groups which find use with the
linking group~, L and L', include functionalities ~uch
as oxy, non-oxo-carbonyl (carboxy carbonyl), oxo-
carbonyl (aldehyde or ketone), the nitrogen or 3ulfuranalogs thereof, e.g. imino, thiono, thio, amidino,
etc., disulfide, amino, diazo, hydrazino, oximino,
phosphate, phosphono, etc.
1 3191 19
The linking group to the hybridizing sequence
may be linked through an oxygen or sulfur present on a
pyrimidine, purine, ~ugar or phosphorous group, to a
carbon atom of a pyrimidine or purine, or to a phos-
phorous atom. The links may be ethers to oxygen andsulfur, esters, both organic and inorganic, to oxygen
and sulfur, amides, both organic and inorganic, to
amines and phosphorous, and alkylamino to amino groups.
Esters include carboxylates, e.g. carboxy esters,
carbamates, carbonates, etc., and phosphates, phos-
phonates, etc. Of particular interest is linking at
the terminal unit of the hybridizing sequence through a
sugar hydroxyl, particularly at the S'-position.
The phosphorous moiety may include phosphates,
phosphoramidates, phosphordiamidate, phosphorothioate,
phosphorothionate, phosphorothiolate, phosphoramidothi-
olate, phosphonates, phosphorimidate, and the like.
~here the phosphorous is bound to other than oxygen of
the sugar, the sugar will be modified by having an
amino or thio functionality at the site of binding, 90
that both amino and thio sugars may be employed to
provide for novel linkages between the phosphorous and
the ~ugar.
K is a chelating agent, having at least 3
heteroatoms, which are oxygen, nitrogen, or sulfur,
usually combinations thereof, more usually having about
6 heteroatoms or more, which~may serve to chelate a
metal ion capable of acting to inactivate, particularly
to enhance cleavage, of a nucleic acid. The function-
alities may include carbonyl, oxy, thiono, amino,amido, mercapto, thioether, imino, where carbonyl oxy-
gens will normally be separated by at least 2 carbon
atoms, usually up to 6 carbon atoms, more usually up to
4 carbon atoms; except for amido, heteroatoms will nor-
mally be separated by at least 2 carbon atoms. Conven-
iently, there will be at lea~t 2 non-oxo-carbonyl
groups frequently at least 3 non-oxo-carbonyl group~
9 1~191 19
and not more than about 6, usually not more than about
5 non-oxo-carbonyl groups. Of particular interest are
alkylene diamines and polyalkylene diamine~ having ~rom
3 to 8, usually 4 to 6 carboxyl groups, usually as
carboxymethylene groups, e.g.,
R2N(CH2)mN(T)((CH)nN(J))X(CH2)pNR2, wherein R is a
carboxyalkylene group of from 2 to 3 carbon atom~ or H,
at least one R on each N being carboxyalkylene, m, n
and p are the same or different and are 2 to 4, usually
2 to 3, and x is O to 2.
Illustrative chelating groups include ethy-
lenediaminetetraacetic acid, dipropyleneaminepenta-
acetic acid, diethylenetriaminepentaacetic acid, 2,3-
bis-(2'-acetamidoethyl)succinic acid, porphyrins,
phthalocyanins tetraacetic acid, and crown ethers.
A wide variety of linking groups may be
employed, depending upon the nature of the terminal
nucleotide, the functionality selected for, whether the
linking group is present during the synthesis of the
oligonucleotide, the functionality present on the
hydrophobic moiety and the like. A number of linking
groups are commercially available and have found exten-
~ive use for linking polyfunctional compound~. The
linking groups include: -OCH2CH2NHCO(CH2)nCONH-;-
OCH2CH2NH-X-(CH2)nNH-;-O-P(O)(OH)NHCO(CH2)nCOHN-;-
OGH2CH2NHCO~S-;-NH(CH2)nNH;-O(CH2)n ; O(CH2C 2 m ;
NH(CH2)nSYN; ~Co(cH2)nco; -SCH2CH2CO-; -CO~NYS-;
-(NCH2CH2)mCH2N-; charged and uncharged homo- and
copolymers of amino acids, such as polyglycine, polyly-
sine, polymethionine, etc. usually o~ about 500 to
2,000 dalton~; wherein O is phenyl; X is 2,5-
quinondiyl, Y is S-(3-succindoyl) to form succinimidyl,
n is usually in the range o~ 2 to 20, more u~ually 2 to
12, and m is 1 to 10, usually 1 to 6.
The amphiphilic character imparting or ~olu-
bility modifying group (M) may be a wide variety of
groups, being aliphatic, aromatic, alicyclic, heterocy-
lo ~3191 19
clic, or combinations thereof, substituted or unsubsti-
tuted, usually of at least 6, more usually at least 12
and not more than about 1000, usually not more than
about 500, more usually not more than about 200 carbon
atoms, having not more than about 1 heteroatom per 2
carbon atoms, being charged or uncharged, including
alkyl of at least 6 carbon atoms and up to about 30
carbon atoms, usually not more than about 24 carbon
atoms, fatty acids of at least about 6 carbon atom~,
usually at least about 12 carbon atoms and up to about
24 carbon atoms, glycerides, where the fatty acids will
generally range from about 12 to 24 carbon atoms, there
being from 1 to 2 fatty acids, usually the 2 or 3
positions or both, aromatic compounds having from 1 to
4 rings, either mono- or polycyclic, fused or un~used,
polyalkyleneglycols where the alkylenes are of from 2
to 8, usually of from 2 to 4 carbon atoms, more usually
2 to 3 carbon atoms, there usually being at least about
6 units more usually at least about 10 units, and
usually fewer than about 500 units, more usually fewer
than about 200 units, preferably fewer than about 100
units, where the alkylene glycols may be homopolymers
or copolymers; alkylbenzoyl, where the alkyl group will
be at least about 6 carbon atoms, usually at least
about 10 carbon atoms, and not more than about 20
carbon atoms; alkyl phosphates or phosphonates, where
the alkyl group will be at least 6 carbon atoms, usu-
ally at least about 12 carbon atoms and not more than
about 24 carbon atoms, usually not more than about 20
carbon atoms, or the like.
