Oligonucleotide analogues

Details Oligonucleotide analogues Oligonucleotide analogues

1998-10-09

2003-05-13

McGarry, Sean (Department: 1635)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C536S023100, C536S024500, C435S091100

Reexamination Certificate

active

06562960

ABSTRACT:

This invention relates to oligonucleotide analogues, their preparation and their use.
In accordance with the invention, oligonucleotide analogues can be prepared which have good hybridisation properties to single- and double-stranded nucleic acids, RNase H-activating properties, good hydrolytic stability and good stability towards cleavage by nucleases, facilitating their use as inhibitors of gene expression, for example by antisense interaction, and as pharmaceuticals in the treatment of diseases such as cancer and viruses such as influenza, herpes and HIV.
Accordingly, the present invention provides an oligonucleotide analogue having 10 to 200 natural and/or synthetic nucleoside units linked by internucleoside linkages, at least one of the internucleoside linkages being of formula
where the indicated methylene group is attached to a 3′ carbon atom of a nucleoside, the indicated oxygen atom is attached to a 5′ carbon atom of an adjacent nucleoside, R
1
is hydrogen, hydroxy, O
−
, thiol, S
−
, —NH
2
or a group of formula R
1
a
, —OR
1
a
, —SR
1
a
, —NHR
1
b
or —NR
1
b
R
1
c
where R
1
a
is an unsubstituted or substituted C
1
to C
10
alkyl, C
2
to C
10
alkenyl, C
3
to C
8
cycloalkyl, C
6
to C
10
aryl or C
7
to C
13
aralkyl group and R
1
b
and R
1
c
are each independently an unsubstituted or substituted C
1
to C
10
alkyl, C
2
to C
10
alkenyl, C
3
to C
8
cycloalkyl, C
6
to C
10
aryl or C
7
to C
13
aralkyl group or R
1
b
and R
1
c
together with the nitrogen atom to which they are attached denote a five- or six-membered hetercyclic ring, and X is oxygen or sulphur.
The number of nucleoside units in the oligonucleotide analogue may vary, for example from 15 to 100, according to the nature of the nucleic acid sequence to which the oligonucleotide analogue is targeted. Preferably, the oligonucleotide analogue has 15 to 40, especially 15 to 25 nucleoside units. The oligonucleotide analogue may more preferably have 15 to 20 nucleoside units for certain targets, 20 to 25 nucleoside units for other targets, 18 to 25 nucleoside units for further targets and 18 to 22 nucleoside units for yet further targets.
In an oligonucleotide analogue of the invention, the number of internucleoside linkages of formula I may vary according to the properties desired. For example, for some purposes one internucleoside linkage of formula I may suffice, while for other purposes all the internucleoside linkages may be of formula I and may be the same or different. For most purposes, up to 75%, for example up to 50%, particularly up to 25%, of the internucleoside linkages may be of formula I.
In some embodiments of the invention, at least two consecutive internucleoside linkages, for example two, three, four, five or six consecutive internucleoside linkages, which may be the same or different, in the oligonucleotide analogue are of formula I. There may be such a sequence of consecutive internucleoside linkages at each end of the oligonucleotide analogue; more usually, there is one such sequence of consecutive internucleoside linkages of formula I between sequences of nucleosides having other internucleoside linkages. In other embodiments of the invention having two or more internucleoside linkages of formula I, internucleoside linkages of formula I may alternate with other internucleoside linkages, for example along the whole length of the oligonucleotide analogue or in a region at one or both ends of the oligonucleotide analogue, or in a region in the middle of the oligonucleotide analogue.
In embodiments of the invention where not all of the internucleoside linkages are of formula I, the remaining internucleoside linkages may be natural phosphodiester linkages or other synthetic substitutes therefor such as phosphorothioate, phosphorodithioate, alkylphosphonate (—O—P(O)(R)O—), phosphoramidate, short chain alkyl, cycloalkyl, short chain heteroatomic, —NHCOCH
