Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2001-05-04
2004-08-31
Lacourciere, Karen A. (Department: 1635)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C536S024300, C536S024310, C536S024330, C435S006120, C435S091100
Reexamination Certificate
active
06784291
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to therapeutic compositions and methods for inhibiting expression of full-length proteins in cells, and in particular to antisense compositions targeted against an MRNA sequence having its 5′ end 1 to about 25 base pairs downstream of a normal splice acceptor junction in a preprocessed mRNA. Such targeting is effective to inhibit natural mRNA splice processing and produce splice variant mRNAs.
BACKGROUND OF THE INVENTION
Inhibition of protein expression by antisense targeting of DNA or RNA coding for the protein has been the subject of extensive study. Many reported procedures have employed phosphorothioate-linked oligonucleotides, which are charged, nuclease-resistant analogs of native DNA. The antisense mechanism involved is based on the activation of RNase, which cleaves the target nucleic acid to which the oligomer is bound. While these compounds have shown high activity, they also tend to show high levels of side effects, i.e. by cleavage of non-target RNA or by non-antisense mechanisms, such as nonspecific binding to proteins.
Another class of antisense oligomers, termed RNase-inactive, do not promote cleavage of bound RNA and are believed to act by sterically blocking the molecular machinery from transcribing, processing, or translating the target sequence. While these compounds tend to produce fewer side reactions, such as nonselective cleavage, than phosphorothioate oligomers, it has generally been necessary to target specific regions of RNA, such as the AUG start codon, for successful inhibition.
More recently, targeting of the splice acceptor junction of nuclear (unspliced) RNA by RNase-inactive oligomers has been reported. Kole and Dominski (U.S. Pat. No. 5,665,593) reported suppression of missplicing of &bgr;-globin RNA, in order to combat variants of &bgr;-thalassemia which result from such aberrant splicing. In this case, the aberrant splice junction was targeted, to direct splicing back to the normal site. R V Giles et al.,
Antisense & Nucleic Acid Drug Dev.
9:213-220 (1999), targeted a splice junction to induce missplicing of c-myc mRNA. In each of these cases, the region targeted is still somewhat restricted, in that the antisense oligomer spans the intron/exon splice junction of the pre-mRNA. Due to the advantages accorded by the use of uncharged, RNase-inactive oligonucleotides, a demonstration of further flexibility in targeting would be quite useful.
SUMMARY OF THE INVENTION
In one aspect, the invention provides an antisense compound, and a corresponding method of inhibiting normal splicing of preprocessed RNA in a eukaryotic cell, by contacting the cell with such an antisense compound. The compound is characterized by:
(a1): an uncharged morpholino backbone;
(a2): a base-sequence length of between 12 and 25 nucleotide bases; and
(a3): a base sequence that is complementary to a target region of a selected preprocessed mRNA coding for a selected protein, where the 5′ end of the target region is 1-25 bases downstream of a normal splice acceptor site in the preprocessed mRNA, and having the properties that:
(b1): the compound is taken up by eukaryotic cells;
(b2): the compound hybridizes to the target region of preprocessed mRNA in such cells, and
(b3): the compound so hybridized to the target pre-mRNA prevents splicing at the normal acceptor splice site, such that the splice mechanism proceeds to a downstream splice acceptor site in the preprocessed mRNA, producing a splice variant processed MRNA with a truncated coding sequence.
In more specific embodiments, the 5′ end of the target region is 2-20 bases, or 2-15 bases, downstream of the normal splice acceptor site. The length of the targeting compound is preferably about 15 to 20 nucleotide bases.
In one embodiment, the compound has intersubunit linkages selected from the group consisting of the structures presented in FIGS.