The "M" group may be charged or uncharged,
preferably being uncharged. Illustrative groups
include polyethylene glycol having from about 40 to S0
units, copolymers of ethylene and propylene glycol,
laurate esters of polyethylene glycols, triphenyl-
methyl, naphthylphenylmethyl, palmitate, distearyl-
glyceride didodecylphosphatidyl, cholesteryl, arachi-
donyl, octadecanyloxy, tetradecylthio, etc.
13191 19
1 1
Functionalities which may be present include
oxy, thio, carbonyl, (oxo or non-oxo), cyano, halo,
nitro, aliphatic unsakuration, etc.
In designing the nucleic acid sequence, it
will be desirable to have a high affinity between the
subject composition and the target single stranded
nucleic acid sequence. Sequences will preferably be
selected having greater than 40~ GC content, more
preferably greater than 50% and may have 60% or more GC
content. For optional selectivity, the melting temper-
ature of the hybrid to be formed should be 5 to 10C
above the ambient temperature at which the hybrid
forms, usually the ambient temperature being 37C in a
mammalian host. For mammalian hosts, the melting
temperature will generally be chosen to be about ~2-
50C. The target sequence should be selected to be
relatively free of ~econdary and tertiary ~tructure.
In many mRNA's, an open region will be present in the
vicinity of the start codon (AUG).
In preparing the subject compositions, various
strategies may be employed, depending upon whether "M"
is present, the nature of "M", the nature of the oligo-
nucleotide and the nature of the linking group. Thus,
so long as care is taken that the addition of the two
different groups, '7M" and the chelating group, do not
interfere with one another, the groups may be added
sequentially.
One technique for providing the chelating
agent may be found in Dryer and Dervan, supra. In thi~
technique, a modified nucleoside is employed during the
synthesis of the oligonucleoti~e. Thymidine may be
modified at the methyl group by providing for a carboxy
alkyl group. The carboxy group may then be further
functionalized with an alkylene diamine, and the amino
group employed for amide formation with a carboxy con-
taining chelating agent. The modified thymidine may
then be employed as a nucleotide reagent in the auto-
mated synthe~i~ of the oligonucleotide.
12 l~ql 19
Alternatively, the final nucleotide adduct in
the synthesis of the oligonucleotide may be functional-
ized in a variety of ways which may serve to act as a
linking unit to the chelating agent. For example,
5 after removal of the trityl protective group an amino-
ethanolphosphoramidite is addecl, as described by the
supplier (Applied 8iosystems, Foster City, CA) to pro-
vide for an available amino group. After deblocking
and removing the oligonucleotide chain from the sup-
10 port, the amino group is then available for linking tothe chelating agent. Alternatively, the oligonucleo-
tide i9 phosphorylated employing a polynucleotide
kinase, followed by formation of a phosphoramidate
using an activating agent, such as 1-methylimidazole or
a water soluble carbodiimide, in the presence of an
alkylene diamine, providing for an amino functionality
(Chu and Orgel, DNA (1985) 4:327-331). A further
alternative is to deblock the oligonucleotide while
retaining the oligonucleotide on the support, followed
20 by treatment with carbonyldiimidazole. After removal
of excess of the carbonyldiimidazole, a diamine may be
added to provide an aminoalkylcarbamate (Wachter et
al., Nucl. Acids ~es. (1986) 14:7985-7994~.
Where "M" is to be added, a mercaptan group
25 may be provided a~ part of the functionalizing agent or
separate from the f`unctionalizing agent. The mercaptan
group may be pa~rt of the linker to the support or may
be part of the functionalizing agent of the oligo-
nucleotide, where both the chelating agent and "M" may
3o be bound to the same linking group. Besides mercaptan
groups, maleimido groups may be employed, where "M" or
the chelating agent may have a mercaptan group to form
a thioether.
Various active functionalities can be employed
35 to produce a covalent linkage, such a~ isocyanate~,
isothiocyanates, diazo groups 3 imino chlorides, imino
e~ters, anhydride~, acylhalide~, sulfinylhalides,
13 131~1 19
sulfonyl chlorides, etc. Conditions for carrying out
the various reactions and joining non-nucleotide
moieties to nucleotide moieties may be found in Chu and
Orgel DNA (1985) 4:327-331; Smith et al. Nucl. Acids
5 Res. (1985) 13:2399-2412.
The linking arms, "Mt', and the chelating
moiety may be added at various times, depending upon
the particular reaction scheme. For the most part, the
chelating agent may be part of a nucleoside and be
included in the synthesis of the oligonucleotide or may
be added after oligonucleotide formation. "Ml' will
normally be added after oligonucleotide formation.
For the most part, reaction conditions will be
mild and will employ polar solvents or combinations of
polar and nonpolar solvents. Solvents will vary and
include water, acetonitrile, dimethylformamide, diethyl
ether, methylene chloride, dimethylsulfoxide, etc.
Reaction conditions will be for the most part in the
range of about -100-60C. Usually, after completion of
the reaction between components of the conjugate, the
resulting product will be subjected to purification.
The manner of purification may vary, depending
upon whether the oligonucleotide is bound to a support.
For example, where the oligonucleotide is bound to a
support, after addition of the linking arm to the
oligonucleotide ! unreacted chains may be degraded, so
as to prevent their contaminating the re~ulting
product. Where the oligonucleotide is no longer bound
to the support, whether only reacted with the linking
arm or as the conjugate to the chelating agent or as
the final product, each of the intermediates or final
product may be purified by conventional techniques,
such as electrophoresiq, solvent extraction, HPLC,
chromatography, or the like. The purified product is
then ready f'or use.