2
—, —CH
2
NHCO—, —CONHCH
2
—, —CH
2
CONH—, —CH
2
NHO—, —CH
2
N(CH
3
)O—, —CH
2
ON(CH
3
)—, —CH
2
N(CH
3
)N(CH
3
)— or —ON(CH
3
) CH
2
— linkages, or combinations of two or more such linkages. Preferably, the remaining internucleoside linkages are phosphodiester, phosphorothioate or phosphorodithioate linkages or a mixture of two or more of these three types, particularly phosphodiester, phosphorothioate or a mixture of phosphodiester and phosphorothioate linkages. In certain especially preferred embodiments the remaining internucleoside linkages are phosphorothioate linkages.
Preferably, not more than 50% of the internucleoside linkages are phosphorothioate linkages.
In certain embodiments of the invention, the oligonucleotide comprises a region having phosphodiester and/or phosphorothioate and/or phosphorodithioate internucleoside linkages between two regions having internucleoside linkages of formula I, or a mixture thereof with phosphorothioate or phosphodiester linkages, particularly a region having phosphorothioate linkages between two regions having internucleoside linkages of formula I or a mixture thereof with phosphorothioate or phosphodiester linkages.
In some especially preferred embodiments, the oligonucleotide analogue of the invention comprises a region of at least 6 nucleosides linked by phosphorothioate linkages between two regions having nucleosides linked only by internucleoside linkages of formula I.
In oligonucleotide analogues of the invention, the nucleoside units may be natural or synthetic nucleosides having a purine or pyrimidine base such as adenine, guanine, cytosine, thymine or uracil, or an analogue of these bases such as 2-aminoadenine, 6-hydroxypurine, 5-methylcytosine, 5-propynylcytosine, 5-fluorouracil, 5-propynyluracil or dihydrouracil, attached to the I′ carbon atom of a furanose sugar. As is well understood by those skilled in the art, when the oligonucleotides are for use in antisense applications, the sequence of nucleosides is chosen to be complementary to a target RNA sequence. For example, the oligonucleotide analogue of the invention may be complementary to a region of mRNA for human c-raf kinase, in which case, a preferred sequence is
5′-TCC CGC CTG TGA CAT GCA TT-3′ SEQ ID NO:1.
described as Seq. ID No. 8 in WO 95/32987 or the oligonucleotide analogue of the invention may be complementary to a region of mRNA for human PKC-&agr;, in which case a preferred sequence is
5′-GTT CTC GCT GGT GAG TTT CA-3′ SEQ ID NO:2
described as Seq. ID No. 2 in WO 95/02069.
In some oligonucleotide analogues of the invention, at least one nucleoside is modified at the 2′ position thereof, for example to increase binding affinity for a given target and/or to increase nuclease resistance. All of the nucleosides may be so modified, or up to 80%, for example up to 70%, up to 60%, up to 50%, up to 40%, up to 30%, up to 20%, or up to 10%, of the nucleosides, may be so modified. Examples of 2′ modifying atoms and groups, i.e. atoms or groups which may be attached to the 2′ position of a nucleoside in place of a hydrogen atom or hydroxy group to effect a modification, include halogen atoms such as fluorine, chlorine and bromine atoms; C
1
to C
10
unsubstituted or substituted alkyl groups such as methyl, trifluoromethyl, ethyl, propyl, butyl, pentyl, hexyl, octyl or decyl; C
6
to C
10
aryl groups such as phenyl, tolyl or xylyl; C
7
to C
13
aralkyl groups such as benzyl; amino, C
1
to C
10
alkyl amino such as methylamino, ethylamino or octylamino; C
1
to C
10
alkylthio such as methylthio, ethylthio or octylthio; azide; nitrate; nitrite; cyanide; cyanate; methanesulphonate; C
1
to C
10
aminoalkylamino; a group of formula —OR
2
where R
2
is a C
1
to C
10
aliphatic group; substituted silyl; an RNA cleaving group; a cholesteryl group; a conjugate; a reporter group; an intercalator, a group for improving the pharmacokinetic properties of an oligonucleotide; and a group for improving the pharmacodynamic properties of an oligonucleotide.
Preferred modifying atoms and groups at the 2′ position are halogen atoms, especially fluori

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