2
AA-
2
EE. In preferred embodiments, the linkages are selected from a phosphorodiamidate linkage as represented at
FIG. 2B-B
, where X=NH
2
, NHR, or NRR′, Y=O, and Z=O, and an alternate phosphorodiamidate linkage as represented at
FIG. 2B-B
, where X=OR, Y=NH or NR, and Z=O. R and R′ are groups which do not interfere with target binding. Preferably, R and R′ are independently selected from alkyl and polyalkyleneoxy (e.g. PEG; (CH
2
CH
2
O)
n
), or a combination thereof. The alkyl/polyalkyleneoxy chain may be substituted, preferably at the distal terminus, by a group selected from hydroxy, alkoxy, amino, alkylamino, thiol, alkanethiol, halogen, oxo, carboxylic acid, carboxylic ester, and inorganic ester (e.g. phosphate or sulfonate). Preferably, the chain (independent of substituents) is from 1 to 12 atoms long, and more preferably is from 1 to 6 atoms long. In selected embodiments, R and R′ are independently methyl or ethyl. In one embodiment, X=N(CH
3
)
2
, Y=O, and Z=O.
NRR′ may also represent a nitrogen heterocycle having 5-7 ring atoms selected from nitrogen, carbon, oxygen, and sulfur, and having at least as many carbon ring atoms as non-carbon ring atoms. Examples include morpholine, pyrrolidine, piperidine, pyridine, pyrimidine, pyrazine, triazine, triazole, pyrazole, pyrrole, isopyrrole, imidazole, oxazole, imidazole, isoxazole, and the like.
When the downstream splice acceptor site is a whole multiple of three bases downstream of the normal splice acceptor site, the splice variant mRNA has a coding sequence in frame with that of the processed mRNA when it is normally spliced.
The protein is preferably selected from the group consisting of myc, myb, rel, fos, jun, abl, bcl, p53, an integrin, a cathedrin, a telomerase, hCG, a receptor protein, a cytokine, a kinase, HIV rev, human papilloma virus, and human parvovirus B19. In selected embodiments, the protein is selected from myc, myb, abl, p53, hCG-&bgr;subunit, androgen receptor protein, and HIV-1 rev.
In further selected embodiments, the selected protein has multiple distinct binding regions, as in most transcription factors, and the truncated coding sequence codes for a variant protein in which one such binding region is disabled. Preferably, the variant protein is a dominant negative protein. One example is human c-myc, where the variant protein is an N-terminal truncated c-myc. In this embodiment, the antisense compound employed has a base sequence selected from the group consisting of SEQ ID NOs: 16 through 32 herein. The variant protein may also be a C-terminal altered c-myc, in which case the antisense compound employed can be an 18- to 20-mer having a base sequence which is a contiguous sequence selected from SEQ ID NO: 34; e.g. SEQ ID NO: 33.
In additional exemplary embodiments, the selected protein and the corresponding antisense base sequence(s) targeting its pre-mRNA are selected from the group consisting of:
(a) human chorionic gonadotropin, &bgr; subunit: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 15; e.g. SEQ ID NO: 14;
(b) human androgen receptor: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 9 or SEQ ID NO: 13; e.g. SEQ ID NO: 8 or 12, respectively;
(c) human p53: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 36; e.g. SEQ ID NO: 35;
(d) human abl: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 38; e.g. SEQ ID NO: 37; and
(e) HIV-1 rev: a contiguous 18- to 20-nucleotide sequence selected from SEQ ID NO: 41; e.g. SEQ ID NO: 40.
These and other objects and features of the present invention will become more fully apparent when the following detailed description of the invention is read in conjunction with the accompanying drawings.
REFERENCES:
patent: 5665593 (1997-09-01), Kole et al.
patent: 5811537 (1998-09-01), Friesen
patent: 6046319 (2000-04-01), Power et al.
patent: 6171798 (2001-01-01), Levine et al.
patent: 6183964 (2001-02-01), Boeke et al.
patent: WO 97/34638 (1997-09-01), None
Green et al. Antisense Oligonucleotides: An Evolving Technology for t
Hudziak Robert
Iversen Patrick L.
AVI BioPharma Inc.
Gorthey LeeAnn
Lacourciere Karen A.
Perkins Coie LLP
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