13191 19
The subject products will be selected to have
an oligonucleotLde sequence complementary to a sequence
of interest. The sequence of interest may be present
in a prokaryotic or eukaryotic cell, a virus, a normal
or neoplastic cell. The sequences may be bacterial
sequences, plasmid sequences, viral sequences, chromo-
somal sequences, mitochondrial sequences, plastid
sequences, etc. The sequences may involve open reading
frames for coding proteins, ribosomal RNA, snRNA,
hnRNA, introns, untranslated 5'- and 3'-sequences
flanking open reading frames, etc. The subject
sequences may therefore be involved in inhibiting the
availability of an RNA transcript, inhibiting expres-
sion of a particular protein, enhancing the expression
of a particular protein by inhibiting the expression of
a repressor, reducing prol~feration of viruses or neo-
plastic cells, etc.
The subject conjugates may be used in culture
or in vi~o for modifying the phenotype of cells, limit-
ing the proliferation of pathogens such as viruses,bacteria, protista, mycoplasma, or the like, or induc-
ing morbidity in neoplastic cells or specific classes
of normal cells. Thus, one can use the subject composi-
tions in therapy, by administering to a host subject in
a diseased state, one or more of the subject composi-
tions to inhibit the transcription and/or expression of
; the n?tive genes of a cell. The subject compo~itions
may be used for protection of a mammalian host from a
variety of pathogens, e.g., enterotoxigenic bacteria,
Pneumococcus, Neisseria, etc.; protists, such as
Giardia, Entamaeba, etc.; neoplastic cells, such as
lymphoma, leukemia, carcinoma, sarcoma etc.; specific
B-cells, specific T-cells, such as helper cells,
supressor cells, CTL, NK, etc.
; 35 The ~ubject compositions will be selected so
as to be capable of inactivating sequencas of interest,
particularly mRNA, or in some circumstances the subject
13191 19
composition can be used with other nucleic acid
moieties, e.g., tRNA, snRNA, DNA, e.g., plasmids,
viru~es, etc. Thu~, the subject compositions may bind
to mRNA and provide for cleavage of the mRNA, so as to
prevent the expression of a product. By employing
sequences which are relatively inert to degradation,
the lifetime of the chelate conjugate may be substan-
tially extended in the host cell, so as to have a rela-
tively high kill ratio per sequence.
The subject se4uences may be complementary to
such sequences as sequences expressing gro~th factors,
lymphokines, immunoglobulins, T-cell receptor sites,
MHC antigens, DNA or RNA polymerases, antibiotic resis-
tance, multiple drug resistance (mdr), gene~ involved
with metabolic processes, in the formation of amino
acids, nucleic acids, or the like, DHFR, etc. as well
as introns or flanking sequences associated with the
open reading frames.
The subject composition~ may be administered
to a host in a wide variety of ways, depending upon
whether the compositions are used in vitro or in vivo.
In vitro, the compositions may be introduced into the
nutrient medium, so a~ to modulate expression of a par-
ticular gene by transferring across the membrane into
the cell interior such as the cytoplasm and nucleus.
The ~ubject compo~itions may find particular use in
; protecting mammalian cells in culture from mycopla~ma,
for modifying phenotype for research purposes, ~or
evaluating the effect of variation of expression on
3~ various metabolic processes, e.g., production of parti-
cular products, variation in product distribution, or
the like. While no particular additives are necessary
for tran~port of the subject compositions intracellu-
larly, the subject compositions may be modified by
being encapsulated in liposomes or other vesicle, may
be used in conjunction with permeabilizing agents,
e.g., non-ionic detergents, Sendai virus, etc~
'``4~
16 ~31~
For in vlvo administratlon, depending upon lts
-
particular purpose, the sub~ect compos~tlons may be
admlnlstered in a varlety of ways, such as in~ectlon,
infu~ion, tablet, etc., ~o that the composltions may be
taken orally, parenterally, intravascularly, intraperi-
toneally, subcutaneou~ly, intrale310nally, or the
llke. The compositions may be ~ormulated in a variety
of ways, being di~persed in variou~ phy~iologically
acceptable media, such as delonized water, water, phos-
phate buffered saline, ethanol, aqueous ethanol, formu-
lated ln the lumen of vesicles, microencap~ulated, etc.
Becau~e of the wide variety of applications
and manners of admlni~tration, no particular composi-
tion can be suggested. Rather, as to each indication,
the subject compositions may be tested in conventional
ways and the appropriate concentration~ determined
empirically. Other additive3 may be included, such as
~tabilizers, buffer~, additional drug~, detergents,
etc. The~e additives are conventional, and would
generally be present in le~s than about 5 wt~, u~ually
les~ than 1 wtS, being pre~ent in an effective do~age,
as appropriate. For filler3 or excipient~, these may
be as high a~ 99.9S of the composition, depending upon
the amount of active materlal necessary.
The following example3 are presented by way of
illustration not by way of limitation.
EXAMPLE 1
Synthe~ls of Diethylenetriamine pentacetic acid
(DTPA) Con~u~ated Oligomer~
Chemical Synthesls of DNA. The chemical ~yn-
thesi~ of DNA was carried out using 311ght modifica-
tion~ of the conventional phosphoramidite methods on an
Applied Biosystems tModel 381) DNA synthesizer. The
method used i~ a modlfication of the technique
described by Caruthers and coworkers (Beaucage and
Carutherq, 1984, European Patent Application
61746 issued March 20, 1985.
~'`'' '~
., ,
I 3 1 9 1 1 9
17
In this technique, nucleoside phosphoramidites
dissolved in anydrous acetonitrile are mixed with
tetrazole and sequentially coupled to the 5' hydroxy
terminal nucleotide of the growing DNA chain bound to
controlled pore glass (CPG) support~ via a succinate
spacer (Matteucci and Caruthers. Tetrahedron Letters
(1980) 21:719-722). Nucleoside addition is followed by
capping of unreacted 5' hydroxyls with acetic
anhydride, iodine oxidation, and 5' detritylation in
trichloroacetic acid-methylene chloride. The resin
bound oligomer i~ then dried by extensive washing in
anhydrous acetonitrile and the process repeated.
Normal cycle times using this procedure are 12 minutes
with condensation efficiencies of >98~ (as judged by
trityl release).
Chemical Synthesis of Amine Linker Arm
Containing DNA Oli~onucleotides Bound to CPG Glass
Beads. The completed fully blocked DNA chains can
subsequently be modified to contain a 5' amino linker
arm while ~till attached to the CPG support. Several
method~ have been used to accomplish this.
Aminolink Procedure for 5' Amine Linker Arm
Attachment. At the end of the synthesis, trityl is
removed from the product oligonucleotide chains and an
aminoethanolphosphoramidite is added to the 5' hydroxyl
u~ing the Aminolink procedure developed and marketed by
- Applied Biosy~tems (Foster City, CA). The resin bound
oligonucleotide i~ then deblocked and released from the
column using a method appropriate to the type of phos-
phate linkage prasent. For normal phosphodie~ter~,
hydrolysis in concentrated ammonia is appropriate. For
DNA triester~ and methylpho~phonateq, ethylene diamine
(EDA) phenol deblocking followed by ammonia or EDA:
ethanol release is appropriate. Result~ indicate that
all of the cyanoethyl-phosphorus and aryl-amide base
blocking groups are removed under these condition~.
1 3 1 9 1 1 9
18
Addltion of_Amine Containlng Llnker Arms U~ing
Phosphoramldate Linkage. One alternative is the use of
the technique of Chu and Orgel, Proc. Natl. Acad. Scl.
USA (1986) 82:963-967 to add linker arms to the 5' end
of any ollgonucleotide. In thls instance, the oligo-
nucleotide is first phosphorylated u~ing polynucleotide
kina~e. After purification by polyacrylamide gel
electrophoresis, the product DNA containing a free 5'-
hydroxyl i~ phosphorylated with the forward reaction of
T4 polynucleotide kinase according to Chu and Orgel,
~upra (1986). Pho~phorylated oligomer~ are ~eparated
from unreacted ATP using a C-18 reverse phase column
(Waters SEP-PAK) according to the direction of the
manufacturer. The phosphorylated oligomer i3 then
treated with 0.1M 1-methyl imidazole, 0.1M 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide, 0.1M alkyl
diamine (e.g., hexane diamine), pH 7, under conven-
tional conditions in an aqueous medium to form the
desired phosphoramidate containing a free amine with
the following structure
oligomer-P-NH-(CH2)n-NH2
~H
Addition of An Amine Linker Arm Usin3 Carbonyl
Diimidazole (CDI). A third alternati~e to the addition
of an amine linker arm to the 5' end of any oli~o-
nucleotide is to use carbonyl-bis (imidazole). In this
technique, the CPC bound, ba3e blocked oligomer (trityl
of~) is fir~t treated with CDI (50 mg/ml) in dry aceto-
ni~rile for 30 to 45 minute~ to form the imidazole
carboxylic acid ester. The exce~s CDI i~ then wa~hed
off with acetonitrile and water and the diamine of
choice added to the column generally in a mixture of
acetonitrile or dioxane and water~ The dlamine form~ a
* Trademark
J
~ .
" ~ ., ~
1319~ 1~
19
stable carbamata linka~e after a brief incubation. The
oligomer can then be deblocked and relea~ed ~rom the
column under conditions approprLate to the type of
pho~phate llnkage present.
Attachment o~ the Clea~age Unlt DTPA. Once
the amine terminated oligomer i9 deblocked and charac-
terlzed, the cleavage unit was added using the ~ollow-
ing method. Ten units of the o:Ligomer were di~ol~ed
in 100 ~1 dimethylformamide containing 0.1 M DTPA (bi~-
anhydride). The mixture was incubated ~or 1-2 hours at
room temperature, and the exces3 DTPA removed by gel
filtration and concentration on Centricon C1U mem-
branes. The final product was dried in ~acuo and dis-
solved to a final concentration of 1 mM in water (ba~ed
on its optical den~ity at 260nM) and stored frozen.
Thi~ solution was ~table ~or at least 1 month. The
compound was homogeneous as judged by polyacrylamide
gel electrophoresi3.
EXAMPLE 2
Synthe~is o~ DTPA Derivative-~ of Normal DNAs U~ing
Imidazole Activated Carboxylic Acid Esters and
Lon~ Chain Aminoalkanes
In thi~ example, a 20 nucleotide DNA comple-
mentAry to the initiation region of mouse bata globin
mRNA was synthe~ized according to the method giYen in
EXAMPL~ 1. After ~ynthe~is, the product material wa~
retained on the synthe~is support wlth trityl removed
from the 5' end of the molecule. The solid material
wa~ then thoroughly wa~hed with anhydrou~ acetonitrile
and blown dry under a stream of dry argon. U~ing a
plastic syringe, 1 cc of 0.3M carbonyldiimidazole di~-
solved in anhydrou~ acetonitrile wa~ pu3hed slowly
through the synthe~is column containing the support
bound ollgomer o~er a period of 45 minutes. The 5'
carbonyllmidazole acti~ated oligomer on the column wa~
then washed free o~ exce3s reagent with 15 ~1 of aceto-
*Trademark
13191 19
nitrile and then treated with 0.2 M decanediamlne lnacetonltrlle:water (10:1) for 30 minutes.
The material on the column was washed free o~
unreacted decane-diamine with acetonitrlle and water,
and then eluted from the column ln concentrated ammon-
ium hydroxlde. After removal from the column, the
ammonium hydroxide ~olution containing the oligomer
conjugate was placed in a sealed Yial and incubated 5
hour~ at 55C.
The product iY then lyophilized several tlmes
from 50% aqueous ethanol and purified via reversed
phase HPLC C-8 silica columns eluting 5 to 50S aceto-
nitrilet25 mM ammonium acetate, pH 6.8 ln a llnear
gradient. If required, the material was further puri-
fied by ion-exchange HPLC on Nucleogen DEAE 60-7 elut-
ing 20~ acetonitrile/25 mM ammonium acetate, pH 6.5.
The recovered product i9 then characterized by gel
electrophoresis in 15S polyacrylamide gel~ carried out
a~ descrlbed by Maxam and Gilbert (Meth Enzymol.
~1980) 68:499-560). Oligonucleotides in finished gels
are visualized using Stains-all.
As a further check on the material, the
pre~ence of a primary amine can be determined by two
methods. Flrst, reactlon with fluore camine produced a
fluorescent product characteri~tic of the presence o~ a
primary amine while no rluorescence i9 observed with
~imilarly treated control oligomers o~ the ~ame type~,
but lacking the amine linker. Second, the decane con-
~ugate was dissolved in 100 ~l 0.1 M ~odium bicarbonate
to which was added 1 mg of fluorescein isothiocyanate.
After 1 hour of incubation, the unreacted FITC was
removed by gel filtration chromatography on Sephadex
G-25 spun columns. The product was then analyzed by
polyacryla~ide gel electrophore~l~ as described above,
and the fluorescent band product vlsualized under UV
illumination. A single fluorescent band is observed
which corresponds to the ol~gomer ~isualized by subse-
quent staining with Stains-all.
*Trademarks
,is,~,j
. . ~
- 1 3 1 9 1 1 9
21
The product of this reaction is an aminoalkyl
carbamate coupled to the 5' end of the oligonucleotide.
The alkylcarbamate i~ stable to moderate exposure to
concentrated ba~e. The free amino group distal to the
carbamate linkage is available for subsequent deriva-
tion which can be accomplished according to the method
given in EXAMPLE 1. The structure of the final con-
jugate synthesized by this method is illustrated as:
0 0
oligomer - 0 - C - NH - (CH2)10 - NH - C - DTPA
EXAMPLE 3
Synthesis of DTPA Derivatives of DNAs Using
Imidazole Activated Carboxylic Acid_Esters
and a Poly-D-lysine Linker
In this example, a 25 nucleotide DNA comple-
mentary to the initiation region of rabbit beta globin
mRNA was synthesized according to the method given in
EXAMPLE 1. After synthesis, the CPG support containing
the oligomer was treated with 80% acetic acid for 30
minutes to remove trityl from the 5' end of the mole-
cule. The solid material was then thoroughly washed
with anhydrous acetonitrile and blown dry under a
~tream of dry argon and treated with 0.3M CDI as in
EXAMPLE 4. The 5' CDI activated oligomer on the column
waq then washed free of exces~ reagent with 15 ml of
acetonitrile and then treated with 0.2 M poly-D-lysine
(MW+1,000) dissolved in 50% acetonitrile containing
3 0.1 M sodium phosphate, pH 8 for 16 hours at room
temperature.
The material on the column was washed free of
~alts and unreacted polyly~ine with water and aceto-
nitrile and then eluted from the column in concentrated
ammonium hydroxide. A~ter removal ~rom the column, the
ammonium hydroxide solution containing the oligomer
22 1 Sl qll 9
con~ugate wa~ incubated for 5 hours at 55C in a sealed
glas~ vial. The product was then lyophilized 3everal
times from 50~ aqueous ethanol and purifled Yia gel
filtration chromatography on TSK G4000SW in 100 mM Tri~
buf~er, pH 7.5. The presence of a primary amlne was
determined by reaction with fluorescamine. No fluores-
cence was observed with control oligomers lacking the
polyamine linker.
The polyamine conjungate cannot ea~ily be
characteri2ed by gel electrophoresis since it is elec-
trostatically and molecularly polydi~perse. In order
to render the polyamine conjugate negatively charged,
the complex was reacted with FITC to label the molecule
and to neutralize the positive charges on the amines.
This wag accomplished by dissolving a portion of the
material in 100 ~1 0.1 M sodium bicarbonate to which
was added 1 mg of fluoresceinisothiocyanate. After 1
hour of incubation, the unreacted FITC was removed by
gel filtration chromatography on SephadeX*G-25 spun
20 columns (Maniatis et al., Molecular Cloning - A
Laboratory Manual, Cold Spring Harbor Lab., Cold Spring
Harbor, New York (1982)). The product was then
analyzed by polyacrylamide gel electrophoresis carried
out as described by Maxam and Gilbert (Meth. Enzymol.
(1980) 60:499-560) and the fluorescent band product
~isualized under UV illumination. A ~ingle broad
~luore~cent band i9 ob3erved which corre3ponds~to the
DNA visualized by Stains-all.
The struoture of this con~ugate may be
illu9trated as having the general formula:
O O O
n n n
oligomer - O - C - NH - (CH - C - NH)n - C-CH-COOH
(1H2)4 (1H2)4
~H 1H2
O ~ ~ - DTPA
*Trademark
... ,~, ~,.,j
13191 19
23
By varying the reaction excess of the DTPA or
the molecular size of polylysine used, it is possible
to construct conjugate~ with varying degrees of substi-
5 tution, si~e and charge. The ability to vary theseproperties of the complex make it po~sible to design
the use of the compound in various applications.
EXAMPLE 4
Synthesis of DTPA Derivatives of
DNA Methylphosphonate~
The chemical synthesis of DNA methylphophon-
ates may be carried out using a modification of the
phosphochloridite method of Letsinger (Letsinger et
al., J. Am. Chem. Soc. (1975) 97 :3278; Letsinger and
Lunsford, J. Am. Chem. Soc. (1976) 98 :3655-3661; Tanaka
and Letsinger, Nucl. Acids Res. (1982) 1~:3249).
However, the preferred method and the one used in this
example uses methyl phosphonamidites (Applied
Biosy3tems, Foster City, CA). The method for
performing the synthesis uses exactly the ~ame 3teps
and reaction times a3 in conventional DNA synthesis,
with the exception that THF rather than acetonitrile is
used to dissolve the phosphonamidites due to their
25 reduced solubility in the latter. Normal cycle times
using thi~ procedure are 15 minute3 with condensation
efficiencies of >94~ (as judged by trityl release).
The ultimate base may be added as the cyanoethyl pho3-
photriester which yields, upon cleavage in base, a 5 '
: 30 terminal pho~phodiester. Thi3 step make~ it po3sible
to radiolabel the oligonucleotide, purify and sequence
the product using gel electrophoresis at intermediate
stages of preparation (Narang et al, Can. J. 8iochem.
(1975) 53:342-394; Miller et al., Nucl. Acids Res.
.... ~ 35 (1983) 11 :6225-6242).
An amine terminated linker arm i3 then added
a~ follows. Trityl i~ removed as before and the resin
24 131~1 19
treated with 0.2 M Aminolin~ (Applied Bio~y tems,
Foster City. CA) di~solved in dry acetonitrlle contain~
ing 0.2M dimethylaminopyrodine for 5 minute~. The
linker arm oligonucleotide is then oxidized in iodine
and wa~hed in acetonitrile as above. Capping with
acetic anhydride i~ not performed ~ince any deblocked
primary amine would be modified to the base stable
acetamide and thu~ unavailable for further reaction.
At the end of the synthe~is, the a~ine termi-
nated linker arm methylpho~phonate oligomer i~ ba~edeblocked a~ follow~. The resin containing the DNA i~
removed from the column and placed in a water jacketed
column and incubated in 1-2 ml phenol:ethylene diamine
(4:1) for 10 hours at 40C. At the end Or the incuba-
1~ tion in phenol:ethylene diamine, the re~in i~ washedfree of the phenol reagent and released ba e protecting
groups using methanol, water, methanol and methylene
chloride in ~ucce~slon. After drying in a stream of
nitrogen, the intact, basa-deblocked chains are cleaved
from the ~upport u~ing EDA:ethanol (1:1) or brier
treatment with room temperature ammonium hydroxide.
PuriYication of the amine terminated DNA
methylphosphonate i~ then performed aY ~ollows. The
material is ~irst lyophilized several times ~rom 50S
aqueous ethanol and purified via re~ersed pha~e HPLC C-
8 311ica column3 eluting 5 to 50% acetonitrile/25 mM
ammonium acetate, pH 6.8 in a linear gradiant. Amine
containing ~raction~ a~ determined by fluore~camine
reactivity are pooled and the product recovered by
3o drying in vacuo and ~urther puriSied by ion-exchange
HPLC on Nucleogen DEAE 60-7 eluting 20% aceonitrile/25
mM ammonium acetate, pH 6.5.
The purlfied product i~ then con~erted to the
DTPA derivative as in EXAMPLE 1. Purification o~ the
complex is then e~rected a~ preriously described.
Alternativelyt unbound ollgonucleotide 19 remoYed by
gel filtration on Sephadex*G-I00 or HPCFC*on TSK
G4000S~ alut i ng 10 mM Tris, pH 7.5.
,~'! * Trade mark
,~
~ .
2s 13191 19
The structure o~ the final product of this
procedure is illustrated as:
O O
,. ..
5 oligomer - P - O - (CH2)2 - NH - C - DTPA
MP
OH
EXAMPLE 5
Synthesis of DTPA DerivatiYes of DNA
Ethyltriesters Using the Phosphoramidite Approach
The synthesis of the title compound triesters
is performed according to the method of Zon and cowork-
ers (Gallo et al., Nucl. Acids Res. (1986) 14:7405;
Summers et al., Nucl. Acids Res. (1986) 14:7421-7436).
The method of synthesis is similar to that which i~
used for in situ production with ethyl triesters as
described by Letsinger (Letsinger et al., J. Am. Chem.
Soc. (1975) 97:3278; Letsinger and Lunsford, J. ~m.
-
20 Chem. Soc. (1976) 98:3655-3661; Tanaka and Letsinger,
Nucl. Acids Res. (1982) 10:3249). In brief, fully
blocked dimethoxytrityl nucleosides are derived by
repeated lyophilization from benzene, dis~olved in
anhydrous acetonitrile/2,6-lutidine (8:2) and added
25 dropwi3e to a stirred solution of chloro diisopropyl-
amino ethoxyphosphine in the same solvent at -70C.
The product is recovered by aqueou~ extraction, in
vacuo drying and silica gel chromatography.
- The chemical synthesi~ of DNA can be carried
30 out using slight modifications of the conventional
phosphoramidite methods. In this technique, nucleoside
phosphoramidites di3solved in anhydrous acetonitrile
are mixed with tetrazole and sequentially coupled to
the 5' hydroxy terminal nucleoside bound to CPG.
35 Nucleoside additinn i~ followed by capping of unreacted
5' hydroxyl~ with acetic anhydride, iodine oxidation,
and 5~ detritylation in trichloroacetic acid-methylene
26 13191 19
chloride. The resin bound oligomer is then dried by
extensive washing in anhydrou~ acetonitrile and the
process repeated. Normal cycle time~ u~ing thi~ proce-
dure are 17 minute~ with condensation efficiencie~ o~
>96~ (as ~udged by trityl release). The terminal re~i-
due is conventionally added a~ a diester in order to
facilitate radiolabeling and purification. The 5'
terminal trityl group is left if HPLC purification is
desired, but generally the 5' terminal trityl i~
removed and the aminolink procedure described in
EXAMPLE 1 is used.
At the end of the synthesi~, the fully blocked
product i~ ba~e-deblocked as follow~. The re~in
containing the fully protected DNA is removed from the
column and placed in a water ~acketed chromatography
column. The re~in is then incubated in 1-2 ml
phenol-ethylene diamine (4:1) for 10 hours at 40C. At
the end of the incubation in phenol:ethylene diamlne,
the resin is washed free of the phenol reagent and
relea~ed base protecting groups using methanol, water,
met~anol and methylene chloride. After drying in a
stream of nitrogen, the intact, base^deblocked chain~
are cleaved from the support u3ing EDA:ethanol (1:1) or
brie~ treatment with room temperature ammonium
hydroxide.
Purification of the aminolink DNA ethyl-
triester product 19 ~hen performed a~ follow~. The
material i3 ~ir~t lyophilized several times from 50S
aqueous ethanol and purified via reversed pha~e HPLC C-
30 8 silica columns eluting 5 to 50S acetonitrile/25 mM
ammonium acetate, pH 6.8 in a linear gradient. Amine
containing fraction~ a~ determined by fluorescamine
reactivity are pooled and the product recovered by
drying in vacuo and further purified by ion-exchange
35 HPLC on Nucleogen DEAE 60-7 eluting 20% acetonitrlle/25
mM ammonium acetate, pH 6.5.
*Trademark
~.
'`' ,~ f~);
1 3 1 q 1 1 q
27
The product olLgonucleotide i9 then suitable
for coupling to DTPA and purification by the techniques
previously described.
The structure of the final product of this
procedure is illustrated as:
O O
,. ..
oligomer - P - O - (CH2)2 - NH - C - DTPA
ETE
OH
EXAMPLE 6
Synthesis of DTPA Derivatives of DNA Alkyl
and Aryltriesters Using the
Phosphate Triester Approach
A preferred method for the production of the
oligonucleotide triesters of variable alkane chain
length is via conventional phosphate triester chemistry
to synthesize the desired sequences as the p-
chlorophenyl phosphate triesters. Upon completion oftha synthesis, the fully protected oligonucleotide
chlorophenyltriesters bound to the synthesis support
are subjected to ester exchange in the presence of
tetrabutylammonium fluoride and the desired alcohol.
This basic method for the construction of DNA oligo-
nucleotides is classical DNA synthesis chemistry and
presents no problems. The essential chemistry i~ well
described (Gait, Oligonucleotide Synthesis: A Practical
Approach IRL Press, Washington, D.C. (1984)) and can be
used with little modification. An alternative phos-
phite based chemistry which is much more rapid and
gives equivalent yields is set forth below.
The chemical synthesis of DNA p- or o-
chlorophenyl phosphotriestars was carried out using a
modification of the phosphochloridite method of
Letsinger (Letsinger et al., J. Am. Chem. Soc. (1975)
; 97:3278; Letsinger and Lunsford, J. Am. Chem. Soc.
28 13191 19
(1976) 98:3655-3661; Tanaka and Letsinger, Nucl. Acids
Res. (1982) _:3249). A programmable, automated DNA
synthesizer used for pho~phomonochloridite based
syntheses (Alvarado-Urbina et al., Science (1981)
21 4:270-273.
Fully blocked and carefully dried nucleosides
dissolved in anhydrous acetonitrile, 2,6-lutidine and
activated in situ with chlorophenoxydichlorophosphine
are sequentially added to the 5' hydroxy terminal
10 nucleotide of the growing DNA chain bound to controlled
pore glass supports via a succinate spacer (Matteucci
and Caruthers, Tetrahedron Lett. (1980) 21 :719-722).
Derivatized glass supports, fully block nucleosides and
other synthesis reagents are commercially available
15 through Applied Biosystems (San Francisco, CA) or
American Bionuclear (Emeryville, CA). Nucleoside
addition is followed by capping of unreacted 5'
hydroxyls with acetic anhydride, iodine oxidation, and
5' detritylation in trichloroacetic acid-methylene
20 chloride.
The resin bound oligomer chlorophenyltriester
is then dried by extensive washing in anhydrous aceto-
nitrile and the process repeated. Normal cycle times
using this procedure are 13 minutes with condensation
25 efficiencies of >92% (as judged by trityl release).
The ultimate base may be added as a methyl phospho-
triester which yields, upon cleavage in base, a 5'
terminal phosphodiester. This step makes it po~sible
to radiolabel the oligonucleotide and to purify and
30 3equence the product using gel electrophoresis (Narang
et al., Can. J. Biochem. (1975) 53:392-394; Miller et
al., Nucl. Acids Res. (1983) 1 :6225-6242).
The fully blocked material bound to the
synthesis qupport is then subjected to ester exchange
35 in the presence of tetrabutylammonium fluoride (TBA~)
and the desired alcohol. This method yields rapid and
quantitative alcohol exchange. The reaction is com-
13191 19
29
plete within 20 minutes for most aryl and alkyl alco-
hols which are capable of forming stable products. With
any alcohol, the presence of trace amounts of water can
affect the overall yield. Thus, care must be taken to
5 used anhydrous alcohols at this step.
In this example, anhydrous n-propanol is used
to dissolve TBAF to a final concentration of 0.2 M.
The solution is then percolated slowly over the resin
containing the oligomer chlorophenyl triester and
10 allowed to react for about 1 hour at room temperature.
The resin is then washed with methanol and acetonitrile
and dried under a stream of dry argon. Amine linker
arm addition, deblocking and purification are then
effected as in EXAMPLE 2. DTPA conjugation is then
15 performed as in EXAMPLE 1. The final yield of conju-
gate is about 10% of the starting equivalents of
nucleoside resin used.
The structure of the final product is illu-
strated as:
O O
,~ ,.
oligomer - C - NH - (CH2)10 ~ NH - C - DTPA
PTE
OH
EXAMPLE 7
Effect of DTPA Conjugate~ on the Synthesis
of Hemoglobin in Mouse MEL Cells
The effectiveness of oligomer DTPA con~ugate
30 mediated HART (Paterson et al., Proc. Natl. Acad. Sci.
USA (1977) 74:4370; Haqtie and Held, Proc. Natl. Acad.
Sci. USA (1978) 75:1217-1221) was determined in
cultured cells incubated in the presence or absence of
the oligomer. The cells used were Friend murine
35 erythroleukemia (M~L) cells which can be induced to
synthesize hemoglobin by a variety of agentq including
DMSO and butyric acid (cf. Gusella and Houseman, Cell
1~191 lq
(1976) 8:263-269). Friend leukemia cells were grown in
culture using well known techniques in a C02 incubator.
Hemoglobin synthesis was induced using 1.5% DMSO.
Induced cells expressing hemoglobin were
5 visualized by benzidine treatment which stains globin
producing cells blue (Leder et al., Science (1975)
190:893). Cells were exposed to selected oligonucleo-
tides and DTPA conjugate at concentrations ranging from
0.1 ~IM to 50 IIM during a 11- to 5-day induction period.
10 Controls included mock-treated cells and cells treated
with unmodified oligomers of the same sequence.
Treated cells were scored for globin production based
on staining intensity and the results compared to
controls. Cell death or damage due to treatment was
5 scored by Trypan blue exclusion.
The following Table (Table I) indicates the
speciIic sequences synthesized.
TABLE I
DNA SEQUENCES SYNTHESIZED AND CO~JUGATED
FOR USE IN ÆLL CULTURE EXPERIMENTS
Probes Synthe~ized
Antisense to Mouse
Beta-globin mRNA ,~ GC Sequence (3' to 5')
_
MBG 15 methylphosphonate 60% g tac cac gtg gac tG
MBG 15 methylphosphonate-
C2amine 60% g tac cac gtg gac tGp-O-(CH2)2-NH2
~G 15 methylphosphonate 60% g tac cac gtg gac ~Gp-~(CH2)2
DTPA conjugate -NH-C(O)-DTPA
MBG 20 antisense 55% G TAC CAC GTG CAC TGA CTA C
MBG 20 antisense 55% G TAC CAC GTG CAC TGA CTA
- C2-amine Cp O (CH2)2 NH2
MBG 20 antisense 55% G TAC CAC GTG GAC TGA CTA
C6-amine C-O-(CO)-NH-(CH2)6-NH2
MBG 20 antisense 55% G TAC CAC GTG CAC TGA CTA
C~,-amine DTPA Cp-O-(CH2)2-NH-C(O)-DTPA
conjuga~e
., _
13191 lq
31
a) Lower case letters represent nucleosldes coupled to the 3'
adjacent nucleoside via a methylphosphonate linkage. Upper case
letters represent 3' adjacent normal phosphodiester 1inkage. C
derivatives are formed from the conden~ation of ethanolamine wi~h
a 5' terminal phosphate via an ester linkage. C6 and C10
derivatives are the corresponding diamines coupled via an alkyl
carbamate linkage to the 5' terminal hydroxyl. DTPA represents
diethylenetriamine pentaacetic acid.
The data in the ~ollowing Table (Table II)
show that DTPA conjugated oligomers were approximately
500 times more effective than control oligomers with or
without the amine linker attached. Significant cyto-
toxicity was observed only at concentrations above
10 ~M, about 100 times the minimum dose for a
significant effect.
3o
13191 19
32
TABLE II
The Effect of Cleaver Conjugate Oligonucleotides
in Preventing the Synthesis of Hemoglobin
in Cultured Cells
_ _ _
% Inhibition
Treatment (1) Conc. ~ Viable Cells Clobin+ Cells
Experiment #1 - ~ffect Compared to Other Constructs.
DMSO Control 46% 0%
10 MBG-20 Antisense 50 ~M 50% 41~
MBG-20-C2amine 50 ~M 61% 41%
MBG-20-DTPA 50 ~M 0% 100%
Conjugate
Experiment #2 - Concentration Dependence of DTPA Conjugate
DMSO Control 1.5% 65% 0%
Solvent Control n.d 0%
EDTA Control 1 ~M 65% 0%
MBG-20-DTPA 1 ~M 50% 98%
Conjugate 57% 96%
500 nM 34% 95%
14% 96%
300 nM 65% 84%
65% 84%
200 nM 78% 58%
100 nM 69% 56g
76% 60%
. - 10 nm 67% 47%
74% 61%
1 nM 70~ 41%
3 0.1 nM 72% 2%
none nd 0%
33 13191 19
Table II
(continued)
Experlment t3 - E~fect o~ ~ethylphosphonate DTPA Con~ugate
5 Treatment (1) Conc. '~ Viable Cell~ S Inhibitlon
DMS0 Control 50~ 0%
MBG 15 methyl- 100 nM 50~ 48%
pho~phonate 10 nM 51~ 43%
DTPA Con~ugate 1 nM 54% 3f%
0.1 nM 54% 37%
(1) C2 derivative~ are formed from the condensation o~
ethanolamine with a 5' terminal phsophate Yia an e~ter
linkage. DTPA repre~ent~ diethylenetriamine pentaacetic
acid.
It is evident from the above result~, that
conjugate~ o~ a chelating agent and an oligonucleotide
~equence mar be used to preferentially inhibit the
- 20 expre~sion o~ a sequence in a viable cell. In this manner,
cell~ can be modified in a variety of ways, ~o a~ to change
the phenotype or to ~electively kill cells. The con~ugate
i9 ~table and does not require that a metal be non-
covalently bound to the chelating agent prior to use in
order to achieve ef~ectivene~s. In addition, the subJect
compo~itions can be used in vi~o or in vitro, allowing ~or
selection of cells, enhancing actiYity of particular cells,
reducing actlvity o~ particular cells, or permitting ~elec-
tion Or a particular clas of cells. A Yariety of con~u-
3 ~ates can be produced, which will ha~e long hal~ live~, ~oa3 to be able to pro~ide for destruction of a large number
Or RNA sequences for each molecule con~ugate.
13191 19
34
Although the foregoing invention has been deæcribed
in some detail by way of illustration and example for
purposes of clarity of understanding, it will be obvious
that certain changes and modifications may be practiced
within the scope of the appended claims.
